( D ) LCL1 . 11 were transfected with NeoR or NucNeoR expression plasmids antigen presentation was assessed 48 h later with NeoR ‐ specific T clone 20 ‐ 4 / A4 . Antigen ‐ transfected LCL1 . 11 were recognized irrespectively of NeoR localization , while untransfected or 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 ‐ NucNeoR were incubated for 48 h with IFN ‐ γ in addition with 7 . 5 mM 3 ‐ MA or 200 µM leupeptin or 100 µM chloroquine for last 20 h . Subsequently , were fixed with 0 . 5 % paraformaldehyde tested with 20 ‐ 4 / A4 in a GM ‐ CSF release . Both NeoR NucNeoR were presented by a pathway that involves autophagy lysosomal processing .

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

(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 transfected with NeoR or NucNeoR expression plasmids were tested likewise . Presentation of endogenous NeoR NucNeoR was blocked by inhibitors , indicating that NeoR NucNeoR presentation is dependent on autophagy 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 inhibitors , LCL1 . 11 were incubated for 20 h with 200 ng / mL recombinant NeoR in presence or absence of inhibitors . Afterwards , residual inhibitors were removed by washing were co ‐ cultured with NeoR ‐ specific CD4 + T clone 20 ‐ 4 / A4 . 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 were transfected with siRNA specific for Atg12 or GFP , Atg12 mRNA were determined 24 h later by qPCR . 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 NucNeoR were expressed in these antigen presentation was assessed 36 h later . In transfected with Atg12 siRNA , but not in mock ‐ transfected or transfected with GFP siRNA , antigen presentation of both NeoR 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 were transfected with NES ‐ GFP ‐ NeoR ‐ GFP ‐ NLS expression construct subcellular localization of fusion was monitored by UV . As compared to untreated ( a ) , treated with 8 nM LMB for 20 h ( b ) showed a strong accumulation of in nucleus . ( a ' ) ( b ' ) are corresponding daylight of transfected .

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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 ‐ NucNeoR ‐ transfected LCL1 . 11 were either left untreated or incubated for 20 h with 8 nM LMB . Subsequently , were fixed with 0 . 5 % paraformaldehyde probed with NeoR ‐ specific T . LMB treatment had no significant effect on presentation of NeoR or NucNeoR on MHC class II , indicating that CRM1 ‐ dependent export is not involved in presentation of this 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 LL37 - induced mice . Bottom right panels , magnified of boxed areas . DAPI ( blue ) indicates localization . Scale bar : 50 μm . Right panel , of relative for pS6 ( Ser240 / 244 ) in control LL37 group ( n = 5 for each group ) . Data represent mean ± . * * P < 0 . 01 . two - unpaired Student ' s t - was used .

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

(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 of lesional skin sections from WT Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group ) . Scale bar : 50 μm . ( E ) Dermal infiltrating 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 ) mRNA expression of Tnf - α , Il6 , Mmp9 Vegf in skin lesions ( n = 6 for each group ) .

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

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


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

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

(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 ) back skins of WT ( Tsc2 + / + ) 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 first LL37 injection . mouse experiments were repeated for three times , 5 mice were included in each group for each time . results of a representative mouse experiment were displayed . Scale bar : 2 mm .

O O O O B-TISSUE O O O B-GENEPROD O O O O B-GENEPROD 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 O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

(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 ) mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10 ) 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 ( blue ) indicates localization . Scale bar : 50 μm . results are representative of at least 3 independent experiments . Data represent mean ± .

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 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

(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 ) 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 were analyzed by microscope . Scale bar : 50 μm .

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 O O O B-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 ) human keratinocytes were incubated with sLL37 - flag or LL37 - flag for 1 h . lysates were immunoprecipitated with anti - flag or anti - TLR2 antibodies , showing an interaction between LL37 TLR2 . Medium , medium of human keratinocytes treated with sLL37 - flag or LL37 - flag . results are representative of at least 3 independent experiments .

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

(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 of dotted areas . Epi , epidermis . Der , dermis . Scale bar : 50 μm . ( F ) of relative 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 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 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 ) mRNA expression of NF - κB family of transcription factors ( Nfκb1 , Nfκb1 , Rela Relb ) in mice skin lesions ( n = 6 for each group ) .

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

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

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

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


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

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

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

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

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


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

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

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(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 ) were incubated in presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for indicated times . were fixed , permeabilized stained with antibodies against TFE3 ( green ) 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 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-GENEPROD 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 were incubated in presence of DMSO ( Control ) or BFA ( 2 . 5 µg / ml ) for 16 h . were fixed , permeabilized stained with antibodies against TFE3 ( green ) CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( G ) of of MEF with TFE3 upon DMSO ( Control ) or BFA 16 h treatment ( mean ± SD of two independent experiments , student ' s t - versus DMSO treated , * * * p < 0 . 001 ; n > 400 per condition ) .

O O O O O O B-CELL_LINE 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 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 B-CELL_LINE 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 B-SMALL_MOLECULE 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 subcellular distribution of TFE3 TFEB in MEF incubated with DMSO ( Ctrl . ) or Tunicamycin ( Tun . ) for 16 h using TFE3 , TFEB , Lamp1 ( as a lysosomal marker ) , H3b ( as a marker ) . Data are representative of three independent experiments .

O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-CELL_LINE 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 O O O B-GENEPROD 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 lysates from wild - type , PERK - KO GCN2 - KO MEF incubated in 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 B-GENEPROD O O B-GENEPROD O O B-CELL_LINE 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 ) , PERK - KO ( D ) MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) as indicated in ( A ) . were fixed , permeabilized stained with antibodies against TFE3 ( green ) CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( E - F ) of of MEF with 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 versus DMSO treated , ns = not significant , * * * p < 0 . 001 ; n > 400 per condition ) .

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 B-CELL_LINE 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 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 B-CELL_LINE 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 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-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 ) of phospho - 4EBP1 in wild - type GCN2 - KO MEF upon starvation for 2 h ( mean ± SD of fold increase of phospho - 4EBP1 to total 4EBP1 from three independent experiments , one - way ANOVA versus starvation treated wild - type , * * * p < 0 . 001 ) .

O O O O O O B-GENEPROD 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 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

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

O O O O O B-CELL_LINE 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 B-SMALL_MOLECULE 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 infected with either adenovirus expressing TFE3 - WT - Myc or TFE3 - S321A - Myc for 16h . were fixed , permeabilized stained with antibodies against Myc . Scale bar , 10 µm . of of ARPE - 19 with TFE3 - Myc ( means ± SD of 3 independent experiments , students t - , * * * * p < 0 . 0001 ; n > 400 per condition ) .

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

(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 lysates from ARPE - 19 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 fractions of ARPE - 19 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 of a GST pull - down from fractions of MEF 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 B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE 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 lysates from ARPE - 19 incubated in 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 ) of of ARPE - 19 with TFE3 upon Tunicamycin ( Tun . ) or Tunicamycin + FK506 treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA versus Tunicamycin treated , * * * p < 0 . 001 ; n > 400 per condition ) .

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

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

(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 lysates from ARPE - 19 depleted of Calcineurin catalytic subunits PPP3CA PPP3CB . Data are representative of three independent experiments .

O O O B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD I-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 of PPP3CA PPP3CB mRNA transcript in ARPE - 19 depleted of Calcineurin catalytic subunits PPP3CA PPP3CB ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analsysis versus siNon - Target ( siNT ) treated , * * * 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 B-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD I-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 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 ) of of ATF4 depleted ARPE - 19 with TFE3 upon Tunicamycin treatment for 16 h ( mean ± SD of two independent experiments , one - way ANOVA versus siNon - target treated within indicated treatment * * * p < 0 . 001 ; n > 400 per condition ) .

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

(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 of TFEB TFE3 mRNA transcript in ARPE - 19 depleted of TFEB TFE3 ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , student ' s t - versus siNon - Target ( siNT ) treated , * * * 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 B-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-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 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 of MCOLN1 , ATP6V1C1 , HEXA UVRAG mRNA transcript in ARPE - 19 depleted of TFEB TFE3 upon incubation with DMSO ( Control ) or Tunicamycin ( 5 µg / ml ) for 16 h ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA versus same treatment condition in siNon - Target ( siNT ) treated or versus DMSO condition in siNon - Target ( siNT ) treated , * 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 B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-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 B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-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) 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 lysates from ARPE - 19 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 ) of ATF4 , CHOP GADD34 in ARPE - 19 treated as indicated in ( A ) ( mean ± SD of fold change of indicated to actin from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(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 of ATF4 , CHOP , GADD34 MCOLN1 mRNA transcript in ARPE - 19 treated as indicated in ( A ) ( mean ± SD of fold change of indicated genes normalized to actin mRNA from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected , * * 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 B-GENEPROD O B-GENEPROD O B-GENEPROD I-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 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 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 of ATF4 ( A ) , ASNS ( B ) , CAT1 ( C ) xCT ( D ) mRNA transcript in ATF4 - depleted ARPE - 19 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 fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected siRNA Non - Target ( siNT ) treated , * 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 B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 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 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 of ATF4 promoter 2000bp upstream of region of interest ( ATF4 - Control ) from MEF 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 . was immunoprecipitated with antibodies for anti - TFE3 . Bar graphs show amount of immunoprecipitated detected by real - time PCR . Values were normalized to input plotted as relative enrichment compared to untreated conditions ( means ± SD of three independent experiments , students t - , * p < 0 . 05 , * * p < 0 . 01 ) .

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

(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 of SUMF1 , DERL1 , ATF6 , APEX1 GPX1 mRNA transcript in ARPE - 19 infected with either adenovirus expressing Null or TFEB - S211A ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , students t - , * * 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 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

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

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

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 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 B-GENEPROD 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 B-GENEPROD O B-GENEPROD 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

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

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 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 B-CELL_LINE 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 ) condensation , an indication of undergoing apoptosis , was assessed in Null - or TFEB / 3 knockout - MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for indicated times . Confocal microscopy were obtained from fixed 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 B-GENEPROD O O O O B-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY 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 lysates from Null - or TFEB / 3 knockout - MEF treated as described in ( A ) . Data are representative of three independent experiments . ( F - H ) of CHOP ( F ) , PUMA ( G ) NOXA ( H ) in Null - or TFEB / 3 knockout - MEF treated as indicated in ( E ) ( mean ± SD of indicated to actin from three independent experiments , student ' s t - versus corresponding time point in Null - MEF , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ) .

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 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 B-GENEPROD 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 B-GENEPROD 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

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

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 B-GENEPROD 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-GENEPROD 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

(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 lysate , purified fraction secreted culture filtrate ( CFP ) for TB healthy donors ( HD ) . ELISA graphs ( left ; TB : black lines ; n = 25 ; HD : red dotted lines ; n = 2 ) under curve ( AUC ) values for 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 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

(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 IgA serum ELISA response against MTB whole lysate , purified fraction secreted culture filtrate ( CFP ) of TB ( n = 25 ) healthy donors ( HD ; n = 9 ) . Median is shown .

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

(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 frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) for one representative TB ( TB24 ) HD ( left ) . Frequency of circulating CD19 + CD27 + + CD38 + plasmablasts of CD19 + B in peripheral blood of TB ( n = 24 ) HD ( n = 8 ) . Dashed indicates threshold for detectable plasmablast populations ( right ) . Median are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-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 B-CELL_TYPE 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 activation markers ( CD86 , CD84 CD24 ) in plasmablasts memory B as measured by flow cytometry .

O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O 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 TB7 ( top ) IgG serum response against MTB lysate of TB7 , TB19 TB39 ( bottom ) one HD at indicated time points before after treatment onset .

O O O B-CELL_TYPE 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

(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 anti - MTB lysate IgG serum ELISA response ( y - axis ) versus frequency of circulating plasmablasts ( x - axis ) for TB .

O O O O O O O O O O B-ORGANISM 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

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

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 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 IGHV , IGKV IGLV segments of IgA IgG plasmablast antibody genes sequenced from TB7 , TB24 TB33 . absolute number of sequences analyzed is indicated below graph . Geometric means with are indicated in grey . SHM means of historic data from sorted CD27 + IgA + or CD27 + IgG + 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 B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-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 B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR 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 memory B from TB7 , TB24 TB33 . PB , plasmablasts ( CD19 + CD27 + + CD38 + ) ; M , memory B ( CD19 + CD27 + ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 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 frequency of HBHA - reactive memory B ( CD19 + CD27 + HBHA + ) in peripheral blood of one representative TB ( TB29 ) , health - care worker ( HCW ) 2 HD , respectively .

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

(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 out of CD27 + memory B in TB ( n = 23 ) HCWs ( n = 7 ) . Mean are indicated . P - value was determined using Wilcoxon - Mann - Whitney ; * * * * p < 0 . 0001 .

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

(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 frequency of resting memory B ( CD19 + CD27 + CD10 - ) out of B in peripheral blood of TB compared to HD .

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

(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 HCW ( black lines ) compared to two representative HD ( red lines ) . Dashed indicates threshold OD405nm for positive reactivity ( left ) . Asterisks indicate serum responses of donors selected for single sorting of HBHA - reactive memory B . Data are representative of two independent experiments .

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

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

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

(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 of clonally expanded B clusters among HBHA - reactive memory B from two TB ( TB35 , TB29 ) HCWs ( HCW1 , HCW 2 ) . in clusters are indicated in grey , single are indicated in . No B 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 IgM isotype distribution of single - - sorted HBHA - reactive memory . Mean are indicated .

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

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

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

(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 MTB fraction for MTB - reactive ( n = 26 ) non - reactive ( n = 14 ) antibodies .

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

(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 as determined by flow cytometry for three representative MTB lysate - reactive antibodies 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 TB33PB123 to LAM purified from MTB as determined by Western Blot . negative isotype control ( mGO53 ) a commercially available anti - LAM antibody ( positive control ) are shown for comparison .

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

(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 LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole ( left ) flow cytometry ( right ) .

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

(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 indicated concentrations . mean ± SD of absorbance was calculated .

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

(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 ) .

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

(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 of TB35 HCW1 . Dashed red indicates threshold OD405nm for positive reactivity . Green indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) .

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

(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 .

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

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


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

O O O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM 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) 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 ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( A ) Representative results for MTB - reactive plasmablast antibodies . Mean are indicated .

O O O O O O O O O O O O O O O O B-CELL_LINE 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 B-ORGANISM O O B-CELL_TYPE 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. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated.


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

O O O O O O O O O O O O O O O O B-CELL_LINE 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 B-ORGANISM O O B-SUBCELLULAR O O O O O B-SUBCELLULAR 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 ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( C ) Representative results for HBHA - reactive memory B antibodies from TB HCW .

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

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

O O O O O O O O O O O O O O O O B-CELL_LINE 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 B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 bronchial epithelial ( hAECB ) lack FcγR ( CD16 , CD64 CD32 ) FcαR ( CD89 ) surface expression as analyzed by flow cytometry . Human THP1 serve as positive control .

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

(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 ) neonatal FcR ( FcRn ) is expressed in both A549 human THP1 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 ( CFUs ) in human THP1 macrophages 1 h after infection with MTB pre - incubated with LAM - reactive IgA1 ( left ) or IgG1 ( right ) antibody TB24PB037 or respective isotype control antibody as indicated . Mean are indicated .

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

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

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

(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 IgG antibodies on epithelial invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney ; * * * * p < 0 . 0001 .

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

(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 indicated strains were spotted on SD plates containing 0 ( C - ) or 1 mM choline ( C + ) 0 ( I - ) or 75 μM inositol ( I + ) incubated at 30oC for 3 d . A representative experiment is shown ( from n = 5 ) .

O O O O 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 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

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 ) , coS # 2 ( I - K ) cultured in C - were analyzed by electron microscopy . In addition , coS # 3 ( H ) coS # 2 ( L ) are shown after culture in C + for 3 generations . arrow heads ( F , I , J ) point to protrusions in envelope . CW , wall ; ER , endoplasmic reticulum ; PM , plasma ; 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 ) .

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

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 strains indicated were spotted on SD C + / - I + / - 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.


lipid TAG content , ergosterolester content ( EE , inset ) per OD600 unit of 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 - t - of indicated bar compared to C + condition . Data information : data were obtained by mass spectrometry are presented as mean ± SD ( n = 3 biological replicates ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - t - of indicated bar compared to cho2opi3 parent unless indicated otherwise .

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

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 , cho2opi3lro1 pLRO1 on choline - free medium as indicated at 30 oC for 7 d .

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

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

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


ACC1 transcript after switching strains indicated from SD C + I + to medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , culture for 24 h at 30 oC . Data were normalized to ACT1 expressed as means of 3 biological replicates relative to corresponding cultured in C + I + , with values indicated .

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

ACC1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.


( F - G ) EM of cho2opi3 that after preculture in SD C + , were transferred to OD600 0 . 05 subsequently cultured to mid - log phase in SD C + ( F ) or SD C - ( G ) both containing 0 . 05 μg / mL SorA . CW , wall ; ER , endoplasmic reticulum ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 500 nm .

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

(F-G) EM analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to mid-log phase in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.


POX1 transcript after switching strains indicated from SD C + I + to medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , culture for 24 h at 30 oC . Data were normalized to ACT1 expressed as means of 3 biological replicates relative to corresponding cultured in C + I + , with values indicated .

POX1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.


Growth of co S ( 2n - 1 ) in absence of choline is lost when GPT2 or DGA1 is overexpressed , restored by SorA . Plasmids pHEYg - 1 - DGA1 , pHEYg - 1 - GPT2 empty vector control ( pEV ) were transformed into strains indicated . After preculture in SD C + , ten - fold serial dilutions were spotted on SD C + / - with or without 0 . 1 μg / mL SorA incubated at 30 oC for 3 d .

B-EXP_ASSAY O O 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-SMALL_MOLECULE 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 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

Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.


encoded GP - of indicated C - Laurdan - stained yeast strains cultured with or without choline , accompanied by corresponding weighted GP - histogram fitted to a single Gaussian function ( blue ) , transmission image . arrows point to plasma membranes exhibiting higher lipid packing than internal , organellar membranes ; blue horizontal arrows indicate highly packed lipid material in co S ( 2n - 1 ) . To deplete cho2opi3 parent of PC , were transferred to SD C - at OD600 0 . 1 , cultured into mid - log phase . scale bars correspond to 10 µm ; red scale bars in transmission correspond to 5 µm .

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

Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.


( A ) Mettl14 cKO leads to perinatal lethality . Newborn cKO pups are smaller with tight shiny skin .

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(A) Mettl14 cKO leads to perinatal lethality. Newborn cKO pups are smaller with tight and shiny skin.


( B ) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO .

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(B) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals.


( C ) H / E of newborn skin sections from WT Mettl14 cKO mice .

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(C) H/E staining of newborn skin sections from WT and Mettl14 cKO mice.


( D ) Expression of p63 in skin epidermis was examined by immunofluorescence in WT cKO mice . ( E ) Number of p63 - positive in WT cKO skin was quantified shown as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O B-GENEPROD O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice. (E) Number of p63-positive cells in WT and cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test).


( F ) Skin stratification in WT cKO skin was determined by immunofluorescence with different antibodies as indicated . Krt14 : Keratin 14 ; Krt10 : Keratin 10 ; β4 : β4 - integrin ; DAPI for nucleus . dashed denotes basement that separates dermis epidermis ( Epi ) .

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

(F) Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14: Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis and epidermis (Epi).


( G ) Wound healing as monitored by wound 8 days post injury . n = 3 ; P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D . ( standard deviation ) .

O O O B-EXP_ASSAY I-EXP_ASSAY 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

(G) Wound healing as monitored by wound size 8 days post injury. n=3; P<0.01 (Student's t-test). Error bar represents S.D. (standard deviation).


H ) Histological of skin sections at wound edges . Halves of wound sections are shown . Note significant reduction of HPE ( hyperproliferative epidermis ) in cKO skin . Es : eschar . Dotted lines denote epidermal boundaries . ( I ) of Ki67 - positive present in wound HPE . plot indicates mean ( open circles within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 19 , P < 0 . 01 ( Student ' s t - ) .

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

H) Histological staining of skin sections at the wound edges. Halves of wound sections are shown. Note significant reduction of HPE (hyperproliferative epidermis) in cKO skin. Es: eschar. Dotted lines denote epidermal boundaries. (I) Quantification of Ki67-positive cells present in wound HPE. The plot indicates the mean (open circles within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=19, P<0.01 (Student's t-test).


( A ) EdU of WT or Mettl14 cKO skin after - chase labeling . Skin samples were counterstained with antibody against β4 - integrin . Note reduced EdU label - retaining in cKO skin epidermis . Arrows indicate Edu - positive . ( B ) Label - retaining in WT or Mettl14 cKO skin were quantified shown as box plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 19 , P < 0 . 01 ( Student ' s t - ) .

O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-TISSUE O O O O O B-TISSUE O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells. (B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test).


( C ) Number of holoclones derived from WT Mettl14 cKO skin was quantified shown as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 24 , P < 0 . 01 ( Student ' s t - ) .

O O O B-EXP_ASSAY O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=24, P<0.01 (Student's t-test).


( D ) of keratinocytes isolated from WT or Mettl14 cKO skin .

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(D) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin.


( E ) Expression of Mettl14 p63 in WT KO was determined by immunoblots with respective antibodies . Immunoblot for GAPDH was used as loading control .

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

(E) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control.


( F ) Proliferation of WT Mettl14 null in vitro was quantified shown as dot plots . n = 3 , P < 0 . 01 ( Student ' s t - ) for Day 6 , 9 , 12 , P < 0 . 05 ( Student ' s t - ) for Day 3 . Error bar represents S . D .

O O O B-EXP_ASSAY 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

(F) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n=3, P<0.01 (Student's t-test) for Day 6, 9, and 12, and P<0.05 (Student's t-test) for Day 3. Error bar represents S.D.


( G - H ) CFE ( colony formation efficiency ) of WT Mettl14 null was determined in vitro . Results were quantified shown as bar graph ( F ) . n = 19 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

O O O O O B-EXP_ASSAY 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 O O O O O O O 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-H) CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (F). n=19, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) microscopy demonstrates different survival capability of WT Mettl14 inducible KO with or without Tamoxifen ( TAM ) treatment . ( J ) of WT Mettl14 inducible KO in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO with TAM treatment compared with WT or KO without TAM stimulation at both Day 7 14 . Error bar represents S . D .

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

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment. (J) Ratio of WT and Mettl14 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D.


B ) Mutations in predicted m6A modification sites can significantly reduce Pvt1 methylation . n = 3 . Error bar represents S . D .

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

B) Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation.n=3. Error bar represents S.D.


C ) Deletion of endogenous Pvt1 by an inducible Cas9 ( iCas9 ) system can significantly reduce Pvt1 level ( from RT - PCR , left panel ) MYC level ( from immunoblot , right panel ) expression . n = 3 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

C) Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT-PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n=3, P<0.01 (Student's t-test). Error bar represents S.D.


D ) CFE of WT , Pvt1 inducible KO , Pvt1 inducible KO rescued with WT or mutant Pvt1 was quantified presented as bar graph . n = 3 , * * : P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

D) CFE of WT, Pvt1 inducible KO, and Pvt1 inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n=3, **: P<0.01 (Student's t-test). Error bar represents S.D.


E ) Skin organoids derived from WT or Pvt1 inducible KO were grafted to nude mice . regenerated skin was analyzed by H / E .

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

E) Skin organoids derived from WT or Pvt1 inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining.