( D ) Representative immunofluorescence images of HeLa cells transfected with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for ACA ( red ) and DNA ( blue ) . ( E ) Scatter graphs illustrating kinetochore intensity of GFP - Cyclin B2 - WT / 4A treated as in D . Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of GFP - Cyclin B2 - WT . Each dot represents one cell ( ≥ 30 cells from three independent experiments ) .

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(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).


( A ) HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were collected and lysed . The samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - Cyclin B2 antibody , anti - Cyclin B1 antibody , and anti - tubulin

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(A) HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were collected and lysed. The samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B2 antibody, anti-Cyclin B1 antibody, and anti-tubulin antibody


( B ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA . Images were acquired at the indicated time points after the start of NEBD . The arrow indicates lagging

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(B) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA. Images were acquired at the indicated time points after the start of NEBD. The arrow indicates lagging chromosomes


( C ) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(C) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B. Bars are means ±SD (≥30 cells from three independent experiments)


( D ) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B ( n = 30 cells )

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(D) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B (n = 30 cells)


( E ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . Images were acquired at the indicated time points after the start of

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(E) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. Images were acquired at the indicated time points after the start of NEBD


( F ) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(F) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E. Bars are means ±SD (≥30 cells from three independent experiments)


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( green ) in D , ACA ( red ) , and DNA ( blue ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( red ) in DNA ( blue )

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Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue)


Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( green ) in D , ACA ( red ) , and DNA ( blue ) E ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( red ) in E , and DNA ( blue )

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Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue)


Scatter graphs illustrating kinetochore intensity o Cdc20 ( F , cells treated as in D and E ) Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity o Cdc20 (F, cells treated as in D and E) Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


( G ) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12

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(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h


( H ) Immunoprecipitation assay performed with Cdc20 antibody . HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were arrested in mitosis , collected , and lysed . Cell lysates were incubated with Protein A / G beads bound with Cdc20 antibody . Following extensive washes , immunoprecipitates were resolved by SAS - PAGE and analyzed by Western blotting using anti - Cdc20 , anti - BubR1 , anti - Bub3 , and anti - Mad2

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(H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies


Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR ‐ and NucNeoR ‐ expressing cells . RCC1 . 24 cells were transfected with the NeoR ( A ) and NucNeoR ( B ) expression constructs . After 2 days , the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c ‐ myc antibody 9E10 . While NeoR was evenly distributed in nucleus and cytoplasm ( a ) , NucNeoR was confined to the cell nucleus ( b ) . Daylight photographs of the stained cells are shown in ( a ' ) and ( b ' ) .

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Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR‐ and NucNeoR‐expressing cells. RCC1.24 cells were transfected with the NeoR (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c‐myc antibody 9E10. While NeoR was evenly distributed in nucleus and cytoplasm (a), NucNeoR was confined to the cell nucleus (b). Daylight photographs of the stained cells are shown in (a') and (b').


( C ) NeoR ‐ and NucNeoR ‐ transfected RCC1 . 24 cells were treated with 100 U / mL IFN ‐ γ for 48 h to induce MHC class II expression . Subsequently , the cells were washed and co ‐ cultured with the CD4 clone 20 ‐ 4 / A4 , which recognizes a peptide derived from NeoR on HLA ‐ DP3 . GM ‐ CSF secretion by the T cells was measured 20 h later by ELISA . Both transfectants were recognized after IFN ‐ γ treatment , indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation .

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(C) NeoR‐ and NucNeoR‐transfected RCC1.24 cells were treated with 100 U/mL IFN‐γ for 48 h to induce MHC class II expression. Subsequently, the cells were washed and co‐cultured with the CD4 clone 20‐4/A4, which recognizes a peptide derived from NeoR on HLA‐DP3. GM‐CSF secretion by the T cells was measured 20 h later by ELISA. Both transfectants were recognized after IFN‐γ treatment, indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation.


( D ) LCL1 . 11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR ‐ specific T cell clone 20 ‐ 4 / A4 . Antigen ‐ transfected LCL1 . 11 cells were recognized irrespectively of NeoR localization , while untransfected cells or cells transfected with a GFP ‐ expressing control plasmid were not recognized .

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(D) LCL1.11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR‐specific T cell clone 20‐4/A4. Antigen‐transfected LCL1.11 cells were recognized irrespectively of NeoR localization, while untransfected cells or cells transfected with a GFP‐expressing control plasmid were not recognized.


( A ) RCC1 . 24 ‐ NeoR and ‐ NucNeoR cells were incubated for 48 h with IFN ‐ γ and in addition with 7 . 5 mM 3 ‐ MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 ‐ 4 / A4 cells in a GM ‐ CSF release assay . Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing .

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(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.


( B ) LCL1 . 11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise . Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors , indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing .

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(B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing.


( C ) To exclude toxic side effects of the inhibitors , LCL1 . 11 cells were incubated for 20 h with 200 ng / mL recombinant NeoR protein in the presence or absence of the inhibitors . Afterwards , residual protein and inhibitors were removed by washing and the cells were co ‐ cultured with the NeoR ‐ specific CD4 + T cell clone 20 ‐ 4 / A4 . The presentation of exogenous NeoR on MHC class II was not affected by 3 ‐ MA , but was impaired when lysosomal processing was blocked with chloroquine or leupeptin .

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(C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.


( D ) LCL1 . 11 cells were transfected with siRNA specific for Atg12 or GFP , and Atg12 mRNA levels were determined 24 h later by qPCR . The relative amount of Atg12 mRNA was reduced approximately fourfold .

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(D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold.


( E ) At 24 h after siRNA transfection , NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later . In cells transfected with Atg12 siRNA , but not in mock ‐ transfected cells or cells transfected with GFP siRNA , antigen presentation of both NeoR and NucNeoR was greatly reduced .

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(E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.


( A ) RCC1 . 24 cells were transfected with the NES ‐ GFP ‐ NeoR ‐ GFP ‐ NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence . As compared to untreated cells ( a ) , cells treated with 8 nM LMB for 20 h ( b ) showed a strong accumulation of the protein in the nucleus . ( a ' ) and ( b ' ) are the corresponding daylight photographs of the transfected cells .

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(A) RCC1.24 cells were transfected with the NES‐GFP‐NeoR‐GFP‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM LMB for 20 h (b) showed a strong accumulation of the protein in the nucleus. (a') and (b') are the corresponding daylight photographs of the transfected cells.


( B ) NeoR ‐ and NucNeoR ‐ transfected LCL1 . 11 cells were either left untreated or incubated for 20 h with 8 nM LMB . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and probed with the NeoR ‐ specific T cells . LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II , indicating that CRM1 ‐ dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II .

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(B) NeoR‐ and NucNeoR‐transfected LCL1.11 cells were either left untreated or incubated for 20 h with 8 nM LMB. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the NeoR‐specific T cells. LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II, indicating that CRM1‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.


( G ) Immunostaining of pS6 ( Ser240 / 244 ) in skin lesions from control and LL37 - induced mice . Bottom right panels , magnified images of boxed areas . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . Right panel , the quantification of relative fluorescence intensity for pS6 ( Ser240 / 244 ) in control and LL37 group ( n = 5 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . two - tailed unpaired Student ' s t - test was used .

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(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.


( D ) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group ) . Scale bar : 50 μm . ( E ) Dermal infiltrating cells were quantified ( n = 6 for each group ) for each high power field ( HPF ) .

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(D) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n=6 for each group). Scale bar: 50 μm. (E) Dermal infiltrating cells were quantified (n = 6 for each group) for each high power field (HPF).


( F ) The mRNA expression levels of Tnf - α , Il6 , Mmp9 and Vegf in skin lesions ( n = 6 for each group ) .

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(F) The mRNA expression levels of Tnf-α, Il6, Mmp9 and Vegf in skin lesions (n = 6 for each group).


( I ) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle . Scale bar : 50 μm . ( J ) Dermal infiltrating cells were quantified ( n = 6 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm. (J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


( A ) The back skins of WT ( Tsc2 + / + ) and Tsc2 + / - mice were intradermally injected with LL37 or control vehicle ( n = 5 for each group ) . Pictures were taken on day 0 . 5 , 2 , 5 , 8 , 12 after the first LL37 injection . The mouse experiments were repeated for three times , and 5 mice were included in each group for each time . The results of a representative mouse experiment were displayed . Scale bar : 2 mm .

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(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm.


( A ) The mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10 ) and patients with rosacea ( n = 15 ) .

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(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15). Data


( G ) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 ( 4 μM ) ± RAPA for 24 h . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM .

O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE 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

(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.


( E ) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC - labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed , then live cells were analyzed by fluorescent microscope . Scale bar : 50 μm .

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

(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.


( F , G ) Primary human keratinocytes were incubated with sLL37 - flag or LL37 - flag for 1 h . Cell lysates were immunoprecipitated with anti - flag or anti - TLR2 antibodies , showing an interaction between LL37 and TLR2 . Medium , the medium of primary human keratinocytes treated with sLL37 - flag or LL37 - flag . All results are representative of at least 3 independent experiments .

O O O O O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

(F, G) Primary human keratinocytes were incubated with sLL37-flag or LL37-flag for 1 h. Cell lysates were immunoprecipitated with anti-flag or anti-TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37-flag or LL37-flag. All results are representative of at least 3 independent experiments.


( E ) Immunostaining of phospho - p65 ( p - p65 ) in skin sections . Bottom right panels , magnified images of dotted line areas . Epi , epidermis . Der , dermis . Scale bar : 50 μm . ( F ) Quantification of relative fluorescence intensity for p - p65 in epidermis ( n = 6 ) .

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

(E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm. (F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6).


( G ) The mRNA expression levels of NF - κB family of transcription factors ( Nfκb1 , Nfκb1 , Rela and Relb ) in mice skin lesions ( n = 6 for each group ) .

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

(G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group).


( I ) IHC of p65 on skin sections from HS and rosacea . Scale bar , 50 μm . ( J ) Percentage of nuclear p65 positive cells in the epidermis from HS ( n = 8 ) and rosacea ( n = 13 ) . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test ( F and G ) or two - tailed unpaired Student ' s t - test ( J ) was used .

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

(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.


( B ) The mRNA expression levels of mouse chemokines ( Cxcl11 , Cxcl12 , Ccl2 and Ccl3 ) in skin lesions ( n = 6 for each group ) .

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

(B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group).


( E ) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA - sequencing . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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

(E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients . ( C ) The change of patients ' CEA score showed in scatter histogram at 0 week and 4th week . ( D ) The change of patients ' IGA score showed in scatter histogram at 0 week and 4th week . " The Change value < 0 " indicates that skin lesions show positive improvement after treatment ; " The Change value > 0 " indicates skin lesions show exacerbation after treatment ; " The Change value = 0 " indicates that skin lesions show no change after treatment ; Data represent the mean ± SEM , and two - tailed unpaired Student ' s t - test was used ( C , D ) .

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

Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. "The Change value < 0" indicates that skin lesions show positive improvement after treatment; "The Change value > 0" indicates skin lesions show exacerbation after treatment; "The Change value = 0" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).


( A ) ARPE - 19 cells were incubated in the presence of DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved ( EBSS ) for 16 h . Cells were fixed , permeabilized and stained with an antibody against TFE3 . Scale bar , 10 µm . Data are representative of three independent experiments .

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

(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments.


( B ) Immunoblot of protein lysates of ARPE - 19 cells treated as shown in ( A ) . Data are representative of three independent experiments .

O O O B-EXP_ASSAY 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 O O O O O

(B) Immunoblot of protein lysates of ARPE-19 cells treated as shown in (A). Data are representative of three independent experiments.


( C ) ARPE - 19 cells incubated with Tunicamycin as indicated in ( A ) were immunostained with antibody against CHOP . Scale bar , 10 µm . Data are representative of three independent experiments .

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

(C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments.


( D ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or starvation ( Strv . ) treatments as indicated in ( A ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Control treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition).


( E ) Wild - type mouse embryonic fibroblast ( MEF ) cells were incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for the indicated times . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments .

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

(E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.


( F ) Wild - type MEF cells were incubated in the presence of DMSO ( Control ) or BFA ( 2 . 5 µg / ml ) for 16 h . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( G ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or BFA 16 h treatment ( mean ± SD of two independent experiments , student ' s t - test analysis versus DMSO treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student's t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition).


( H ) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO ( Ctrl . ) or Tunicamycin ( Tun . ) for 16 h using TFE3 , TFEB , Lamp1 ( as a lysosomal membrane marker ) , and H3b ( as a nuclear marker ) . Data are representative of three independent experiments .

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

(H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.


( A ) Immunoblot of protein lysates from wild - type , PERK - KO and GCN2 - KO MEF cells incubated in the presence of DMSO ( Control ) , Tunicamycin ( 0 . 1 µg / ml ) , or starved in EBSS for 16 h . Data are representative of three independent experiments .

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

(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.


( B - D ) Wild - type ( B ) , GCN2 - KO ( C ) , and PERK - KO ( D ) MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) as indicated in ( A ) . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( E - F ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) , Tunicamycin treatments for 16 h ( E ) , or starvation for 2 h ( F ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated cells , ns = not significant , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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 B-CELL_LINE 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 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 B-CELL_LINE O O O B-GENEPROD 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 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-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).


( G ) Quantification of phospho - 4EBP1 protein levels in wild - type and GCN2 - KO MEF cells upon starvation for 2 h ( the mean ± SD of the fold increase of the phospho - 4EBP1 to total 4EBP1 ratio from three independent experiments , one - way ANOVA analysis versus starvation treated wild - type cells , * * * p < 0 . 001 ) .

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

(G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001).


( H ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or Torin - 1 ( 250 nM ) treatments for 1 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated wild - type cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).


( A ) ARPE - 19 cells infected with either adenovirus expressing TFE3 - WT - Myc or TFE3 - S321A - Myc for 16h . Cells were fixed , permeabilized and stained with antibodies against Myc . Scale bar , 10 µm . Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 - Myc ( means ± SD of 3 independent experiments , students t - test , * * * * p < 0 . 0001 ; n > 400 cells per condition ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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 O 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) ARPE-19 cells infected with either adenovirus expressing TFE3-WT-Myc or TFE3-S321A-Myc for 16h. Cells were fixed, permeabilized and stained with antibodies against Myc. Scale bar, 10 µm. Quantification of the percentage of ARPE-19 cells with nuclear TFE3-Myc (means ± SD of 3 independent experiments, students t-test, ****p < 0.0001; n>400 cells per condition).


( B ) Immunoblots of protein lysates from ARPE - 19 cells infected as indicated in ( A ) . Data are representative of three independent experiments .

(B) Immunoblots of protein lysates from ARPE-19 cells infected as indicated in (A). Data are representative of three independent experiments.


( C ) Immunoblots of TFE3 - Ser321 phosphorylation state in nuclear fractions of ARPE - 19 cells incubated with DMSO ( Ctrl . ) , Tunicamycin ( Tun . ) , or Torin - 1 . Data are representative of three independent experiments .

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

(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.


( D ) Immunoblots of protein of a GST pull - down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin ( 5 µg / ml ) ( Tun . ) for 16 h . Data are representative of three independent experiments .

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

(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.


( E ) Immunoblots of protein lysates from ARPE - 19 cells incubated in the presence of DMSO ( Ctrl . ) , Tunicamycin ( 5 µg / ml ) ( Tun . ) , or starved in EBSS ( Strv . ) for 16 h . Data are representative of three independent experiments .

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

(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.


( F ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Tunicamycin + FK506 treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Tunicamycin treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).


( G ) Quantification of the percentage of calcineurin depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Starvation ( Strv . ) treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).


( H ) Immunoblots of protein lysates from ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB . Data are representative of three independent experiments .

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

(H) Immunoblots of protein lysates from ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.


( I ) Relative Quantitative Real - Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analsysis versus the siNon - Target ( siNT ) treated cells , * * * p < 0 . 001 ) .

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

(I) Relative Quantitative Real-Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analsysis versus the siNon-Target (siNT) treated cells, ***p < 0.001).


( J ) Quantification of the percentage of ATF4 depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h ( mean ± SD of two independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).


( A ) Relative Quantitative Real - Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , student ' s t - test versus siNon - Target ( siNT ) treated cells , * * * p < 0 . 001 ) .

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

(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student's t-test versus siNon-Target (siNT) treated cells, ***p < 0.001).


( B ) Relative Quantitative Real - Time PCR analysis of MCOLN1 , ATP6V1C1 , HEXA and UVRAG mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 upon incubation with DMSO ( Control ) or Tunicamycin ( 5 µg / ml ) for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the same treatment condition in the siNon - Target ( siNT ) treated cells or versus the DMSO condition in siNon - Target ( siNT ) treated cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( A ) Immunoblots of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null , TFEB - S211A , or TFE3 upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) or starved in EBSS for 16 h . Data are representative of five independent experiments . ( B ) Quantification of ATF4 , CHOP and GADD34 protein levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( C ) Relative Quantitative Real - Time PCR analysis of ATF4 , CHOP , GADD34 and MCOLN1 mRNA transcript levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).


( A - D ) Relative Quantitative Real - Time PCR analysis of ATF4 ( A ) , ASNS ( B ) , CAT1 ( C ) and xCT ( D ) mRNA transcript levels in ATF4 - depleted ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved in EBSS for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected and siRNA Non - Target ( siNT ) treated cells , * p < 0 . 05 , * * * p < 0 . 001 ) .

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

(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).


( E ) ChIP - qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest ( ATF4 - Control ) from MEF cells that were untreated ( control ) , starved for 2 h , or treated with Tunicamycin ( 0 . 1 µg / ml ) for 16 h . Amplification regions are indicated by arrows . Chromatin DNA was immunoprecipitated with antibodies for anti - TFE3 . Bar graphs show the amount of immunoprecipitated DNA detected by the real - time PCR assay . Values were normalized to the input and plotted as relative enrichment compared to untreated conditions ( means ± SD of three independent experiments , students t - test , * p < 0 . 05 , * * p < 0 . 01 ) .

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

(E) ChIP-qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest (ATF4-Control) from MEF cells that were untreated (control), starved for 2 h, or treated with Tunicamycin (0.1 µg/ml) for 16 h. Amplification regions are indicated by arrows. Chromatin DNA was immunoprecipitated with antibodies for anti-TFE3. Bar graphs show the amount of immunoprecipitated DNA detected by the real-time PCR assay. Values were normalized to the input and plotted as relative enrichment compared to untreated conditions (means ± SD of three independent experiments, students t-test, *p < 0.05, **p < 0.01).


( F ) Relative Quantitative Real - Time PCR analysis of SUMF1 , DERL1 , ATF6 , APEX1 and GPX1 mRNA transcript levels in ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , students t - test , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(F) Relative Quantitative Real-Time PCR analysis of SUMF1, DERL1, ATF6, APEX1 and GPX1 mRNA transcript levels in ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, students t-test, **p < 0.01, ***p < 0.001).


( G ) Immunoblots of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A . Data are representative of three independent experiments . ( H ) Quantification of IRE1α and APEX1 protein levels in ARPE - 19 cells infected as indicated in ( G ) ( mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments , students t - test , * * * p < 0 . 001 ) .

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

(G) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A. Data are representative of three independent experiments.(H) Quantification of IRE1α and APEX1 protein levels in ARPE-19 cells infected as indicated in (G) (mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments, students t-test, ***p < 0.001).


( A ) Immunoblots of protein lysates from Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for the indicated times . Data are representative of three independent experiments . ( B ) Quantification of ATF4 protein levels in Null - or TFEB / 3 knockout - MEF cells treated as indicated in ( A ) ( mean ± SD of the ATF4 to actin protein ratio from three independent experiments , student ' s t - test versus the corresponding time point in Null - MEF cells , ns = not significant , * * p < 0 . 01 ) .

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

(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student's t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01).


( C ) Cell viability was determined in Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for the indicated times ( mean ± SD of the percentage of viable cells compared to control from three independent experiments , one - way ANOVA analysis versus the corresponding time point in Null - MEF cells * * * p < 0 . 001 ) .

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

(C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001).


( D ) Chromatin condensation , an indication of cells undergoing apoptosis , was assessed in Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for the indicated times . Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342 . Scale bar , 20 µm . Data are representative of three independent experiments .

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-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY 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

(D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments.


( E ) Immunoblots of protein lysates from Null - or TFEB / 3 knockout - MEF cells treated as described in ( A ) . Data are representative of three independent experiments . ( F - H ) Quantification of CHOP ( F ) , PUMA ( G ) and NOXA ( H ) protein levels in Null - or TFEB / 3 knockout - MEF cells treated as indicated in ( E ) ( mean ± SD of the indicated protein to actin ratio from three independent experiments , student ' s t - test analysis versus the corresponding time point in Null - MEF cells , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ) .

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

(E) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells treated as described in (A). Data are representative of three independent experiments.(F-H) Quantification of CHOP (F), PUMA (G) and NOXA (H) protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (E) (mean ± SD of the indicated protein to actin ratio from three independent experiments, student's t-test analysis versus the corresponding time point in Null-MEF cells, ns= not significant, *p < 0.05, **p < 0.01).


( I ) adenovirus of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null , TFEB - S211A or TFE3 . Data are representative of three independent experiments . ( J ) Quantification of PUMA protein levels in ARPE - 19 cells infected as indicated in ( I ) ( mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments , one - way ANOVA analysis versus the adenovirus - Null infected cells , * p < 0 . 05 , * * * p < 0 . 001 ) .

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

(I) adenovirus of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A or TFE3. Data are representative of three independent experiments.(J) Quantification of PUMA protein levels in ARPE-19 cells infected as indicated in (I) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the adenovirus-Null infected cells, *p < 0.05, ***p < 0.001).


( A ) Serum IgG ELISA reactivity with MTB ( H37Rv ) whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) for TB patients and healthy donors ( HD ) . ELISA graphs ( left ; TB : black lines ; n = 25 ; HD : red dotted lines ; n = 2 ) and area under the curve ( AUC ) values for all tested samples ( right ; TB : n = 25 ; HD : n = 17 ) are shown . Median is shown .

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

(A) Serum IgG ELISA reactivity with MTB (H37Rv) whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) for TB patients and healthy donors (HD). ELISA graphs (left; TB: black lines; n=25; HD: red dotted lines; n=2) and area under the curve (AUC) values for all tested samples (right; TB: n=25; HD: n=17) are shown. Median is shown.


( B ) AUC values for the IgA serum ELISA response against MTB whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) of TB patients ( n = 25 ) and healthy donors ( HD ; n = 9 ) . Median is shown .

O O O O O O O B-SUBCELLULAR B-TISSUE B-EXP_ASSAY 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 O O

(B) AUC values for the IgA serum ELISA response against MTB whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) of TB patients (n=25) and healthy donors (HD; n=9). Median is shown.


( C ) Flow cytometry gating strategy and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) for one representative TB patient ( TB24 ) and HD ( left ) . Frequency of circulating CD19 + CD27 + + CD38 + plasmablasts of all CD19 + B cells in peripheral blood of TB patients ( n = 24 ) and HD ( n = 8 ) . Dashed line indicates threshold for detectable plasmablast populations ( right ) . Median and SEM are shown .

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

(C) Flow cytometry gating strategy and frequency of circulating plasmablasts (CD19+CD27++CD38+) for one representative TB patient (TB24) and HD (left). Frequency of circulating CD19+CD27++CD38+plasmablasts of all CD19+ B cells in peripheral blood of TB patients (n=24) and HD (n=8). Dashed line indicates threshold for detectable plasmablast populations (right). Median and SEM are shown.


( D ) Expression of CD19 and activation markers ( CD86 , CD84 and CD24 ) in plasmablasts and memory B cells as measured by flow cytometry .

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

(D) Expression of CD19 and activation markers (CD86, CD84 and CD24) in plasmablasts and memory B cells as measured by flow cytometry.


( E ) Plasmablast response of patient TB7 ( top ) and IgG serum response against MTB cell lysate of patient TB7 , TB19 and TB39 ( bottom ) and one HD at the indicated time points before and after treatment onset .

O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR B-TISSUE 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

(E) Plasmablast response of patient TB7 (top) and IgG serum response against MTB cell lysate of patient TB7, TB19 and TB39 (bottom) and one HD at the indicated time points before and after treatment onset.


( F ) Dots indicate AUC values for the anti - MTB cell lysate IgG serum ELISA response ( y - axis ) versus frequency of circulating plasmablasts ( x - axis ) for individual TB patients .

O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR B-TISSUE B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O

(F) Dots indicate AUC values for the anti-MTB cell lysate IgG serum ELISA response (y-axis) versus frequency of circulating plasmablasts (x-axis) for individual TB patients.


( A ) Gating strategy , phenotype and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) isolated by flow cytometric cell sorting from 3 TB patients ( TB7 , TB24 , TB33 ) in comparison to one representative HD . Boxes indicate sort gates . The plasmablast frequency is indicated .

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

(A) Gating strategy, phenotype and frequency of circulating plasmablasts (CD19+CD27++CD38+) isolated by flow cytometric cell sorting from 3 TB patients (TB7, TB24, TB33) in comparison to one representative HD. Boxes indicate sort gates. The plasmablast frequency is indicated.


( B ) Absolute number of somatic hypermutations ( SHM ) in the IGHV , IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7 , TB24 and TB33 . The absolute number of sequences analyzed is indicated below the graph . Geometric means with SEM are indicated in grey . SHM means of historic data from sorted CD27 + IgA + or CD27 + IgG + cells from peripheral blood of HD are indicated in red for comparison ( Berkowska et al , 2015a ; Tiller et al , 2007 ) .

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(B) Absolute number of somatic hypermutations (SHM) in the IGHV, IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7, TB24 and TB33. The absolute number of sequences analyzed is indicated below the graph. Geometric means with SEM are indicated in grey. SHM means of historic data from sorted CD27+IgA+ or CD27+IgG+ cells from peripheral blood of HD are indicated in red for comparison (Berkowska et al, 2015a; Tiller et al, 2007).


( C ) Isotype distribution of plasmablast and memory B cells from TB7 , TB24 and TB33 . PB , plasmablasts ( CD19 + CD27 + + CD38 + ) ; M , memory B cells ( CD19 + CD27 + ) .

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

(C) Isotype distribution of plasmablast and memory B cells from TB7, TB24 and TB33. PB, plasmablasts (CD19+CD27++CD38+); M, memory B cells (CD19+CD27+).


( D ) Gating strategy , phenotype and frequency of HBHA - reactive memory B cells ( CD19 + CD27 + HBHA + ) in peripheral blood of one representative TB patient ( TB29 ) , health - care worker ( HCW ) 2 and HD , respectively .

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(D) Gating strategy, phenotype and frequency of HBHA-reactive memory B cells (CD19+CD27+HBHA+) in peripheral blood of one representative TB patient (TB29), health-care worker (HCW) 2 and HD, respectively.


( E ) Dots indicate frequency of HBHA - reactive memory B cells out of all CD27 + memory B cells in individual TB patients ( n = 23 ) and HCWs ( n = 7 ) . Mean and SEM are indicated . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * p < 0 . 0001 .

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(E) Dots indicate frequency of HBHA-reactive memory B cells out of all CD27+ memory B cells in individual TB patients (n=23) and HCWs (n=7). Mean and SEM are indicated. P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


( F ) Dots indicate the frequency of resting memory B cells ( CD19 + CD27 + CD10 - ) out of all B cells in peripheral blood of individual TB patients compared to HD .

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(F) Dots indicate the frequency of resting memory B cells (CD19+CD27+CD10-) out of all B cells in peripheral blood of individual TB patients compared to HD.


( G ) Anti - HBHA serum IgG ELISA reactivity for TB patients and HCW ( black lines ) compared to two representative HD ( red lines ) . Dashed line indicates the threshold OD405nm for positive reactivity ( left ) . Asterisks indicate serum responses of donors selected for single sorting of HBHA - reactive memory B cells . Data are representative of two independent experiments .

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(G) Anti-HBHA serum IgG ELISA reactivity for TB patients and HCW (black lines) compared to two representative HD (red lines). Dashed line indicates the threshold OD405nm for positive reactivity (left). Asterisks indicate serum responses of donors selected for single sorting of HBHA-reactive memory B cells. Data are representative of two independent experiments.


( H ) Absolute number of somatic hypermutations ( SHM ) in the IGHV ( IGHA , IGHG and IGHM ) , IGKV or IGLV segments of sorted anti - HBHAmemory cells from TB patients and HCWs . Geometric means with SEM are indicated in grey . For comparison , red lines indicate the historic SHM means for randomly sorted CD27 + IgA + , CD27 + IgG + or CD27 + IgM + cells from peripheral blood of HDs ( Berkowska et al , 2015a ; Tiller et al , 2007 ; Tsuiji et al , 2006 ) .

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(H) Absolute number of somatic hypermutations (SHM) in the IGHV (IGHA, IGHG and IGHM), IGKV or IGLV segments of sorted anti-HBHAmemory cells from TB patients and HCWs. Geometric means with SEM are indicated in grey. For comparison, red lines indicate the historic SHM means for randomly sorted CD27+IgA+, CD27+IgG+ or CD27+IgM+ cells from peripheral blood of HDs (Berkowska et al, 2015a; Tiller et al, 2007; Tsuiji et al, 2006).


( I ) Number and size of clonally expanded B cell clusters among HBHA - reactive memory B cells from two TB patients ( TB35 , TB29 ) and HCWs ( HCW1 , HCW 2 ) . Cells in clusters are indicated in grey , single cells are indicated in white . No B cell clusters were shared between donors .

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(I) Number and size of clonally expanded B cell clusters among HBHA-reactive memory B cells from two TB patients (TB35, TB29) and HCWs (HCW1, HCW 2). Cells in clusters are indicated in grey, single cells are indicated in white. No B cell clusters were shared between donors.


( J ) IgA , IgG and IgM isotype distribution of single - cell - sorted HBHA - reactive memory cells . Mean and SEM are indicated .

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(J) IgA, IgG and IgM isotype distribution of single-cell-sorted HBHA-reactive memory cells. Mean and SEM are indicated.


( A , B ) Representative ELISA graphs show reactivity of antibodies from patient TB7 to ( A ) MTB whole cell lysate or ( B ) MTB bacteria ( left ) . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) . AUC ( area under curve ) values indicate the reactivity to ( A ) MTB cell lysate or ( B ) whole MTB bacteria for antibodies from TB7 , TB24 , TB33 . Total numbers of analyzed antibodies are indicated ( right ) .

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(A, B) Representative ELISA graphs show reactivity of antibodies from patient TB7 to (A) MTB whole cell lysate or (B) MTB bacteria (left). Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). AUC (area under curve) values indicate the reactivity to (A) MTB cell lysate or (B) whole MTB bacteria for antibodies from TB7, TB24, TB33. Total numbers of analyzed antibodies are indicated (right).


( D ) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB - reactive ( n = 26 ) and non - reactive ( n = 14 ) antibodies .

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(D) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB-reactive (n=26) and non-reactive (n=14) antibodies.


( E ) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate - reactive antibodies and one non - reactive antibody ( isotype control , mGO53 ) .

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(E) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate-reactive antibodies and one non-reactive antibody (isotype control, mGO53).


( A ) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot . The negative isotype control ( mGO53 ) and a commercially available anti - LAM antibody ( positive control ) are shown for comparison .

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(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison.


( B ) Anti - BCG specificity of the LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole bacteria ( left ) and flow cytometry ( right ) .

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(B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right).


( C ) Anti - LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations . The mean ± SD of the absorbance was calculated .

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(C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated.


( D ) Antibody TB24PB037 binds to MTB - LAM but not to LAM from M . smegmatis . ELISA performed as described in ( C ) .

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(D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C).


( E ) Representative HBHA ELISA for antibodies cloned from HBHA - reactive memory B cells of patient TB35 and HCW1 . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) .

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(E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)).


( F ) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity .

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(F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity.


( G ) Fluorescence microscopy shows BCG - and MTB - reactivity of representative anti - HBHA antibodies compared to a non - reactive isotype control antibody . Scale bar 10 μm ( top / bottom ) .

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(G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom).


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( A ) Representative results for individual MTB - reactive plasmablast antibodies . Mean and SEM are indicated .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( B ) Direct comparison of all tested MTB - reactive IgA ( n = 12 ) and IgG ( n = 26 ) antibodies . Mean and SEM are indicated .

O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-ORGANISM O O O O O O B-ORGANISM 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 O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( C ) Representative results for individual HBHA - reactive memory B cell antibodies from TB patients and HCW .

O O 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-ORGANISM O O O O O O B-ORGANISM 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 O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O

Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( D ) Same as in ( B ) but with HBHA - reactive antibodies as shown in ( C ) . ( D ) Direct comparison all tested HBHA - reactive IgA ( n = 6 ) and IgG ( n = 7 ) antibodies .

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

Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies.


Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( E ) Direct comparison of the indicated MTB - reactive antibodies expressed as IgA and IgG . Mean and SEM are indicated . P - values were determined using non - parameric t - test , * p < 0 . 1 ; * * p < 0 . 01 , ns : p > 0 . 5 .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5.


( A ) Human lung epithelial A549 cells and primary bronchial epithelial cells ( hAECB ) lack FcγR ( CD16 , CD64 and CD32 ) and FcαR ( CD89 ) cell surface expression as analyzed by flow cytometry . Human THP1 cells serve as positive control .

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(A) Human lung epithelial A549 cells and primary bronchial epithelial cells (hAECB) lack FcγR (CD16, CD64 and CD32) and FcαR (CD89) cell surface expression as analyzed by flow cytometry. Human THP1 cells serve as positive control.


( B ) The neonatal FcR ( FcRn ) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting . Data are representative of at least two independent experiments .

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(B) The neonatal FcR (FcRn) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting. Data are representative of at least two independent experiments.


( C ) Absolute bacterial counts ( CFUs ) in human THP1 macrophages 1 h after infection with MTB pre - incubated with the LAM - reactive IgA1 ( left ) or IgG1 ( right ) antibody TB24PB037 or the respective isotype control antibody as indicated . Mean and SEM are indicated .

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(C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated.


( A ) Absolute bacterial counts ( CFUs ) in human lung epithelial A549 cells 1 h after infection with MTB pre - incubated with purified serum IgA ( left ) or IgG ( right ) antibodies from TB patients ( TB ) compared to HD as indicated . Mean and SEM are indicated . Data are representative of three independent experiments with three technical replicates each .

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(A) Absolute bacterial counts (CFUs) in human lung epithelial A549 cells 1 h after infection with MTB pre-incubated with purified serum IgA (left) or IgG (right) antibodies from TB patients (TB) compared to HD as indicated. Mean and SEM are indicated. Data are representative of three independent experiments with three technical replicates each.


( B ) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * p < 0 . 0001 .

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(B) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in (A). P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


Ten - fold serial dilutions of 1 OD600 unit / mL of the indicated strains were spotted on SD plates containing 0 ( C - ) or 1 mM choline ( C + ) and 0 ( I - ) or 75 μM inositol ( I + ) and incubated at 30oC for 3 d . A representative experiment is shown ( from n = 5 ) .

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Ten-fold serial dilutions of 1 OD600 unit/mL of the indicated strains were spotted on SD plates containing 0 (C-) or 1 mM choline (C+) and 0 (I-) or 75 μM inositol (I+) and incubated at 30oC for 3 d. A representative experiment is shown (from n=5).


A - L Wild type cultured in C - ( A ) , cho2opi3 cultured in C + ( B ) , coS # 4 ( C - D ) , coS # 3 ( E - G ) , and coS # 2 ( I - K ) cultured in C - were analyzed by electron microscopy . In addition , coS # 3 ( H ) and coS # 2 ( L ) are shown after culture in C + for 3 generations . The arrow heads ( F , I , J ) point to protrusions in the nuclear envelope . CW , cell wall ; ER , endoplasmic reticulum ; PM , plasma membrane ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 200 nm ( A , B , D , F , G ) or 500 nm ( C , E , H , I , J , K , L ) .

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A-L Wild type cultured in C- (A), cho2opi3 cultured in C+ (B ), coS#4 (C-D), coS#3 (E-G), and coS#2 (I-K) cultured in C- were analyzed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L).


Serial dilutions of the strains indicated were spotted on SD C + / - I + / - and incubated at 30 oC for 4 d .

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Serial dilutions of the strains indicated were spotted on SD C+/- I+/- and incubated at 30 oC for 4 d.


Membrane lipid and TAG content , and ergosterolester content ( EE , inset ) per OD600 unit of the yeast strains indicated after culture to mid - log phase in SD with or without 1 mM choline ( C ) ; * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed t - test of the indicated bar compared to the C + condition . Data information : All data were obtained by mass spectrometry and are presented as mean ± SD ( n = 3 biological replicates ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed t - test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise .

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Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise.


Generation of suppressors of choline auxotrophy of cho2opi3 , cho2opi3lro1 , and cho2opi3lro1 pLRO1 on choline - free medium as indicated at 30 oC for 7 d .

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Generation of suppressors of choline auxotrophy of cho2opi3, cho2opi3lro1, and cho2opi3lro1 pLRO1 on choline-free medium as indicated at 30 oC for 7 d.


Growth ( 30 oC for 6 d ) and phospholipid composition of co acc1N / H lro1 pLRO1 cultured in SD C - ura - , containing glucose / galactose mixtures ( 2 % , w / v ) as carbon source with the percentage of galactose indicated . Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated .

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

Growth (30 oC for 6 d) and phospholipid composition of co acc1N/H lro1 pLRO1 cultured in SD C- ura-, containing glucose/galactose mixtures (2%, w/v) as carbon source with the percentage of galactose indicated. Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated.