( B and C ) Quantification of the number of total GFP - LC3 puncta ( B ) and GFP - LC3 puncta colocalized with TOM20 ( C ) in the panel ( A ) . ( D ) Quantitative analysis of the area of GFP - LC3 puncta ( white ) representing merge of GFP - LC3 puncta , TOM20 , and Parkin in the panel ( A ) .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O

(B and C) Quantification of the number of total GFP-LC3 puncta (B) and GFP-LC3 puncta colocalized with TOM20 (C) in the panel (A). (D) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A).


( B ) Huh7 cells transiently expressing Mito - mRFP - EGFP were transfected with HBV , HBx - flag , and HBV - ΔX constructs , respectively , for 48 h . Cells were immunostained with antibodies specific to HBsAg and flag ( white ) , respectively . Nuclei were stained with DAPI ( blue ) . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , fluorescence signals indicate the expression of Mito - mRFP - EGFP targeting mitochondria : yellow color , no mitophagy ; red color , mitophagy .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O

(B) Huh7 cells transiently expressing Mito-mRFP-EGFP were transfected with HBV, HBx-flag, and HBV-ΔX constructs, respectively, for 48 h. Cells were immunostained with antibodies specific to HBsAg and flag (white), respectively. Nuclei were stained with DAPI (blue). Transfected (+) and untransfected (−) cells are marked. In the zoomed images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color, no mitophagy; red color, mitophagy.


( C ) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel ( B ) .

O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B).


( D ) GFP - LC3 - expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells prestained with LysoTracker ( Lyso , red ) were immunostained with antibodies against HBsAg ( white ) and TOM20 ( blue ) . Nuclei are demarcated with white dot circles . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoom images , the arrows denoting white puncta indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and lysosome . ( E ) Quantification of the colocalization of GFP - LC3 puncta containing lysosome with mitochondria in the panel ( D ) .

O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O

(D) GFP-LC3-expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).


( A - C ) Parkin silencing accelerates HBV - induced mitochondrial apoptotic signaling . HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non - targeting ( NT ) and Parkin siRNA , respectively , for 72 h . Cells were used for Western blot analysis using antibodies specific to the indicated proteins ( A ) , caspase - 3 / 7 activity assay ( B ) , and TUNEL assay ( C )

O O O O O B-GENEPROD O O B-ORGANISM O O B-SUBCELLULAR O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O

(A-C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C)


( B - C ) Representative images ( B ) and quantification ( C ) of Oil Red O staining of hypoxia - and normoxia - cultured cells , 9 days after induction of adipogenesis . n = 5 biologically independent replicates .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

(B-C) Representative images (B) and quantification (C) of Oil Red O staining of hypoxia- and normoxia-cultured cells, 9 days after induction of adipogenesis. n=5 biologically independent replicates.


( D - E ) Representative images ( D ) and quantification ( E ) of Alizarin Red S staining of hypoxic , normoxic and reversed hypoxic ( R _ 2 % O2 ) cells , 12 days after induction of osteogenesis . Cells were exposed to 21 % O2 for 7 days and then moved back to 2 % O2 , where osteogenesis was induced after 4 days . n = 3 for hypoxic cells and n = 5 biologically independent experiments for normoxic and reversed - hypoxic cells and merged results are shown in ( E ) .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Representative images (D) and quantification (E) of Alizarin Red S staining of hypoxic, normoxic and reversed hypoxic (R_2% O2) cells, 12 days after induction of osteogenesis. Cells were exposed to 21% O2 for 7 days and then moved back to 2% O2, where osteogenesis was induced after 4 days. n=3 for hypoxic cells and n=5 biologically independent experiments for normoxic and reversed-hypoxic cells and merged results are shown in (E).


( C - D ) Representative images ( C ) and quantification ( D ) of Mean Fluorescence Intensity ( MFI ) after immunostaining against H3ac and H3 of hypoxic and normoxic cells . Mean Fluorescence Intensity ( MFI ) of histone H3 was used as internal control , for normalization . Nuclei were stained with DAPI . Quantification of H3ac / H3 MFI from n = 67 hypoxic and n = 56 normoxic individual cells from a representative experiment out of 3 biologically independent experiments is shown in D . Distribution of data points in ( D ) is shown as a violin plot , where the mean is indicated by a solid line and the quartiles are indicated with dashed lines .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Representative images (C) and quantification (D) of Mean Fluorescence Intensity (MFI) after immunostaining against H3ac and H3 of hypoxic and normoxic cells. Mean Fluorescence Intensity (MFI) of histone H3 was used as internal control, for normalization. Nuclei were stained with DAPI. Quantification of H3ac/H3 MFI from n=67 hypoxic and n=56 normoxic individual cells from a representative experiment out of 3 biologically independent experiments is shown in D. Distribution of data points in (D) is shown as a violin plot, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines.


( J ) Representative immunoblots for ACS and ACLY in hypoxia - and normoxia - cultured cells . α - tubulin was used as loading control .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(J) Representative immunoblots for ACS and ACLY in hypoxia- and normoxia-cultured cells. α-tubulin was used as loading control.


( A - B ) Representative images after immunostaining of hypoxia - and normoxia - cultured cells against acetyl - Lysine and TOMM20 ( A ) , and assessment of the acetyl - Lysine signal localization , after manual assignment into three categories : exclusively nuclear , exclusively mitochondrial and nuclear / mitochondrial ( B ) . Nuclei were stained with DAPI . n = 3 biologically independent experiments . Results are shown as mean ± S . E . M and statistical significance was determined using two - sided unpaired t - test . In magnified insets intensity of the acetyl - Lysine signal was adjusted similarly to all samples , for visualization purposes .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

(A-B) Representative images after immunostaining of hypoxia- and normoxia-cultured cells against acetyl-Lysine and TOMM20 (A), and assessment of the acetyl-Lysine signal localization, after manual assignment into three categories: exclusively nuclear, exclusively mitochondrial and nuclear/mitochondrial (B). Nuclei were stained with DAPI. n=3 biologically independent experiments. Results are shown as mean ± S.E.M and statistical significance was determined using two-sided unpaired t-test. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.


( B - C ) Representative images ( B ) and quantification ( C ) of Nile Red staining of hypoxic control and BTA - treated cells and of normoxic control and acetate - treated cells . Treatments were done for three days with 1mM BTA or 5mM acetate .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

(B-C) Representative images (B) and quantification (C) of Nile Red staining of hypoxic control and BTA-treated cells and of normoxic control and acetate-treated cells. Treatments were done for three days with 1mM BTA or 5mM acetate.


Representative images after immunostaining of hypoxic control and BTA - treated cells and normoxic control and acetate - treated cells against acetyl - Lysine and TOMM20 ( D ) , assessment of the acetyl - Lysine signal localization , as described above ( E ) Nuclei were stained with DAPI . In magnified insets intensity of the acetyl - Lysine signal was adjusted similarly to all samples , for visualization purposes .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

Representative images after immunostaining of hypoxic control and BTA-treated cells and normoxic control and acetate-treated cells against acetyl-Lysine and TOMM20 (D), assessment of the acetyl-Lysine signal localization, as described above (E) Nuclei were stained with DAPI. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.


Representative images after immunostaining of hypoxic control and BTA - treated cells quantification of nuclear acetyl - lysine MFI ( F ) . Nuclei were stained with DAPI . Quantification of nuclear acetyl - Lysine MFI from n = 67 hypoxic , n = 71 hypoxic _ BTA , n = 44 normoxic and n = 63 normoxic _ acetate individual cells from a representative experiment out of 3 biologically independent experiments is shown in ( F ) . In magnified insets intensity of the acetyl - Lysine signal was adjusted similarly to all samples , for visualization purposes .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O

Representative images after immunostaining of hypoxic control and BTA-treated cells quantification of nuclear acetyl-lysine MFI (F). Nuclei were stained with DAPI. Quantification of nuclear acetyl-Lysine MFI from n=67 hypoxic, n=71 hypoxic_BTA, n=44 normoxic and n=63 normoxic_acetate individual cells from a representative experiment out of 3 biologically independent experiments is shown in (F). In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.


( G - H ) Representative images and quantification of Alizarin Red S staining of control hypoxic and normoxic cells and of normoxic acetate - treated cells , 12 days after induction of osteogenesis . Merged results of n = 5 biologically independent experiments for hypoxic and normoxic cells and n = 4 biologically independent experiments for acetate - treated normoxic cells are shown in ( H ) .

O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(G-H) Representative images and quantification of Alizarin Red S staining of control hypoxic and normoxic cells and of normoxic acetate-treated cells, 12 days after induction of osteogenesis. Merged results of n=5 biologically independent experiments for hypoxic and normoxic cells and n=4 biologically independent experiments for acetate-treated normoxic cells are shown in (H).


( A ) Wild ‐ type and rho0 cells harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to amino ‐ acid or nitrogen starvation ( ‐ N ) medium supplemented with indicated carbon sources . Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α ‐ GFP and α ‐ Cdc11 antibodies . Quantification of ATG8 induction is shown in the lower left panel . Total GFP signals ( GFP ‐ Atg8 and free GFP ) were quantified and normalized to Cdc11 signals . Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation . Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals ( GFP ‐ Atg8 and free GFP ) after 6 h starvation . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Fluorescence microscopical analysis . Wild ‐ type , rho0 , and Δatg7 cells expressing prATG8 ‐ GFP ‐ ATG8 were grown as described in ( A ) and exposed to amino ‐ acid ( left panel , galactose ) or nitrogen starvation ( right panel , glucose ( ‐ N ) ) for 6 h . Vacuoles were visualized by over night FM4 ‐ 64 ( 1 μM ) staining ( red ) . Arrowhead indicates a punctate GFP ‐ Atg8 structure . Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries . Cellular localization of GFP signal was analysed in at least 150 cells ( n ⩾ 150 ) for each strain and condition . Scale bars represent 1 . 5 μm .

O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.


Autophagic response in cells with compromised respiratory chain complex III , IV , or V activity during amino ‐ acid starvation . Wild ‐ type , rho0 , atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III ( CIII : cbs1 ) , complex IV ( CIV : mss51 , pet111 , pet122 ) , or the F1Fo ‐ ATP synthase ( complex V ; CV : atp10 ) were exposed to amino ‐ acidstarvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type cells were exposed to antimycin A ( AA ) or oligomycin ( O ) during the amino ‐ acid starvation period . All strains expressed prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) . Samples were analysed as described in Figure 1A . The means and s . d . of five ( n = 5 ) independent experiments are indicated .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.


( A , B ) Wild ‐ type , rho0 , and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type cells were exposed to antimycin A ( AA ) or oligomycin ( O ) during the amino ‐ acid starvation period . ATP and protein from total cells ( upper panels ) or isolated mitochondria ( lower panels ) were determined at indicated time points .

O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points.


( C ) Wild ‐ type and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with acetate or galactose for 3 h . Wild ‐ type cells were exposed to antimycin A ( AA ) , oligomycin ( O ) , or CCCP ( 50 μM ) during the amino ‐ acid starvation period when indicated . Cells were treated with the mitochondrial membrane potential ‐ dependent dye DiOC6 ( 3 ) and examined by fluorescence microscopy . Average pixel intensities for at least 10 mitochondrial tubules ( n = 10 ) in 5 representative cells were determined for each condition . Values for wild ‐ type mitochondria in the presence of acetate were defined as 100 % and relative values were calculated accordingly . The means and s . d . are shown .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.


( A ) Wild ‐ type , rho0 , Δnpr2 , and Δnpr2 rho0 cells expressing prATG8 ‐ GFP ‐ ATG8 ( upper panel ) or prATG8 ‐ GFP ( lower panel ) were exposed to amino ‐ acid starvation medium supplemented with galactose in the absence or presence of rapamycin . Samples were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Wild ‐ type , rho0 , Δnpr2 , and Δatg7 cells expressing prNPR1 ‐ NPR1 ‐ HA were exposed to amino ‐ acid starvation medium supplemented with galactose in the absence ( upper panels ) or presence ( lower panels ) of rapamycin . The hyperphosphorylated ( Npr1 ‐ P ) and dephosphorylated ( Npr1 ) forms of Npr1 are indicated . Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α ‐ HA and α ‐ Cdc11 antibodies .

O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O

(B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies.


( C ) Wild ‐ type , rho0 , Δnpr2 , and Δatg7 cells were exposed to amino ‐ acid starvation medium supplemented with galactose . Additionally , wild ‐ type cells were treated with rapamycin during starvation ( left panel ) . Phosphorylated ( Atg13 ‐ P ) and dephosphorylated ( Atg13 ) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α ‐ Atg13 and α ‐ Cdc11 antibodies .

O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O

(C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.


( A ) PKA ‐ dependent regulation of the autophagic response under amino ‐ acid starvation . Wild ‐ type , rho0 , pka , and ras2G19V ‐ expressing cells harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to amino ‐ acid starvation medium supplemented with galactose . PKA activity in pka was inhibited by addition of 1NM ‐ PP1 ( PP1 ; 1 μg / ml ) . Samples were analysed as described in Figure 1A ; autophagic flux was determined after 3 and 6 h .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h.


( B ) In vivo activity of PKA . Wild ‐ type , rho0 , pka , and ras2G19V ‐ expressing cells harbouring 6xMYC ‐ cki12 − 200 ( S125 / 130A ) ( Cki1 ) were grown in galactose medium . When indicated , wild ‐ type cells were grown in galactose medium in the presence of antimycin A ( AA ) or oligomycin ( O ) for 6 h . PKA ‐ dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α ‐ Myc antibody ( upper panels ) . Ratio of phosphorylated ( Cki1 ‐ P ) and non ‐ phosphorylated ( Cki1 ) forms of Cki1 relative to wild ‐ type cells ( wt = 1 ) ( lower panels ) .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O

(B)In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels).


( C ) PKA inhibition restores autophagic flux , but not ATG8 induction in the presence of mitochondrial dysfunction . Wild ‐ type , rho0 , pka , and pka rho0 cells expressing prATG8 ‐ GFP ‐ ATG8 were treated as described in ( A ) . Samples were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated in ( A - C ) .

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild‐type, rho0, pka, and pka rho0 cells expressing prATG8‐GFP‐ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (A-C).


( A ) ATG8 induction is independent of the Atg1 - Atg13 complex and autophagic flux . Wild ‐ type , rho0 , Δatg1 , Δatg7 , Δatg9 , and Δatg11 cells harbouring prATG8 ‐ GFP ‐ ATG8 were analysed as described in Figure 1A . The means and s . d . of four ( n = 4 ) independent experiments are indicated .

O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A)ATG8 induction is independent of the Atg1-Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function . Wild ‐ type and rho0 cells harbouring prATG8 ‐ GFP ‐ ATG8 and prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed to amino ‐ acid starvation medium supplemented with galactose for 3 h . Wild ‐ type cells were treated with antimycin A ( AA 30 ′ ) after 2 . 5 h of starvation for 30 min or with oligomycin ( O ) for 3 h of starvation . Arrowheads indicate the position of GFP ‐ Atg8 puncta . Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries . Scale bar represents 1 . 5 μm .

O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm.


( C ) Steady ‐ state levels of Atg1 ‐ and Atg13 ‐ mCherry during amino ‐ acid starvation . Wild ‐ type , rho0 , and Δatg7 cells expressing prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed and treated as described in ( B ) and analysed by whole cell extraction and western blot analysis using α ‐ dsRed and α ‐ Cdc11 antibodies .

O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O

(C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.


Geometric mean - centered , hierarchical cluster heat map from microarray data . 1077 annotated lncRNAs ( p < 0 . 05 ) were represented in liver CSC ( CD13 + CD133 + ) compared with non - CSC ( CD13 - CD133 - ) cells sorted from three HCC primary cells of three patients . Top ten upregulated lncRNAs in CSCs are shown .

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-DISEASE O O O O O O O O O O O O O O O

Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown.


In situ hybridization of HAND2 - AS1 in HCC tumor tissues . HAND2 - AS1 staining is shown in tumor tissues ( left panel ) . Quantitation of HAND2 - AS1 + cells in liver tissues was examined in 10 high - power fields ( HPF ) of sections from ten different HCC patients ( right panel ) . Results are shown as means ± SEM . Scale bar , 100 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O

In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.


Macroscopic appearance of livers in 12 - month - old DEN - treated lncHand2 + / + and lncHand2 knockout ( lncHand2 - / - ) mice ( left panel ) . Black arrows indicate liver tumors . Quantitation of tumor foci numbers and areas in DEN - treated livers ( right panel ) . Results are shown as means ± SD ( n = 12 ) .

O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O O

Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).


Representative H & E and immunohistological staining with Ki67 antibody of liver sections of 12 - month - old DEN - treated lncHand2 + / + and lncHand2 - / - mice . Scale bar , 100 μm .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O

Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.


Quantitation of Ki67 - positive cells in non - treated and DEN - treated mice . Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice . Data are shown as means ± SD .

O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O

Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.


Representative immunofluorescence staining of livers in 5 . 5 - month - old DEN - treated and untreated lncHand2RFP reporter mice for indicated molecules ( lower panel ) . Scale bar , 100 μm . Upper panel : scheme of targeting strategy for IRES - RFP knockin allele .

O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O

Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.


HAND2 - AS1 knockout causes a declined oncosphere - forming capacity in HCC cells . The right panel represents statistical results as means ± SD ( n = 3 per group ) . Overexpression of HAND2 - AS1 ( oelnc ) rescued the sphere formation reduced by HAND2 - AS1 deletion . Scale bar , 100 μm .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

HAND2-AS1 knockout causes a declined oncosphere-forming capacity in HCC cells. The right panel represents statistical results as means ± SD (n=3 per group). Overexpression of HAND2-AS1 (oelnc) rescued the sphere formation reduced by HAND2-AS1 deletion. Scale bar, 100 μm.


CD13 + CD133 + ( CSC ) subpopulations were detected in HAND2 - AS1 knockout cells by FACS analysis . Results are shown as means ± SD ( n = 4 per group ) .

B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

CD13+CD133+ (CSC) subpopulations were detected in HAND2-AS1 knockout cells by FACS analysis. Results are shown as means ± SD (n=4 per group).


Limiting diluted HAND2 - AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB / c nude mice . n = 12 for each group .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O O O O O O O O B-ORGANISM O O O O O O O O

Limiting diluted HAND2-AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB/c nude mice. n=12 for each group.


Estimated frequency of TICs in HAND2 - AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis . Data are shown as the mean and 95 % confidence interval . ( n = 12 grafted tumors per dilution ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

Estimated frequency of TICs in HAND2-AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis. Data are shown as the mean and 95% confidence interval. (n=12 grafted tumors per dilution).


Estimated frequency of TICs in HAND2 - AS1 deficiency and control HCC cells during serial transplantations . Grey area indicates the 95 % CI .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O O O O O O O O O O O O

Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.


Estimated frequency of TICs in different populations after the first transplantation ( n = 15 grafted tumors per dilution ) . Data represent the mean and 95 % confidence interval .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

Estimated frequency of TICs in different populations after the first transplantation (n=15 grafted tumors per dilution). Data represent the mean and 95% confidence interval.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or HAND2 - AS1 KO # 1 or KO # 2 vectors . Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B - NSG mice . Data are shown as means ± SD . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 KO #1 or KO #2 vectors. Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD. One-way ANOVA with Dunnett's correction for multiple comparisons.


HAND2 - AS1 expression levels ( normalized to 18S rRNA ) in livers from BALB / c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg − 1 ASOs on days 15 , 20 , 25 , 30 and 35 ( n = 5 per group ) . Three ASOs ( 1 , 2 , 3 ) against HAND2 - AS1 were used to treat mice and showed similar depletion effects . Results are shown as means ± SD . n = 6 mice per group .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O B-ORGANISM O O B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O O O

HAND2-AS1 expression levels (normalized to 18S rRNA) in livers from BALB/c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg−1 ASOs on days 15, 20, 25, 30 and 35 (n = 5 per group). Three ASOs (1, 2, 3) against HAND2-AS1 were used to treat mice and showed similar depletion effects. Results are shown as means ± SD. n=6 mice per group.


Mice were killed at the 40th day after HCC cell injection , and the tumors were excised and weighed . Representative tumors are shown . Scale bar , 1 cm . Statistical data are shown as means ± SD ( n = 6 mice per group ) ( right panel ) .

B-ORGANISM O O O O O O O B-DISEASE O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O

Mice were killed at the 40th day after HCC cell injection, and the tumors were excised and weighed. Representative tumors are shown. Scale bar, 1 cm. Statistical data are shown as means ± SD (n = 6 mice per group) (right panel).


HAND2 - AS1 overexpression enhances the capacity of oncosphere formation . Scale bar , 100 μm . oe : overexpression . Right panel : Data are shown as means ± SD ( n = 3 per group ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).


Estimated frequency of TICs in HAND2 - AS1 overexpression and control HCC cells during serial transplantations .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O O O O

Estimated frequency of TICs in HAND2-AS1 overexpression and control HCC cells during serial transplantations.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or HAND2 - AS1 overexpression vectors . Quantification for tumor numbers one month after tumor cells orthotopically implanted to B - NSG mice . ( n = 5 mice per group ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O

Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 overexpression vectors. Quantification for tumor numbers one month after tumor cells orthotopically implanted to B-NSG mice. (n = 5 mice per group).


Biotin - RNA pulldowns were performed with nuclear extracts of oncosphere cells using full - length HAND2 - AS1 transcript ( sense ) , antisense , and one HAND2 - AS1 intron control , followed by mass spectrometry . Band 1 : INO80 , band 2 : RUVBL2 .

B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O

Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.


Two core components of the INO80 complex were confirmed by immunoblotting .

O O O O O B-GENEPROD O O O O B-EXP_ASSAY O

Two core components of the INO80 complex were confirmed by immunoblotting.


Immunoprecipitation assays of biotin - labeled CHIRP probes incubated with liver CSC lysates .

B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-TISSUE O O O

Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.


HAND2 - AS1 was visualized by RNA FISH , followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells . Scale bar , 100 μm .

B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-DISEASE O O O O O O O O O O

HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm.


Mapping analysis of INO80 - binding domains of HAND2 - AS1 . Schematic diagram of HAND2 - AS1 full - length and truncated fragments ( top panel ) ; Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2 - AS1 fragments ( middle panel ) ; Different HAND2 - AS1 fragments ( bottom panel ) .

O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel).


EMSA of biotin - labeled HAND2 - AS1 ( nt 83 - 242 ) probes incubated with INO80 protein .

B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O

EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein.


Binding affinity of HAND2 - AS1 with INO80 was determined by five independent EMSA assays . Non - linear regression curves were generated by GraphPad Prism . Results are shown as means ± SD .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD.


Interaction regions of INO80 with HAND2 - AS1 mutations at nt 83 - 242 of exon 2 were detected using RNA pulldown assays . LR , loop region .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or INO80 shRNA # 1 or shRNA # 2 vectors . Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B - NSG mice . Data are shown as means ± SD ( n = 5 mice per group ) . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors. Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons.


Macroscopic appearance of livers in 10 . 5 - month - old DEN - treated Ino80flox / flox ( Ino80 + / + ) and Ino80 knockout ( Ino80 - / - ) ( left panel ) . Black arrows indicate tumors . Quantitation of tumor foci numbers and areas in DEN - treated livers . Results are shown as means ± SD ( n = 10 ) .

O O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O

Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).


Heatmap results for HAND2 - AS1 or INO80 deficiency in HCC cells .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-DISEASE O O

Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells.


Based on GSEA results , significantly changed genes due to depletion of HAND2 - AS1 and INO80 were attributed to the BMP signaling in HCC primary cells . NES , normalized enrichment score ; FWER , familywise error rate .

O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O O O O

Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate.


ChIRP - seq analysis of HAND2 - AS1 genomic binding at target sites in liver CSCs , using LacZ probes as negative control . A 10 - kb interval centered on the called HAND2 - AS1 peak is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown.


Representative ChIRP - seq of HAND2 - AS1 binding of BMPR1A .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O

Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A.


Gel analysis of HAND2 - AS1 RNA binding BMPR1A promoter in liver CSCs . LacZ and PVT1 served as negative controls .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY B-GENEPROD O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O

Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.


HAND2 - AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-DISEASE O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O

HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.


ChIRP - immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2 - AS1 - knockout liver CSCs .

B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O

ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.


EMSA analysis of the interaction of HAND2 - AS1 , INO80 and RUVBL2 with the BMPR1A promoter region .

B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O

EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.


BMPR1A promoter binding region to HAND2 - AS1 / INO80 was deleted ( BMPR1A - p - KO ) in liver CSCs using lenti - Cas9 , followed by HAND2 - AS1 and INO80 overexpression ( oeHAND2 - AS1 , oeINO80 ) for 36 h . BMPR1A mRNA levels were examined by realtime PCR . Data are shown as means ± SD . n = 5 . * * P < 0 . 01 by two - tailed Student ' s t test .

B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.


BMP signaling was examined in INO80 - silenced liver CSCs by Western blot .

B-GENEPROD O O O O B-GENEPROD O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O

BMP signaling was examined in INO80-silenced liver CSCs by Western blot.


BMPR1A is highly expressed in HCC tumor tissues provided by Wang ' s cohort ( GSE14520 ) .

B-GENEPROD O O B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O

BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).


Kaplan - Meier analyses of liver cancer outcomes in the cancer genome atlas ( TCGA ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank test .

O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Representative immunohistochemistry staining of BMPR1A was detected in HCC samples . Scale bar , 100 μm .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-DISEASE O O O O O O O O

Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.


BMPR1A overexpression rescues sphere formation ability reduced by HAND2 - AS1 depletion in HCC samples . Representative sphere formation is shown on the left panel . Scale bar , 100 μm . Percentages of sphere - forming cells were calculated as means ± SD ( right panel ) . n = 4 .

B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.


BMPR1A depletion reduces tumor - initiating capacity . n = 12 mice per group .

B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O

BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.


Representative whole - body imaging of Huh7 - Luc cells transduced with scramble ( Ctrl ) or BMPR1A shRNAs . Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 mice per group ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O

Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).


Macroscopic appearance of livers from 10 - month - old mice treated by DEN or vehicle . Scale bar , 1 cm . Black arrows indicates tumors .

O O O B-TISSUE O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O

Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.


Representative H & E and immunohistological staining with Ki67 antibody in liver sections of 10 - month - old DEN - treated WT and Bmpr1a - / - mice . Scale bar , 100 μm . Right : Quantitation of Ki67 - positive cells in DEN - treated mice . Data are shown as means ± SD . n = 5 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O

Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.


BMP signaling inhibitor noggin decreased tumor sphere formation . Scale bar , 100 μm . Right : Data are shown as means ± SD ( n = 4 ) .

B-GENEPROD O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).


Representative whole - body imaging of Huh7 - Luc cells in different groups . Mice treated with scramble ASOs and / or siRNAs obtained similar results to vehicle treatment . n ≥ 5 mice in each group .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.


Quantification of tumor numbers treated with ASOs and / or siRNAs after Huh7 - Luc tumor cells orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).


Tumors were excised from livers of PDX liver cancer models . Scale bar , 0 . 5 cm .

B-EXP_ASSAY O O O B-TISSUE O O B-DISEASE I-DISEASE O O O O O O O O O O

Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.


Representative H & E staining in liver sections from tumor - bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC # 50 and HCC # 51 . Scale bar , 100 μm .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O B-DISEASE O O O B-DISEASE O O O O O O O O O

Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.


Representative immunohistological staining with Ki67 antibody of tumor - bearing mice treated with ASO1 and siBMPR1A . Scale bar , 100 μm . Lower panel : Quantitation of Ki67 - positive cells in different groups . Data are shown as means ± SD ( n = 5 per group ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).


Northern blotting of HAND2 - AS1 and immunobloting of BMPR1A in tumors from PDX HCC # 50 after treatment .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O B-DISEASE O O O O O

Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.


Kaplan - Meier survival analysis of B - NSG mice orthotopically transplanted with PDXs HCC # 50 ( I ) and HCC # 51 ( J ) . Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone ( n = 10 per group ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O B-DISEASE O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


A Number of differentially up - and down - regulated genes ( DEG ) in RNAi - ASCO1 seedlings as compared to wild type ( WT ) according to the RNA - seq data ( FDR < 0 . 01 , | log2FC | > = 0 . 75 ) . B Fraction of DEG found in each transcript class as defined in the Araport11 gene annotation . AS stands for antisense .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Number of differentially up- and down-regulated genes (DEG) in RNAi-ASCO1 seedlings as compared to wild type (WT) according to the RNA-seq data (FDR < 0.01, |log2FC| >= 0.75). B Fraction of DEG found in each transcript class as defined in the Araport11 gene annotation. AS stands for antisense.


D Representative picture of 14 - day - old plants grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . The scale bar representing 0 . 6cm is included in the picture . E Lateral root density of WT and two independent RNAi - ASCO lines 9 days after transfer in 1 / 2MS supplemented with 0 . 1 µM or 1 µM flg22 .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O

D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture. E Lateral root density of WT and two independent RNAi-ASCO lines 9 days after transfer in 1/2MS supplemented with 0.1 µM or 1 µM flg22.


D Representative picture of 14 - day - old plants grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . The scale bar representing 0 . 6cm is included in the picture .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O

D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture.


F Representative picture of root apical meristems after cell wall staining , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center . G Root apical meristem size of WT and RNAi - ASCO1 ( eg distance from QC to TZ in µm ) .

O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O O O O

F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center. G Root apical meristem size of WT and RNAi-ASCO1 (eg distance from QC to TZ in µm).


F Representative picture of root apical meristems after cell wall staining , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center .

O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O

F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center.


B Number of genes containing at least one differential RNA processing event ( as defined by RNAprof p . adj < 0 . 001 ) in CDS , introns , 5 ' UTR and 3 ' UTR between RNAi - ASCO1 and WT . Up and Down fractions correspond to increase or decrease , respectively , of RNA - seq coverage in RNAi - ASCO1 for each specified gene feature .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O

B Number of genes containing at least one differential RNA processing event (as defined by RNAprof p.adj < 0.001) in CDS, introns, 5'UTR and 3'UTR between RNAi-ASCO1 and WT. Up and Down fractions correspond to increase or decrease, respectively, of RNA-seq coverage in RNAi-ASCO1 for each specified gene feature.


B Scatter plot showing the respective gene expression fold change in RNAi - ASCO and 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO and 35S : ASCO are highlighted as yellow and blue dots , respectively .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O

B Scatter plot showing the respective gene expression fold change in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO and 35S:ASCO are highlighted as yellow and blue dots, respectively.


C Scatter plots showing the respective Percent Spliced In difference ( dPSI ) in RNAi - ASCO and 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO , 35S : ASCO or in both lines are highlighted as red , green or blue dot , respectively . Gray dots represent all AS events .

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

C Scatter plots showing the respective Percent Spliced In difference (dPSI) in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO, 35S:ASCO or in both lines are highlighted as red, green or blue dot, respectively. Gray dots represent all AS events.


Analyses of RT - PCR products of SR34 transcripts on 8 % acrylamide gel . Quantification of the ratio of SR34 isoforms detected in the gel in ( B ) RNAs were extracted from WT and RNAi - ASCO1 14 - day - old plants . The asterisk ( * ) indicates a significant difference as determined by Student ' s T test ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analyses of RT-PCR products of SR34 transcripts on 8% acrylamide gel. Quantification of the ratio of SR34 isoforms detected in the gel in (B) RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.


Analyses of RT - PCR products of NUDT7 transcripts on 8 % acrylamide gel . Quantification of the ratio of NUDT7 isoforms detected in the gel in ( E ) respectively . RNAs were extracted from WT and RNAi - ASCO1 14 - day - old plants . The asterisk ( * ) indicates a significant difference as determined by Student ' s T test ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analyses of RT-PCR products of NUDT7 transcripts on 8% acrylamide gel. Quantification of the ratio of NUDT7 isoforms detected in the gel in (E) respectively. RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.


A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA . The fold enrichment was calculated between ODD or EVEN samples against LacZ . These samples were used for protein precipitation and Mass Spectrometry analyses ( ChIRP - MS ) , from which PRP8a was identified as a potential ASCO partner .

O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O

A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA. The fold enrichment was calculated between ODD or EVEN samples against LacZ. These samples were used for protein precipitation and Mass Spectrometry analyses (ChIRP-MS), from which PRP8a was identified as a potential ASCO partner.


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY

B Validation of PRP8a-ASCO interaction by PRP8a-RIP. U5 RNA was used as a positive control. Data information: In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT -

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY


C Immunoblot analysis was performed during PRP8a immunoprecipitation ( IP ) . The same volume of input , unbound fraction was loaded as well as 20 % of the eluted IP fraction . α - PRP8a and α - IgG : IP performed with anti - PRP8a or control rabbit IgG antibody , respectively .

O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O

C Immunoblot analysis was performed during PRP8a immunoprecipitation (IP). The same volume of input, unbound fraction was loaded as well as 20 % of the eluted IP fraction. α-PRP8a and α-IgG : IP performed with anti-PRP8a or control rabbit IgG antibody, respectively.


: In D , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR , and IgG RIP was used as a negative control .

O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O

: In D, the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR, and IgG RIP was used as a negative control.


A ASCO transcript levels in WT and prp8 - 7 mutants .

O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O

A ASCO transcript levels in WT and prp8-7 mutants.


prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


C prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

C prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


D ASCO transcript levels in smd1b mutant . In A and D RNAs were extracted from WT , prp8 - 7 and smd1b 14 - day - old plants .

O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

D ASCO transcript levels in smd1b mutant. In A and D RNAs were extracted from WT, prp8-7 and smd1b 14-day-old plants.


E SmD1b can bind ASCO in vivo . U6 RNA was used as a positive control and a housekeeping gene ( HKG2 , AT4G26410 ) RNA as a negative control .

O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

E SmD1b can bind ASCO in vivo. U6 RNA was used as a positive control and a housekeeping gene (HKG2, AT4G26410) RNA as a negative control.


F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO . In E and F the results were expressed as % INPUT in SmD1b - GFP RIP and IgG RIP used as a negative control .

O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O

F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO. In E and F the results were expressed as % INPUT in SmD1b-GFP RIP and IgG RIP used as a negative control.


smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


H smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

H smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


Example of a 384 - well plate treated with compounds . The panel shows the staining with filipin ( free cholesterol in green pseudo - colour ) and anti - LBPA antibodies ( red ) . The leftmost column shows control wells treated with DMSO only , and the right penultimate column shows wells treated with U18666A .

O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O

Example of a 384-well plate treated with compounds. The panel shows the staining with filipin (free cholesterol in green pseudo-colour) and anti-LBPA antibodies (red). The leftmost column shows control wells treated with DMSO only, and the right penultimate column shows wells treated with U18666A.


Effects of U18666A , thioperamide and trimeprazine , compared with controls . Cells treated with the indicated compounds or with DMSO alone for 18h at 37 ° C , were processed for immunofluorescence microscopy . Low magnification views are shown after staining with anti - LBPA antibodies ( red ) , filipin ( green pseudo - color ) and DAPI ( blue ) . The scale bar is 20 µm .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

Effects of U18666A, thioperamide and trimeprazine, compared with controls. Cells treated with the indicated compounds or with DMSO alone for 18h at 37°C, were processed for immunofluorescence microscopy. Low magnification views are shown after staining with anti-LBPA antibodies (red), filipin (green pseudo-color) and DAPI (blue). The scale bar is 20 µm.


Screen data plot . The plot shows the integrated intensity of LBPA vs . filipin ( cholesterol ) staining in all HeLa cells of each well analyzed in the screen ( each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound ) . Only compounds that showed less than 20 % toxicity are plotted . The cells treated with U18666A , thioperamide maleate and trimeprazine tartrate are indicated , as well as the DMSO controls .

O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O

Screen data plot. The plot shows the integrated intensity of LBPA vs. filipin (cholesterol) staining in all HeLa cells of each well analyzed in the screen (each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound). Only compounds that showed less than 20% toxicity are plotted. The cells treated with U18666A, thioperamide maleate and trimeprazine tartrate are indicated, as well as the DMSO controls.


Integrated intensity of cholesterol and LBPA staining . The integrated intensity of cholesterol ( filipin in green ) and LBPA ( red ) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls . ( n = 3 independent experiments , 64 images analysed per experiments , error bars = SD , two ways ANOVA ; * * = p < 0 . 01 ; * * * = p < 0 . 005 ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Integrated intensity of cholesterol and LBPA staining. The integrated intensity of cholesterol (filipin in green) and LBPA (red) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls. (n=3 independent experiments, 64 images analysed per experiments, error bars = SD, two ways ANOVA; **=p<0.01; ***=p<0.005).


Thioperamide treatment of tissue culture cells . BHK , CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C . Low magnification views are shown after staining with anti - LBPA antibodies ( green ) and DAPI ( blue ) . n = 3 independent experiments with > 200 images acquired and analysed automatically .

B-SMALL_MOLECULE O O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C. Low magnification views are shown after staining with anti-LBPA antibodies (green) and DAPI (blue). n=3 independent experiments with > 200 images acquired and analysed automatically.


Cholesterol quantification by mass spectrometry . A431 cells were treated with DMSO alone , pitolisant 10µM ( Pito ) , thioperamide 10µM ( thio ) or U18666A 10µM ( U18 ) for 18h at 37 ° C . After extraction , free cholesterol , cholesteryl esters and total cholesterol were quantified by mass spectrometry , and normalized to the DMSO controls . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * * * * = p < 0 .

B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10µM (Pito), thioperamide 10µM (thio) or U18666A 10µM (U18) for 18h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry, and normalized to the DMSO controls. (n=3 independent experiments, error bars = SD, one-way ANOVA, ****=p<0.0001


Screen data plot of the cholesterol content of LBPA - endosomes . Automated unbiased quantification of the filipin integrated fluorescence signal ( cholesterol content ) in LBPA - containing endosomes , after treatment with each compound of the Prestwick library . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The fluorescence signal of LBPA - endosomes was used to segment the imaged and generate a mask , which was then applied on the micrographs to quantify the integrated intensity of filipin staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated .

O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O

Screen data plot of the cholesterol content of LBPA-endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA-containing endosomes, after treatment with each compound of the Prestwick library. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The fluorescence signal of LBPA-endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.


Immunogold labelling with anti - LBPA antibodies . HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy . Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A - gold . Multivesicular endosomes are pseudocoloured ( see Fig EV1 for uncoloured images ) . Bars , 200nm . The data in ( A ) were quantified in a double - blind analysis of two sets of 16 micrographs for each condition ; the number of gold particles per endosome is shown in a scatter plot ( B ) for each endosome identified without any bias in each micrograph of the two control ( DMSO ) and thioperamide - treated ( THIO ) samples .

B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O

Immunogold labelling with anti-LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A-gold. Multivesicular endosomes are pseudocoloured (see Fig EV1 for uncoloured images). Bars, 200nm. The data in (A) were quantified in a double-blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide-treated (THIO) samples.


Distribution of LBPA - containing endosomes in the perinuclear region . Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells , after treatment with each compound of the Prestwick library as in Fig 2C . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell , calculated from the nuclei DAPI - staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated . Number of perinuclear LBPA endosomes . The number of individual LBPA - positive structures was measured in the perinuclear region as in ( C ) . The z - factors are shown to evaluate the distribution of endosomes containing LBPA ; color code as in ( C ) .

O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O

Distribution of LBPA-containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig 2C. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI-staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. Number of perinuclear LBPA endosomes. The number of individual LBPA-positive structures was measured in the perinuclear region as in (C). The z-factors are shown to evaluate the distribution of endosomes containing LBPA; color code as in (C).


Effect of Pitolisant on LBPA . HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37 ° C were labeled with DAPI and anti - LBPA antibodies and analysed by fluorescence microscopy ( quantification in fig EV3A ) .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A).


LBPA intensity in a mixed population of cells expressing HRH3 - GFP . After transfection with HRH3 - GFP , uncloned stably expressing cells ( C ) were labelled with anti - LBPA antibodies and analysed by automated microscopy ( C ) . Unbiased quantification ( D ) shows the inverse correlation between HRH3 - GFP expression ( high expressing cells in green ) and the endosome integrated intensity of LBPA staining ( high LBPA labelling in red ) . ( n = 2 independent experiments with 500 , 000 cells analysed automatically , error bars = SD ) .

B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD).


LBPA intensity in cells expressing or not HRH3 - GFP . HeLa MZ cells stably expressing HRH3 - GFP grown in 96 - well plates were separately treated , with 4 different siRNAs that target HRH3 ( siHRH3 # 1 ; siHRH3 # 2 ; siHRH3 # 3 ; siHRH3 # 4 ) or with control non - target ( siNT ) siRNA ( E ) . HRH3 - GFP was analysed by fluorescence microscopy in 4 rows of cells per condition ; micrographs are stitched together in the montage . Panel F shows an example of cells treated with siHRH3 # 2 or siNT , and then labelled with DAPI and antibodies against LBPA . Cells treated as in ( E ) and labelled with DAPI and antibodies against LBPA as in ( F ) , were analysed by automated microscopy and the integrated intensities of LBPA ( left panel ) and HRH3 - GFP ( right panel ) signals are compared ( G ) . ( n = 2 independent experiments with 192 images acquired and analysed automatically , error bars = SD ) .

B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).


Treatment of NPC fibroblasts and NPC null mice with thioperamide . Fibroblast lines obtained from patients with well - established heterozygote mutations in the NPC1 ( GM17912 line : NPC1 P1007A / T1036M ; GM17911 line : NPC1 I1061T / T1036M ) or with a homozygote mutation in the NPC2 gene ( GM17910 line : C93F / C93F ) were treated or not for 72h with thioperamide 10µM , stained with filipin ( cholesterol ) and analysed by fluorescence automated microscopy .

O O B-DISEASE B-CELL_TYPE O B-GENEPROD O B-ORGANISM O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Treatment of NPC fibroblasts and NPC null mice with thioperamide. Fibroblast lines obtained from patients with well-established heterozygote mutations in the NPC1 (GM17912 line: NPC1 P1007A / T1036M; GM17911 line: NPC1 I1061T / T1036M) or with a homozygote mutation in the NPC2 gene (GM17910 line: C93F / C93F) were treated or not for 72h with thioperamide 10µM, stained with filipin (cholesterol) and analysed by fluorescence automated microscopy.


Quantification of cholesterol and LBPA staining in NPC fibroblasts . Cells as in ( A ) were treated or not with thioperamide for 48h or 72h , labelled with filipin ( cholesterol ) and anti - LBPA antibodies , and analysed by fluorescence automated microscopy . The panels show the integrated intensity of the filipin ( left ) and LBPA ( right ) signals were quantified after 48 ( top ) and 72h ( bottom ) . The colour code of each fibroblast cell line is as in ( A ) . ( n = 3 independent experiments with 144 images acquired and analysed automatically , > 2000 cells per experiment , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ; * * * = p < 0 . 001 ) .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of cholesterol and LBPA staining in NPC fibroblasts. Cells as in (A) were treated or not with thioperamide for 48h or 72h, labelled with filipin (cholesterol) and anti-LBPA antibodies, and analysed by fluorescence automated microscopy. The panels show the integrated intensity of the filipin (left) and LBPA (right) signals were quantified after 48 (top) and 72h (bottom). The colour code of each fibroblast cell line is as in (A). (n=3 independent experiments with 144 images acquired and analysed automatically, >2000 cells per experiment, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001).