( C ) p62 immunostaining of tibialis anterior ( TA ) or quadriceps ( Quad ) muscle from 9 - mo - old control , VCP - WT , or one of two VCP - RH transgenic lines ( RH9 or RH12 ) . Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions .

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

(C) p62 immunostaining of tibialis anterior (TA) or quadriceps (Quad) muscle from 9-mo-old control, VCP-WT, or one of two VCP-RH transgenic lines (RH9 or RH12). Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions.


( D ) Histochemistry and immunohistochemistry of quadriceps muscle from VCP - RH - expressing transgenic mice . Hematoxylin and eosin ( H & amp ; amp ; E ) staining of a 15 - mo - old animal with an RV , p62 immunostaining of an RV from a 12 - mo - old animal , LC3 immunostaining of an RV , and subsarcolemmal accumulations of LC3 from a 12 - mo - old animal . The bracket highlights one LC3 - positive myofiber . A single muscle fiber is outlined in white . ( C and D ) Arrows denote vacuoles or accumulations of p62 or LC3 . Bars , 30 µm .

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

(D) Histochemistry and immunohistochemistry of quadriceps muscle from VCP-RH-expressing transgenic mice. Hematoxylin and eosin (H&amp;E) staining of a 15-mo-old animal with an RV, p62 immunostaining of an RV from a 12-mo-old animal, LC3 immunostaining of an RV, and subsarcolemmal accumulations of LC3 from a 12-mo-old animal. The bracket highlights one LC3-positive myofiber. A single muscle fiber is outlined in white. (C and D) Arrows denote vacuoles or accumulations of p62 or LC3. Bars, 30 µm.


( A ) Lysates from scrambled siRNA or VCP - targeted siRNA - treated U20S cells immunoblotted with antibodies to VCP , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control .

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

(A) Lysates from scrambled siRNA or VCP-targeted siRNA-treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control.


( B ) Fluorescent microscopy images of siRNA scrambled control ( Scr ) or VCP - targeted siRNA KD cells expressing GFP - LC3 . Basal numbers of GFP - LC3 puncta / cell were counted .

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

(B) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted.


( C ) Lysates from control or myc - tagged VCP - WT , ATPase - inactive VCP - EQ , or one of two IBMPFD mutants , VCP - RH or - AE , expressed for 16 h from stably transfected tetracycline - inducible U20S cells and immunoblotted with antibodies to myc , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . Note the increase in LC3II and p62 in VCP siRNA KD and mutant - expressing cells .

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

(C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells.


( D ) Fluorescent microscopy images of tetracycline - inducible U20S cells expressing GFP - LC3 treated with and without Baf for 4 h ( DMSO control [ left ] or Baf + [ right ] ) . Cells are control U20S cells and VCP - WT , - RH , - AE , and - EQ . ( E ) The number of GFP - LC3 puncta / cell was counted for control U20S and VCP - WT , - EQ , - RH , and - AE with and without Baf for 4 h ( DMSO or Baf ) . Note the increase in basal GFP - LC3 puncta in mutant - expressing cells . ( A - C and E ) Error bars represent the standard error from four independent experiments . * , P < 0 . 01 ; and * * , P < 0 . 001 . Bars , 25 µm .

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

(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.


( A ) Epifluorescent images for GFP and mRFP in U20S or VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with mRFP - GFP - LC3 ( tfLC3 ) and treated with rapamycin for 2 h to induce autolysosome formation . ( B ) siRNA control ( Scr ) or VCP - KD - or Baf - treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation . ( A and B ) Open arrows denote autophagosomes ( both GFP and mRFP fluorescence ) , whereas closed arrows highlight autolysosomes ( mRFP only fluorescence ) . ( C ) The graph represents Pearson ' s coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD 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 O O O 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) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.


( D ) Lysates from U20S or tetracycline - inducible VCP - WT , - RH , or - AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α - tubulin . Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant ( RH and AE ) - expressing cells . Bars , 15 µm .

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

(D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.


( A ) Lysates from siRNA - treated U20S cells ( scramble control [ Scr ] or VCP siRNA KD ) after autophagic induction via nutrient deprivation for 0 , 2 , or 4 h and immunoblotting for LC3 and α - tubulin . LC3II degrades over time in control but not in KD cells . One of two experiments is shown .

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

(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown.


( B ) Lysates from U20S or tetracycline - inducible VCP - WT , - EQ , - RH , or - AE cells after autophagic induction via nutrient deprivation for 0 , 2 , 4 , or 6 h and immunoblotting for LC3 and α - tubulin . LC3II degrades in control and VCP - WT but degrades less efficiently in mutant ( EQ , RH , or AE ) - expressing cells . ( C ) Graphical representation of densitometric evaluation of LC3II and α - tubulin at each time point from three independent experiments .

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

(B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.


( A ) Epifluorescent images for GFP - LC3 ( LC3 ) and LTR of VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Open arrows highlight autolysosomes ( GFP and LTR colocalized ) , and closed arrows show autophagosomes ( GFP only ) . ( B ) Pearson ' s coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments .

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

(A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments.


( C ) Epifluorescent images for GFP - LC3 and endogenous Lamp1 immunohistochemistry of U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells or U20S cells cotreated with Baf transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Arrows highlight autolysosomes ( GFP and Lamp1 colocalized ) . ( A and C ) The boxed regions in the merge field are enlarged in the adjacent panels . ( D ) Pearson ' s coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments . ( B and D ) Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 .

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

(C) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.


( E ) Electron microscopy of tetracycline - inducible control or VCP - WT - , VCP - RH - , or VCP - AE - expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h . Note the accumulation of autophagic structures in the mutant - expressing cell lines . Arrows denote autophagic structures .

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

(E) Electron microscopy of tetracycline-inducible control or VCP-WT-, VCP-RH-, or VCP-AE-expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures.


( A ) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or - 2 antibodies . Quadriceps section from a 9 - mo - oldVCP - WT mouse , age - matched VCP - RH transgenic , 12 - mo - oldVCP - RH transgenic , and IBMPFD patientmuscle biopsy are shown . Open arrows highlight p62 and Lamp1 / 2 colocalization , and closed arrows show regions of only p62immunofluorescence . Single myofibers containing RVs are outlined in the merge panels .

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

(A) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or -2 antibodies. Quadriceps section from a 9-mo-oldVCP-WT mouse, age-matched VCP-RH transgenic, 12-mo-oldVCP-RH transgenic, and IBMPFD patientmuscle biopsy are shown. Open arrows highlight p62 and Lamp1/2 colocalization, and closed arrows show regions of only p62immunofluorescence. Single myofibers containing RVs are outlined in the merge panels.


( B and C ) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines ( B shows RH12 , and C shows RH9 lines ) of 12 - mo - old VCP - RH - expressing transgenic mice . Note the large vacuolated structures that are perinuclear or subsarcolemmal . Bars : ( A ) 30 µm ; ( B and C ) 500 nm .

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

(B and C) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines (B shows RH12, and C shows RH9 lines) of 12-mo-old VCP-RH-expressing transgenic mice. Note the large vacuolated structures that are perinuclear or subsarcolemmal. Bars: (A) 30 µm; (B and C) 500 nm.


( A ) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH - expressing mice ( RH12 ) 2 d after electroporation ( baseline ) or after 24 h of nutrient deprivation ( starvation ) of polyQ80 ( Q80 ) - or polyQ19 - luciferase ( Q19 ) in the right and left tibialis anterior , respectively . The ratio of polyQ80 - luciferase / polyQ19 - luciferase is indicated below each image set . Values are in × 104 photons per second per square centimeter per steradian . ( B ) Box and whisker plot of the change ( Δ ) in the ratio of polyQ80 - luciferase / polyQ19 - luciferase activity in the left and right tibialis anterior muscle of control , VCP - WT , or one of two VCP - RH ( RH12 or RH9 ) transgenic mouse lines after 24 h of starvation . The graph is representative of three animals per group . The p - value for RH12 was 0 . 05 and 0 . 06 when compared with control animals or VCP - WT transgenic . The p - value for RH9 was 0 . 03 when compared with either control or VCP - WT transgenic . The p - value was 0 . 01 and 0 . 02 when combined RH12 and RH9 animals were compared with control or VCP - WT groups , respectively . There was no statistical difference between control or VCP - WT groups .

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

(A) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.


( A ) Quantitation of mCherry - TDP - 43 distribution ( nuclear only or cytoplasmic ) in control U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells , or control U20S cells treated with 10 µM Baf or 50 µM chloroquine ( Chlq ) for 4 h from 10 different fields from two independent experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 02 when compared with VCP - WT - expressing cells . ( B ) Epifluorescent images for mCherry - TDP - 43 ( red ) and DAPI ( blue ) in control U20S , VCP - WT - , VCP - RH - , or VCP - AE - expressing cells , or control U20S cells treated with Baf or chloroquine for 4 h . Arrows denote cytosolic TDP - 43 and perinuclear TDP - 43 inclusions .

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(A) Quantitation of mCherry-TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT-expressing cells. (B) Epifluorescent images for mCherry-TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT-, VCP-RH-, or VCP-AE-expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions.


( C , top ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of mCherry - TDP - 43 - transfected VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells . Note the increase in cytosolic TDP - 43 from IBMPFD mutant - and EQ - expressing cells . ( bottom ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry - TDP - 43 or similarly transfected cells treated with 30 µg / ml ( cq1 ) or 120 µg / ml ( cq2 ) chloroquine diphosphate or 200 ng / ml Baf . Data are representative of three independent experiments . Bar , 15 µm .

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

(C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry-TDP-43-transfected VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant- and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry-TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.


( A ) TDP - 43 immunostaining of quadriceps skeletal muscle from 15 - mo - old control ( Cont ) or VCP - WT , - RH9 , or - RH12 transgenic mice . A single myofiber is outlined in white .

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

(A) TDP-43 immunostaining of quadriceps skeletal muscle from 15-mo-old control (Cont) or VCP-WT, -RH9, or -RH12 transgenic mice. A single myofiber is outlined in white.


( B ) TDP - 43 immunostaining of quadriceps skeletal muscle from 3 - mo - old mice treated with saline or 50 mg / kg / d intraperitoneal chloroquine ( Chlq ) for 4 wk . ( A and B ) Insets show one myonuclei from each field . Bars , 30 µm .

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

(B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.


( A ) Muscular exophers contain organelles and large protein complexes . Arrows : white - exopher , blue - muscle cell , green - mitochondria , red - proteasome foci . MOM - mitochondrial outer membrane . ( B ) BMW actively releases significant amounts of exopher . The image shows the middle part of the worm ' s body with muscles marked with dashed lines . Arrows indicate representative exophers , and the asterisk indicates the position of the vulva .

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(A) Muscular exophers contain organelles and large protein complexes. Arrows: white - exopher, blue - muscle cell, green - mitochondria, red - proteasome foci. MOM - mitochondrial outer membrane. (B) BMW actively releases significant amounts of exopher. The image shows the middle part of the worm's body with muscles marked with dashed lines. Arrows indicate representative exophers, and the asterisk indicates the position of the vulva.


( H ) Knockdown of two autophagy genes , atg - 7 and lgg - 1 significantly reduce the number of generated exophers . n = 91 - 103 ; N = 3 .

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(H) Knockdown of two autophagy genes, atg-7 and lgg-1 significantly reduce the number of generated exophers. n = 91-103; N = 3.


( A ) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals . Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life . Starting n = 90 hermaphrodites and 150 males ; N = 3 .

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(A) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals. Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life. Starting n = 90 hermaphrodites and 150 males; N = 3.


( B ) Feminized hermaphrodites of thermosensitive a fem - 1 mutant strain do not produce exophers regardless of growth temperature . This phenotype can be partially rescued by mating fem - 1 mutants with males . n = 10 - 26 ; N = 2 .

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(B) Feminized hermaphrodites of thermosensitive a fem-1 mutant strain do not produce exophers regardless of growth temperature. This phenotype can be partially rescued by mating fem-1 mutants with males. n = 10-26; N = 2.


( D ) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal . n = 118 and 112 animals ; N = 3 .

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(D) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal. n = 118 and 112 animals; N = 3.


( E ) Males and sterile hermaphrodites ( via FUdR treatment ) show the formation of spherical structures in the BWM that resemble mature exophers . MOM - mitochondrial outer membrane .

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(E) Males and sterile hermaphrodites (via FUdR treatment) show the formation of spherical structures in the BWM that resemble mature exophers. MOM - mitochondrial outer membrane.


( B ) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production . n = 50 - 60 ; N = 2 .

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(B) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production. n = 50-60; N = 2.


( F ) Increased eggshell permeability caused by emb - 27 knockdown elevates exopher production . n = 103 and 89 ; N = 3 .

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(F) Increased eggshell permeability caused by emb-27 knockdown elevates exopher production. n = 103 and 89; N = 3.


( C , D ) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled ( C ) and by representative worms traces on the plate ( D ) . n = 11 - 34 ; N = 3 .

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(C, D) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled (C) and by representative worms traces on the plate (D). n = 11-34; N = 3.


( E ) RNAi knockdown of the egg yolk precursor protein VIT - 1 increases the number of muscular exophers . n = 20 ; N = 2 .

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(E) RNAi knockdown of the egg yolk precursor protein VIT-1 increases the number of muscular exophers. n = 20; N = 2.


( F ) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT - 2 . Representative images of embryos with endogenous VIT - 2 : : GFP levels from mothers with different exopheresis activity are shown above the graph . n = 15 and 14 , N = 5 and 6 . Scale bar is 10 µm .

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(F) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT-2. Representative images of embryos with endogenous VIT-2::GFP levels from mothers with different exopheresis activity are shown above the graph. n = 15 and 14, N = 5 and 6. Scale bar is 10 µm.


( G ) RNAi knockdown of RME - 2 yolk receptor abolish exopher production . n = 110 - 123 ; N = 3 .

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(G) RNAi knockdown of RME-2 yolk receptor abolish exopher production. n = 110-123; N = 3.


( H ) Muscle - produced VIT - 2 is released from muscles via exophers . The image shows the midbody of worms expressing the proteasome subunit RPN - 5 tagged with RFP in BWM and VIT - 2 : : GFP endogenous expression . Arrows : white - exopher , blue - muscle cell . Dashed lines mark embryos present in the uterus . Scale bar is 10 µm .

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(H) Muscle-produced VIT-2 is released from muscles via exophers. The image shows the midbody of worms expressing the proteasome subunit RPN-5 tagged with RFP in BWM and VIT-2::GFP endogenous expression. Arrows: white - exopher, blue - muscle cell. Dashed lines mark embryos present in the uterus. Scale bar is 10 µm.


( G ) Tumour growth ( in mm3 ) of FBXW7 + / + and FBXW7 - / - xenografts in nude mice ( n = 10 animals per group ) . Treatment with either vehicle , paclitaxel ( 1 , 5mg / kg ) or tigecycline ( 50mg / kg ) started at day 6 post - tumour - injection , and was administered three times per week . Error bars indicate SEM . ( H ) Representative images of the xenografts defined in ( G ) at day 15 .

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

(G) Tumour growth (in mm3) of FBXW7+/+ and FBXW7-/- xenografts in nude mice (n=10 animals per group). Treatment with either vehicle, paclitaxel (1,5mg/kg) or tigecycline (50mg/kg) started at day 6 post-tumour-injection, and was administered three times per week. Error bars indicate SEM. (H) Representative images of the xenografts defined in (G) at day 15.


( D ) WB illustrating the levels of ATF4 in DLD - 1 cells treated as in ( C ) . GAPDH levels are shown as a loading control .

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(D) WB illustrating the levels of ATF4 in DLD-1 cells treated as in (C). GAPDH levels are shown as a loading control.


B ) Localization of the GFP - tagged PE synthesizing enzyme ( Psd ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM choline . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) ; LD , lipid droplets ; MM , mitochondrial matrix . A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image . C ) Localization of the GFP - tagged PC synthesizing enzyme ( Pmt ) targeted to the indicated organelles in choppΔ cells , grown in SD medium supplemented with 10 mM ethanolamine . Images shown are either a single Z - slice ( ER construct ) or a maximum intensity projection of several Z - sections ( other constructs ) . A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image .

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

B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.


B ) Five - fold serial dilutions of strains of the indicated genotypes ( wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes ) . Cells were grown on SD - HIS - LEU ( - HL ) medium ( KennedyOFF ) or SD - HL containing ethanolamine and choline ( KennedyON ) .

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

B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON).


C ) Thin layer chromatography ( TLC ) analysis of steady - state phospholipid profiles of cells of the indicated genotypes . Where Psd and / or Pmt are missing ( - ) , choppΔ cells were grown in the presence of ethanolamine and / or choline . Bands corresponding to the phospholipids PA , PS , PE , PI and PC are indicated .

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

C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.


C ) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes . Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions . This comparison included all six libraries where the enzymes are targeted to endosomes and / or mitochondria ( top left panel ) or six libraries with enzymes targeted to other organelles ( bottom left panel ) . Data - points for VPS13 are highlighted in red . Schematic illustrating Vps13 - mediated lipid transport at endosomes and mitochondria contact sites ( right panel ) .

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

C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel).


B ) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions . Genes significantly required in KennedyOFF conditions are highlighted in red ( p - value < 0 . 05 , log2 ( fold change ) < - 0 . 5 ) . Genes highlighted in the clusterogram in A ( top inset ) are indicated with blue arrows .

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B) Volcano plot comparing the number of transposon insertions per gene of all 12 libraries in KennedyON and OFF conditions. Genes significantly required in KennedyOFF conditions are highlighted in red (p-value < 0.05, log2(fold change) < -0.5). Genes highlighted in the clusterogram in A (top inset) are indicated with blue arrows.


B ) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries . Left panel , schematic depicting PE and cardiolipin ( CL ) synthesis in mitochondria . The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow . Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM . Right panel , transposon numbers ( normalized to wt libraries ) for the indicated ( set of ) libraries for MDM31 , PGS1 , and CRD1 .

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B) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries. Left panel, schematic depicting PE and cardiolipin (CL) synthesis in mitochondria. The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow. Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM. Right panel, transposon numbers (normalized to wt libraries) for the indicated (set of) libraries for MDM31, PGS1, and CRD1.


B ) Five - fold serial dilutions of strains of the indicated genotypes grown at 30 ° C on SD medium supplemented with 10 mM ethanolamine and / or 10 mM choline . Growth assays are representative images of biological replicates .

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B) Five-fold serial dilutions of strains of the indicated genotypes grown at 30°C on SD medium supplemented with 10 mM ethanolamine and/or 10 mM choline. Growth assays are representative images of biological replicates.


A ) Top : Co - localization of Csf1 - GFP cells in log phase with genomically integrated Pex10 - mCherry ( peroxisome marker ) and mtBFP plasmid ( mito marker ) . Bottom : Co - localization of log growing Csf1 - GFP cells with mCherry - Ubc6 ( tail - anchor ) ( ER marker ) and mtBFP plasmid ( mito marker ) . Scale bar , 2 µm .

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

A) Top: Co-localization of Csf1-GFP cells in log phase with genomically integrated Pex10-mCherry (peroxisome marker) and mtBFP plasmid (mito marker). Bottom: Co-localization of log growing Csf1-GFP cells with mCherry-Ubc6 (tail-anchor) (ER marker) and mtBFP plasmid (mito marker). Scale bar, 2 µm.


C ) Five - fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures , in the presence or absence of 10 mM ethanolamine ( etn ) .

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C) Five-fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures, in the presence or absence of 10 mM ethanolamine (etn).


E ) Bar graphs depicting the fraction of more saturated species ( defined as fatty acid chains with one double bond for ≤ 30 carbon chains or with two double bonds for ≥ 32 ) , and less unsaturated species ( defined as fatty acid chains with zero double bond for ≤ 30 carbon chains or with one double bond for ≥ 32 ) for each phospholipid class with different head groups . Bars represent the means of two or three biological replicates ( individual measurements are shown as points and triangles ) . p - values are calculated with a two - sample t - test .

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 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 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

E) Bar graphs depicting the fraction of more saturated species (defined as fatty acid chains with one double bond for ≤30 carbon chains or with two double bonds for ≥32), and less unsaturated species (defined as fatty acid chains with zero double bond for ≤30 carbon chains or with one double bond for ≥32) for each phospholipid class with different head groups. Bars represent the means of two or three biological replicates (individual measurements are shown as points and triangles). p-values are calculated with a two-sample t-test.


Figure 1 . Cytoplasmic conical structures in Thermococcus kodakaraensis . ( A ) A tomographic slice shows a side view of a conical structure ( c ) in the cytoplasm , rotated and enlarged in ( B ) . ( C ) A tomographic slice shows a side view of the cone in another cell , highlighting the subunit texture along the edge of the cone ( arrowheads ) . ( D , E ) Top views of a cone at different heights show the inner ring ( r ; enlarged in inset to highlight 19 - subunit structure ) and outer cone . ( F - H ) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone . ( I , J ) Different views of a 3D segmentation of the cone shown in ( A ) , embedded in a tomographic slice . s , S - layer ; m , membrane ; a , archaella ; rib , ribosomes ; rez , ribosome - excluding zone . Scale bars 100 nm ; scale bar in ( D ) applies to ( D , E ) ; scale bar in ( F ) applies to ( F - H ) ; segmentation not to scale .

O O O O O O O B-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-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

Figure 1. Cytoplasmic conical structures in Thermococcus kodakaraensis. (A) A tomographic slice shows a side view of a conical structure (c) in the cytoplasm, rotated and enlarged in (B). (C) A tomographic slice shows a side view of the cone in another cell, highlighting the subunit texture along the edge of the cone (arrowheads). (D, E) Top views of a cone at different heights show the inner ring (r; enlarged in inset to highlight 19-subunit structure) and outer cone. (F-H) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone. (I, J) Different views of a 3D segmentation of the cone shown in (A), embedded in a tomographic slice. s, S-layer; m, membrane; a, archaella; rib, ribosomes; rez, ribosome-excluding zone. Scale bars 100 nm; scale bar in (D) applies to (D, E); scale bar in (F) applies to (F-H); segmentation not to scale.


Figure 2 . Cones are associated with chemosensory arrays , ribosome - excluding zones and filament bundles . Tomographic slices show side ( A ) and top ( B , C ) views of cones ( c ) in three cells , highlighting associated chemosensory arrays ( ca ) , ribosome - excluding zones ( rez ) and filament bundles ( f ) . For a slice - by - slice view through the tomogram shown in ( A ) , see Movie S1 . ( D ) and ( E ) show different views of a 3D segmentation of the structures shown in ( C ) , with the conical structure in blue and the filament bundle in red . s , S - layer ; m , membrane ; a , archaellum ; o , other filaments . Scale bar 100 nm ( applies to A - C ) ; segmentation not to scale .

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

Figure 2. Cones are associated with chemosensory arrays, ribosome-excluding zones and filament bundles. Tomographic slices show side (A) and top (B, C) views of cones (c) in three cells, highlighting associated chemosensory arrays (ca), ribosome-excluding zones (rez) and filament bundles (f). For a slice-by-slice view through the tomogram shown in (A), see Movie S1. (D) and (E) show different views of a 3D segmentation of the structures shown in (C), with the conical structure in blue and the filament bundle in red. s, S-layer; m, membrane; a, archaellum; o, other filaments. Scale bar 100 nm (applies to A-C); segmentation not to scale.


Figure 3 . Archaellum orientation with respect to the cell envelope . ( A - D ) show tomographic slices through side views of cones . In the bottom row , white lines show the angle of the archaellum with respect to the surface layer , and red dashed lines show the conserved distance from the archaellum at the membrane to the cone . Scale bar 100 nm ( applies to A - D ) .

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

Figure 3. Archaellum orientation with respect to the cell envelope. (A-D) show tomographic slices through side views of cones. In the bottom row, white lines show the angle of the archaellum with respect to the surface layer, and red dashed lines show the conserved distance from the archaellum at the membrane to the cone. Scale bar 100 nm (applies to A-D).


Figure 4 . Structure of the T . kodakaraensis archaellum . ( A ) A sub - tomogram average of the archaellum reveals structural features , including four layers of density in the cytoplasm ( L1 - L3 , cone ) . CM , cytoplasmic membrane . The speculated identity of densities in the archaellum is proposed : archaellum fiber = FlaA / B flagellins ; integral membrane density = FlaJ ; L1 = FlaI ; L2 / L3 / cone = FlaH / FlaC / D / E . Scale bar 10 nm ( applies to A , B ) .

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

Figure 4. Structure of the T. kodakaraensis archaellum. (A) A sub-tomogram average of the archaellum reveals structural features, including four layers of density in the cytoplasm (L1-L3, cone). CM, cytoplasmic membrane. The speculated identity of densities in the archaellum is proposed: archaellum fiber = FlaA/B flagellins; integral membrane density = FlaJ; L1 = FlaI; L2/L3/cone = FlaH/FlaC/D/E.Scale bar 10 nm (applies to A, B).


Figure 4 . Structure of the T . kodakaraensis archaellum . ( B ) For comparison , a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown ( adapted with permission from [ 8 ] ) . Arrows indicate components with recognized homology . OM , outer membrane ; IM , inner membrane . Scale bar 10 nm ( applies to A , B ) .

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Figure 4. Structure of the T. kodakaraensis archaellum.(B) For comparison, a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown (adapted with permission from [8]). Arrows indicate components with recognized homology. OM, outer membrane; IM, inner membrane. Scale bar 10 nm (applies to A, B).


B . Growth of DiCre - 3D7 transgenic lines following LDH1 , HAD4 , and Lipin depletion with 100 nM rapamycin and 2 . 5 mM glucosamine was assessed relative to uninduced parasite cultures . The untransfected DiCre - 3D7 parasite line was used as a negative control . Data are presented as the mean ± standard error of the mean ( SEM ) from three independent replicates .

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

B. Growth of DiCre-3D7 transgenic lines following LDH1, HAD4, and Lipin depletion with 100 nM rapamycin and 2.5 mM glucosamine was assessed relative to uninduced parasite cultures. The untransfected DiCre-3D7 parasite line was used as a negative control. Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.


C . Untargeted LC - MS analysis of LDH1 depletion ( one cycle of rapamycin and glucosamine treatment ) indicated minimal but selective metabolic changes . m / z feature intensities are plotted as the log2 ratio of treated / untreated and the Benjamini - Hochberg corrected P values across six biological replicates plotted as - log10 ( P ) . Below , LDH1 substrate and product ( pyruvate and lactate respectively ) abundance plotted as the ratio of treated / untreated from biological replicates ( mean ± SEM ) and the parental DiCre line presented as the negative control .

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

C. Untargeted LC-MS analysis of LDH1 depletion (one cycle of rapamycin and glucosamine treatment) indicated minimal but selective metabolic changes. m/z feature intensities are plotted as the log2 ratio of treated/untreated and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). Below, LDH1 substrate and product (pyruvate and lactate respectively) abundance plotted as the ratio of treated/untreated from biological replicates (mean±SEM) and the parental DiCre line presented as the negative control.


D . Loss of Lipin ( PF3D7 _ 0303200 ) leads to accumulation of various lipid species ( three cycles of rapamycin and glucosamine treatment ) . Data are presented as the mean log2 fold change Lipin depleted ( treated ) enriched trophozoite - stage iRBCs to untreated controls from three to six biological replicates ( ± SD ) . The schematic depicts the proposed lipid phosphatase activity of Lipin .

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

D. Loss of Lipin (PF3D7_0303200) leads to accumulation of various lipid species (three cycles of rapamycin and glucosamine treatment). Data are presented as the mean log2 fold change Lipin depleted (treated) enriched trophozoite-stage iRBCs to untreated controls from three to six biological replicates (± SD). The schematic depicts the proposed lipid phosphatase activity of Lipin.


E . Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis . Data are represented as the mean log2 fold change of enriched trophozoite - stage iRBCs treated for three cycles compared to untreated controls from three / four biological replicates . Three nucleotide - nucleoside pairs are depicted in the top inset panels , HAD4 disruption leads to accumulation of the nucleotide - monophosphate and depletion of the nucleoside . The schematic depicts lower glycolysis and triose - phosphate interconversion , with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset .

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

E. Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis. Data are represented as the mean log2 fold change of enriched trophozoite-stage iRBCs treated for three cycles compared to untreated controls from three/four biological replicates. Three nucleotide-nucleoside pairs are depicted in the top inset panels, HAD4 disruption leads to accumulation of the nucleotide-monophosphate and depletion of the nucleoside. The schematic depicts lower glycolysis and triose-phosphate interconversion, with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset.


B . Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption . Rapamycin / glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated . Data represents the mean relative parasitemia ± SEM from three biological replicates ( y - axis ) across seven replication cycles ( x - axis ) .

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

B. Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption. Rapamycin/glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated. Data represents the mean relative parasitemia ± SEM from three biological replicates (y-axis) across seven replication cycles (x-axis).


C . Metabolite profile following three cycles of rapamycin and glucosamine treatment . The abundance of isoprenoid biosynthestic intermediates ( 1 - deoxy - xylulose - 5 - P ( DOXP ) , methyl - erithritol - 4 - P ( MEP ) , methyl - erithritol - cyclo - 2 , 4 - PP ( MEcPP ) , and isopentyl - PP ) , and hemoglobin - like peptides are presented as the mean log2 fold change from four biological replicates ( ± SD ) .

C. Metabolite profile following three cycles of rapamycin and glucosamine treatment. The abundance of isoprenoid biosynthestic intermediates (1-deoxy-xylulose-5-P (DOXP), methyl-erithritol-4-P (MEP), methyl-erithritol-cyclo-2,4-PP (MEcPP), and isopentyl-PP), and hemoglobin-like peptides are presented as the mean log2 fold change from four biological replicates (±SD).


C . HA - tagged SHMT - M detection via immunofluorescence microscopy . DAPI was used to visualise the nucleus , and MitoTracker CMXros used to illuminate the mitochondrion . Anti - HA signal partially colocalises with the mitochondrion .

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

C. HA-tagged SHMT-M detection via immunofluorescence microscopy. DAPI was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the mitochondrion. Anti-HA signal partially colocalises with the mitochondrion.


D . Pyrimidine biosynthetic intermediates accumulate when SHMT - M is disrupted compared to the DiCre parental line ( C ) following rapamycin / glucosamine treatment ( mean ± SEM ) . Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase ( DHODH ) inhibition and blockage of the mitochondrial electron transport chain ( mETC ) . Ubiquinone ( Q ) is the necessary electron carrier for the mETC .

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

D. Pyrimidine biosynthetic intermediates accumulate when SHMT-M is disrupted compared to the DiCre parental line (C) following rapamycin/glucosamine treatment (mean ±SEM). Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase (DHODH) inhibition and blockage of the mitochondrial electron transport chain (mETC). Ubiquinone (Q) is the necessary electron carrier for the mETC.


E . Asexual growth when SHMT - M is depleted . Parasitemia was determined across seven replication cycles ( x - axis ) via flow cytometry and presented as the mean ± SEM from three independent experiments . Decylubiquinone ( Dub ) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling .

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

E. Asexual growth when SHMT-M is depleted. Parasitemia was determined across seven replication cycles (x-axis) via flow cytometry and presented as the mean ± SEM from three independent experiments. Decylubiquinone (Dub) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling.


IES retention PCR ( cropped inverted images ) . Four maternally - controlled IES and four non - maternally controlled IESs are shown . The IES + band represents retained IES ; the IES - band represents an excised IES ; additional bands are likely PCR artefacts . IRS is IES retention Score for the IESs calculated after whole genome sequencing .

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

IES retention PCR (cropped inverted images). Four maternally-controlled IES and four non-maternally controlled IESs are shown. The IES+ band represents retained IES; the IES- band represents an excised IES; additional bands are likely PCR artefacts. IRS is IES retention Score for the IESs calculated after whole genome sequencing.


( A ) Schematic drawing of the life cycle stages of Paramecium tetraurelia . MIC and parental MAC are represented in red , representing the DAPI signal , and developing MAC ( dm ) is represented in green until fully developed , representing the GFP signal . ( B ) ISWI1 - tagged C - terminally with GFP localizes in the developing MAC as soon as developing new MACs ( panel Early Development ) become visible and remain there throughout late MAC development ( panel Late Development ) . Red : DAPI , Green : ISWI1 - GFP . Blue arrows identify developing MAC ;

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

(A) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC;


( C ) Western blot analysis using anti - HA antibody after coimmunoprecipitation of ISWI1 - 3XFlagHA fusion protein . Non - transformed cells ( WT ) of the same strain were used as the negative control . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 12 % SDS gel .

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

(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.


( D ) Volcano plot illustrating the distribution of proteins identified in label - free MS in WT Vs ISWI1 - 3XFlagHA . Significantly abundant proteins ( fold change ≥ 4 ) are highlighted in orange .

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-GENEPROD O O O O O O O O O O O O O O O O O

(D) Volcano plot illustrating the distribution of proteins identified in label-free MS in WT Vs ISWI1-3XFlagHA. Significantly abundant proteins (fold change ≥ 4) are highlighted in orange.


( E ) Western blot analysis using anti - HA and anti - GFP antibodies after coimmunoprecipitation of Ptiwi01 - 3XFlagHA fusion protein co - transformed with ISWI1 - GFP . Non - transformed cells ( WT ) of the same strain and ISWI1 - GFP fusion protein transformation were used as negative controls . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 10 % SDS gel .

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

(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.


( b ) A53T α - synuclein ( α - syn ) expression was induced in stable PC12 cells for 48 h , then switched off for 24 h . Cells were treated with DMSO ( control ) , or with 0 . 94 μM , 4 . 7 μM or 47 μM of SMER10 ; 0 . 86 μM , 4 . 3 μM or 43 μM of SMER18 ; or 0 . 9 μM , 4 . 7 μM or 47 μM of SMER28 added in the switch - off period . A53T α - syn levels were analyzed by immunoblotting with antibody against HA ( left ) and densitometry analysis relative to actin ( right ) . Error bars denote s . e . m . P = 0 . 0917 , P = 0 . 009 , P = 0 . 0001 ( for increasing concentrations of SMER10 ) ; P = 0 . 0068 , P = 0 . 0023 , P = 0 . 0002 ( for increasing concentrations of SMER18 ) ; P = 0 . 0016 , P 0 . 0001 , P 0 . 0001 ( for increasing concentrations of SMER28 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_LINE 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 O 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 O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD 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 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-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) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).


( c ) COS - 7 cells transfected with EGFP - HDQ74 construct were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( Rap ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , and we assessed the proportions of apoptotic or aggregate - containing transfected cells . Error bars : 95 % confidence interval . P 0 . 0001 ( Rap and SMER28 ) , P = 0 . 013 ( SMER10 ) , P = 0 . 019 ( SMER18 ) ( aggregation ) ; P 0 . 0001 ( Rap , SMER18 and SMER28 ) , P = 0 . 004 ( SMER10 ) ( cell death ) .

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

(c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death).


( d ) Wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs were transfected with EGFP - HDQ74 and treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h , and we assessed the percentages of aggregate - containing transfected cells . The control ( DMSO - treated ) values were fixed at 1 for both cell lines . Error bars : 95 % confidence interval . P = 0 . 039 ( SMER10 ) , P 0 . 0001 ( SMER18 and SMER28 ) ( in Atg5 + / + cells ) ; P = 0 . 092 ( SMER10 ) , P = 0 . 271 ( SMER18 ) , P = 0 . 358 ( SMER28 ) ( in Atg5 − / − cells ) . Note that EGFP - HDQ74 aggregation was higher in Atg5 − / − than in Atg5 + / + cells ( Supplementary Fig . 3a ) .

O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 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 B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O 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 B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O

(d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a).


e ) The percentages of EGFP - positive COS - 7 cells with EGFP - HDQ74 aggregates as in c , treated with DMSO ( control ) , 10 μM lactacystin ( proteasome inhibitor ) or both 10 μM lactacystin along with either 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , expressed as odds ratios . Error bars : 95 % confidence interval . P 0 . 0001 ( control versus Lact ) , P = 0 . 014 ( SMER10 versus Lact ) , P 0 . 0001 ( SMER18 versus Lact ) , P = 0 . 001 ( SMER28 versus Lact ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) COS - 7 cells transfected with EGFP - LC3 construct for 4 h were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( positive control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 16 h , and analyzed by fluorescence microscopy . The effects of treatment on the percentage of EGFP - positive cells with > 5 EGFP - LC3 vesicles are shown . Error bars denote s . e . m . P 0 . 0001 ( all SMERs ) .

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

(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs).


( b ) HeLa cells stably expressing EGFP - LC3 were treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h . Confocal sections show cells containing EGFP - positive autophagic vesicles . Nuclei are stained with DAPI . Bar , 20 μM .

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

(b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM.


( c ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with DMSO ( control ) or 200 nM bafilomycin A1 ( Baf ) , or with 200 nM bafilomycin A1 and 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 . Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1 . Levels of EGFP - LC3 - II were determined by immunoblotting with antibody against EGFP ( above ) and densitometry analysis relative to actin ( below ) . Error bars denote s . e . m . P = 0 . 0259 ( baf ) , P 0 . 0001 ( SMER10 ) , P = 0 . 0003 ( SMER18 and SMER28 ) versus control ; P = 0 . 0025 ( SMER10 ) , P = 0 . 0218 ( SMER18 ) , P = 0 . 0195 ( SMER28 ) versus bafilomycin A1 . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.


( a - c ) Flies treated with 100 μM SMER10 ( a ) , 200 μM SMER18 ( b ) or 100 μM SMER28 ( c ) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO ( control ) alone ( 2 d after eclosion ) . Rhabdomere counts from all three independent experiments are included . n = 600 ommatidia ( SMER10 ) , n = 1 , 500 ommatidia ( SMER18 ) and n = 600 ommatidia ( SMER28 ) . Mann - Whitney test values P 0 . 0001 ( all SMERs ) . Student ' s t - test ( 1 - tailed ) P = 0 . 005 ( SMER10 ) , P = 0 . 004 ( SMER18 ) , P = 0 . 03 ( SMER28 ) comparing means of distributions from independent experiments . These SMER concentrations cause no overt toxicity to flies ( see Supplementary Methods ) . Distributions of DMSO - treated flies may vary when SMERs are treated in different experiments at different times . For instance , an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time - dependent .

O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O 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 B-SUBCELLULAR O O 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 O B-TISSUE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O B-TISSUE 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 B-SMALL_MOLECULE O O O O 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 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 O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O

(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( a ) , 43 μM SMER18 ( b ) or 47 μM SMER28 ( c ) ) or both for the 8 h switch - off period - was analyzed by immunoblotting with antibody against HA ( above ) and densitometry analysis relative to actin ( below ) . The concentration of rapamycin is saturating for its effect on the clearance of A53T α - synuclein . Error bars denote s . e . m . P = 0 . 0025 ( Rap ) , P = 0 . 0018 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 0001 ( Rap or SMER10 versus SMER10 + Rap ) ( a ) ; P = 0 . 0069 ( Rap ) , P = 0 . 0498 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 0038 ( Rap versus SMER18 + Rap ) , P = 0 . 0007 ( SMER18 versus SMER18 + Rap ) ( b ) ; P 0 . 0001 ( Rap , SMER28 , Rap versus SMER28 + Rap , SMER28 versus SMER28 + Rap ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)) or both for the 8 h switch-off period-was analyzed by immunoblotting with antibody against HA (above) and densitometry analysis relative to actin (below). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote s.e.m. P = 0.0025 (Rap), P = 0.0018 (SMER10), P 0.0001 (SMER10 + Rap), P 0.0001 (Rap or SMER10 versus SMER10 + Rap) (a); P = 0.0069 (Rap), P = 0.0498 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.0038 (Rap versus SMER18 + Rap), P = 0.0007 (SMER18 versus SMER18 + Rap) (b); P 0.0001 (Rap, SMER28, Rap versus SMER28 + Rap, SMER28 versus SMER28 + Rap) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates ( above ) and cell death ( below ) in COS - 7 cells , as in Figure 1c - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( d ) , 43 μM SMER18 ( e ) or 47 μM SMER28 ( f ) ) or both for 24 h - were expressed as odds ratios . Error bars : 95 % confidence interval . Aggregation ( top panels ) : P = 0 . 248 ( Rap ) , P = 0 . 217 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 001 ( Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 248 ( Rap ) , P = 0 . 543 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 008 ( Rap versus SMER18 + Rap ) , P = 0 . 002 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 248 ( Rap ) , P = 0 . 002 ( SMER28 ) , P 0 . 0001 ( SMER28 + Rap ) , P 0 . 0001 ( Rap versus SMER28 + Rap ) , P = 0 . 012 ( SMER28 versus SMER28 + Rap ) ( f ) . Cell death ( bottom panels ) : P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER10 , SMER10 + Rap , Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 002 ( Rap ) , P = 0 . 948 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 015 ( Rap versus SMER18 + Rap ) , P 0 . 0001 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER28 , SMER28 + Rap , Rap or SMER28 versus SMER28 + Rap ) ( f ) . Note that we have treated cells for a shorter time in this experiment ( 24 h ) , compared with Figure 1c ( 48 h ) . This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O 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 O O O O O B-SMALL_MOLECULE O O 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 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 B-SMALL_MOLECULE O O O O O O O O O O O B-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 B-SMALL_MOLECULE 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 O O O O O O O O B-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 B-SMALL_MOLECULE 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 B-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 O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-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 B-SMALL_MOLECULE 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 O O O O O O O B-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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-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 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 O O O O O O O O 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-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated for 24 h with either DMSO ( control ) , or with 47 μM SMER10 and its analogs ( SMER10a - c ) ( a ) , 43 μM SMER18 and its analogs ( SMER18a - l ) ( b ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( c ) - was analyzed by immunoblotting with anti - HA antibody ( above ) and densitometry analysis relative to actin ( below ) . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars denote s . e . m . P = 0 . 0058 ( SMER10a ) , P = 0 . 6736 ( SMER10b ) , P = 0 . 9507 ( SMER10c ) , P = 0 . 0481 ( SMER10 ) ( a ) ; P = 0 . 0006 ( SMER18a ) , P = 0 . 0249 ( SMER18b ) , P = 0 . 0167 ( SMER18c ) , P = 0 . 0117 ( SMER18d ) , P = 0 . 0269 ( SMER18e ) , P = 0 . 0165 ( SMER18f ) , P = 0 . 0148 ( SMER18g ) , P = 0 . 0011 ( SMER18h ) , P = 0 . 7369 ( SMER18i ) , P = 0 . 0012 ( SMER18j ) , P = 0 . 1531 ( SMER18k ) , P = 0 . 0006 ( SMER18l ) , P = 0 . 0001 ( SMER18 ) ( b ) ; P = 0 . 0014 ( SMER28a ) , P = 0 . 0002 ( SMER28b ) , P = 0 . 0001 ( SMER28d ) , P = 0 . 0048 ( SMER28h ) , P = 0 . 0002 ( SMER28i ) , P = 0 . 0162 ( SMER28j ) , P 0 . 0001 ( SMER28c , SMER28e - g , SMER28k , SMER28 ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates in COS - 7 cells as in Figure 1c - treated for 48 h with either DMSO ( control ) , or with 47 μM SMER10 and its analog ( SMER10a ) ( d ) , 43 μM SMER18 and its analogs ( SMER18a , SMER18c - h , SMER18j , SMER18l ) ( e ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( f ) - were expressed as odds ratios . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars : 95 % confidence interval . P = 0 . 003 ( SMER10a ) , P = 0 . 004 ( SMER10 ) ( d ) ; P 0 . 0001 ( SMER18a , SMER18c , SMER18d , SMER18f , SMER18h ) , P = 0 . 009 ( SMER18e ) , P = 0 . 001 ( SMER18g ) , P = 0 . 382 ( SMER18j ) , P = 0 . 067 ( SMER18l ) , P = 0 . 031 ( SMER18 ) ( e ) ; P 0 . 0001 ( SMER28a , SMER28c , SMER28e - g , SMER28i - k , SMER28 ) , P = 0 . 574 ( SMER28b ) , P = 0 . 041 ( SMER28d ) , P = 0 . 002 ( SMER28h ) ( f ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( B ) Immunofluorescence ( IF ) validation of mintbody specificity . Mouse MC12 cells , which stably express H3K27me3 - mintbody ( sfGFP ) , are labelled with antibodies specific for H3K27me3 ( Cy5 ) and H3K9me3 ( Cy3 ) . DNA is stained with Hoechst33342 . Single confocal sections are shown . Arrows mark Xi . Scale bar = 10 μm .

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

(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.


( D ) ESCs as in ( B ) were engineered to express H3K27me3 - mintbody ( SNAP / JF646 ) and H4K20me1 - mintbody ( mCherry ) . Cells were cultured in the absence of LIF for 2 days . Time - lapse images for 12 z - plane confocal stack were acquired every 1 h . Maximum intensity projection images are shown with elapse time ( hh : mm ) . Arrowheads mark Xi . Scale bar = 10

O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O 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-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 B-SUBCELLULAR O O O O O

(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm


( E ) H4K20me1 ( blue ) and H3K27me3 ( red ) accumulation across the Xi after 12 h of DOX treatment . The black line is a locally estimated scatterplot smoothing ( LOESS ) regression on all 10 - kb windows ( dots ) . Below each plot shown is the Xist locus ( green bar ) and Xist entry sites ( black bars ) .

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

(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).


( A ) Genome browser tracks showing H3K27me3 ( top ) and H4K20me1 ( bottom ) accumulation at a gene silenced rapidly ( Rnf12 ) or more slowly ( Pgk1 ) . Allele - specific tracks were overlaid ( B6 in red and Cast in blue ) . Note strong H4K20me1 bi - allelic pre - marking at gene bodies .

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

(A) Genome browser tracks showing H3K27me3 (top) and H4K20me1 (bottom) accumulation at a gene silenced rapidly (Rnf12) or more slowly (Pgk1). Allele-specific tracks were overlaid (B6 in red and Cast in blue). Note strong H4K20me1 bi-allelic pre-marking at gene bodies.