Total intracellular glutathione ( GSH + GSSG ) as shown by glutathione reductase ( GR ) re - cycling in H1299 R175H ( tet - off ) ( + / - doxycycline ) H1299 - / - 24h after seeding ( n = 3 ) . Indicated values are fold change compared to mutant TP53 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by GR re - cycling in untreated HCT116 ( R248W , WT - / - ) 48h after seeding ( n = 3 , 5 or 2 respectively ) . Indicated values are fold change to mutant TP53 . Part of this data is shown in Fig . EV4 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Thiol tracker by flow cytometry 48h after seeding of indicated untreated ( n = 2 ) Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Correlation between 14C accumulation ( cpm / mg x ml - 1 ) after treatment with 25 µM 14C - APR - 246 IC50 ( µM ) values for APR - 246 in 11 lines ( n ≥ 3 ) ( r = - 0 . 66 , p = 0 . 03 , Pearson correlation ) . Each dot represents one , 14C accumulation data are shown in Fig 3B values n for lines are listed in Table S1 S3 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


ABCC1 mRNA Expression , RSEM ( Batch normalized from Illumina HiSeq _ RNASeqV2 ) in lung cancer ( luad & lusc studies , * p < 0 . 0001 ) colon adenocarcinoma ( coad study , * p = 0 . 005 ) grouped into having no alterations or putative driver missense mutations in TP53 . Statistical by Mann Whitney , n is indicated in figure . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


14C accumulation ( cpm / mg x ml - 1 ) in H1299 R175H ( + / - doxycycline ) H1299 - / - after 24h treatment with 14C - APR - 246 ( n = 2 ) . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

B-MEASURED_VAR O O O 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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Growth suppression in H1299 R175H ( + / - doxycycline ) H1299 - / - treated for 72h with APR - 246 as shown by WST - 1 ( n = 3 ) . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG per 106 ) , as determined by GR re - cycling after 24h MK - 571 treatment in six different lines ( n ≥ 3 , except HCT116 WT n = 2 ) . * p = 0 . 014 , Paired t . Each dot represents one , see Fig . EV6A for lines n .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n.


Intracellular oxidized glutathione ( GSSG per 106 ) as shown by LC - MS in OVCAR - 3 ( R248Q TP53 ) at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular reduced glutathione ( GSH per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Western blot of MRP1 ( Signaling ) , xCT GAPDH of OVCAR - 3 treated with APR - 246 + / - MK - 571 for 24h .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h.


Western blot of MRP1 ( Signaling ) , xCT GAPDH of HCT116 WT R248W treated with APR - 246 + / - MK - 571 for 24h .

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.


Intracellular cystine ( CySS per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular cysteine ( Cys per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT R248W mutant TP53 + - transfected with MRP1 or xCT siRNA following 24h of 14C - APR - 246 treatment + / - MK - 571 . Indicated values are mean values from four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) two control siRNAs ( n ≥ 3 ) . Mean 14C - accumulation after treatment n of siRNAs is shown in Table S4 . Data for control MRP1 siRNA after 14C - APR - 246 treatment is also shown in Fig 3D .

B-MEASURED_VAR O O O 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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.


Growth suppression in HCT116 WT R248W transfected with MRP1 or xCT siRNA after 48h of APR - 246 treatment as shown by WST - 1 . Indicated values are mean values of four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) two control siRNAs ( n ≥ 3 ) . Data for control MRP1 siRNA after APR - 246 single treatment are also included in Fig 1I . Values n for siRNA shown in Fig . S7C .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.


Total intracellular glutathione ( GSH + GSSG per 106 ) in HCT116 WT as shown by GR re - cycling 48h post transfection of siRNA . Indicated values are means from four MRP1 siRNAs ( n = 3 ) , two xCT siRNA ( n = 2 - 3 ) two control siRNA ( n = 2 - 3 ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).


Total intracellular glutathione ( GSH + GSSG per 106 ) in HCT116 WT R248W measured by GR re - cycling after 24h of MK - 571 or sulfasalazine ( SSZ ) treatment ( n ≥ 3 , except SSZ where n = 2 , n indicated by dots ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT R248W at 24h treatment with 14C - APR - 246 , combined with MK - 571 or sulfasalazine ( SSZ ) , ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) . Data for 14C - APR - 246 + / - MK - 571 is also shown in Fig 3C S3D .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.


of HCT116 WT R248W assessed by DCTM at 24h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) , or untreated ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots )

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)


OVCAR - 3 ( R248Q TP53 ) confluence by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalzine ( SSZ ) ( n = 3 ) . Part of data are shown in Fig 1G . Growth suppression in OVCAR - 3 as determined by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) depicted as heatmaps ( n = 3 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).


a , 2 - bp deletion identified in exon 9b of 1 . b , T - to - A nonsense mutation in exon 4 of 2 ( stop codon is underlined ) . c , Upper panel , intronic mutation in 3 ; lower panel , sequencing of RT - PCR product in 3 , showing exon - 6 skipping . d , Upper panel , G - to - A point mutation at splice - donor site in intron 5 of 6 ; lower panel , sequencing of RT - PCR product in 6 , showing a 6 - bp insertion at junction between exons 5 6 . inserted ( underlined ) is derived from first six nucleotides of intron 5 ( underlined ) , which creates a stop codon ( dotted ) . Most probably , nucleotides 7 8 , GT ( double underlined ) , in intron 5 are alternatively recognized as a splice - donor site . e , Upper panel , 10 - bp deletion in 9 . This mutation deletes four nucleotides from intron 5 six from exon 6 . Lower panel , of RT - PCR product in 9 reveals deletion of 10 nucleotides at 5 ′ end of exon 6 . ' AG ' ( double underlined ) in exon 6 immediately after genomic deletion appears to be recognized as an alternative splice - acceptor site .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, The 2-bp deletion identified in exon 9b of patient 1. b, The T-to-A nonsense mutation in exon 4 of patient 2 (stop codon is underlined). c, Upper panel, intronic mutation in patient 3; lower panel, sequencing of the RT-PCR product in patient 3, showing exon-6 skipping. d, Upper panel, The G-to-A point mutation at the splice-donor site in intron 5 of patient 6; lower panel, sequencing of the RT-PCR product in patient 6, showing a 6-bp insertion at the junction between exons 5 and 6. The inserted sequence (underlined) is derived from the first six nucleotides of intron 5 (underlined), which creates a stop codon (dotted). Most probably, nucleotides 7 and 8, GT (double underlined), in intron 5 are alternatively recognized as a splice-donor site. e, Upper panel, the 10-bp deletion in patient 9. This mutation deletes four nucleotides from intron 5 and six from exon 6. Lower panel, sequence of the RT-PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5′ end of exon 6. The 'AG' (double underlined) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice-acceptor site.


Figure 2 Representation of mutant LAMP - 2b . ' Normal ' represents LAMP - 2b - acid . Because 1 , who developed full DD symptoms , has a mutation in exon 9b , LAMP - 2b isoform is most probably responsible for DD . Residue 265 , one of putative disulphide - bond - creating cysteines , two glycosylation sites are lost by exon - 6 skipping in 3 , which probably results in a significant change in secondary structure . Black boxes represent loops created between two cystein residues flanking each of four loop regions ; Y depicts a glycosylation site ; hatched boxes show altered caused by frameshift mutations ; dashes represent missing due to exon - 6 skipping in 3 . of other mutant lack transmembrane domain ( grey bar ) because of nonsense or frameshift mutations . Thus , those molecules cannot function as lysosomal ' ' .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.


a , Skeletal muscle . Muscle extracts from 1 - 6 10 a control were blotted labelled with antibodies against LAMP - 2 ( top panel ) β - actin ( bottom panel ) . LAMP - 2 is deficient in examined except sample from 1 that showed a trace amount of , which is probably LAMP - 2a isoform . In support of this idea , LAMP - 2b is principal LAMP - 2 species in skeletal muscle6 . Numbers denote as in Table 1 ; C , control . b , Heart . was extracted from cardiac muscle of 4 a control , blotted onto a nitrocellulose , labelled with antibodies against LAMP - 2 ( too panel ) β - actin ( bottom panel ) . LAMP - 2 is deficient also in cardiac muscle . C , control ; 4 , 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.


Serial transverse sections of biopsiedmuscle from six DD , samples from a XMEA a normal control , were immunostained with antibodies against LAMP - 2 limp - I . LAMP - 2 is absent in DD ( 6 shown here ) , whereas it is present in XMEA muscle . antibody against limp - I highlighted vacuoles in both DD XMEA . LAMP - 1 antibody faintly stained both DD XMEA , suggesting that expression level of LAMP - 1 might be lower in skeletal muscle than LAMP - 2 or limp - I ( data not shown ) . muscle from without a lamp - 2 mutation also showed LAMP - 2 deficiency on immunohistochemistry similar to genetically proven DD with LAMP - 2 deficiency ( data not shown ) . immunohistochemical stains of LAMP - 2 , limp - I , LAMP - 1 in normal muscle shows numerous tiny particles accentuates type 1 fibres . H & amp ; amp ; E , haematoxylin eosin , original magnification × 150 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;E, haematoxylin and eosin, original magnification ×150.


( B ) Representative immunofluorescence of HeLa in various mitotic stages . were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.


( C ) Representative immunofluorescence of HeLa that were fixed co - stained for Cyclin B2 ( green ) , mAb414 ( red ) , ( blue ) . Scale bar , 10 µm .

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(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.


Representative immunofluorescence of HeLa treated with an indicated siRNA were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


Representative immunofluorescence of HeLa treated with an indicated various drugs ( E ) . were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


( F ) HeLa were synchronized by double thymidine , then released collected at selected time points . lysate samples were resolved by SDS - PAGE analyzed by Western blotting using anti - Cyclin B1 antibody , anti - Cyclin B2 antibody , or anti - β - Actin antibody .

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(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.


( B ) Immunoprecipitation performed with two different rabbit Cyclin B2 antibodies . Nocodazole - arrested mitotic were collected lysed . Immunoprecipitation was accomplished using control IgG , anti - Cyclin B2 - 1 ( Abcam , ab185622 ) , anti - Cyclin B2 - 2 ( Proteintech , 21644 - 1 - AP ) . Immunoprecipitation samples were resolved by Western blotting using a mouse anti - Cyclin B2 antibody ( Santa Cruz , sc - 28303 ) a mouse anti - Mad2 antibody ( Santa Cruz , sc - 47747 )

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(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747)


( C D ) Flag immunoprecipitation . 293T were co - transfected with Flag - C - Mad2 GFP - Cyclin B1 / B2 ( C ) , with Flag - Mad1 - 1 - 215 GFP - Cyclin B1 / B2 ( D ) . After 36 h , were collected lysed , immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitates were resolved by SDS - PAGE analyzed by Western blotting using anti - Flag antibody anti - GFP

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(C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody


( E ) GST pulldown . Purified GST , GST - Cyclin B1 - 165 - 433 , GST - Cyclin B2 - 130 - 398 - bound agarose beads were used as affinity matrices to absorb purified Trx - 6 × His - tagged C - Mad2 recombinant . Absorbed were analyzed by SDS - PAGE Coomassie Brilliant Blue

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(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining


( F - I ) Representative immunofluorescence of HeLa stably expressing GFP - Cyclin B2 ( F ) or GFP - Cyclin B1 ( H ) . were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h . Then were fixed co - stained for Mad2 ( red ) ( blue ) . Scale bar , 10 µm . Scatter graphs illustrating kinetochore of GFP - Cyclin B2 ( G ) or GFP - Cyclin B1 ( I ) in treated as in F H , respectively . Bars represent mean kinetochore ( ± SD ) normalized to values of siControl group . Each dot represents one ( ≥ 30 from three independent experiments )

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(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)


( B ) Flag immunoprecipitation . Flag - tagged Cyclin B2 - WT , Cyclin B2 - 4A , or Cyclin B1 were expressed in 293T purified with anti - Flag M2 beads . Subsequently , beads were incubated with mitotic HeLa lysates for 2 h , , following extensive washes , immunoprecipitatates were resolved by SDS - PAGE analyzed by Western blotting using anti - Flag antibody anti - Mad2 antibody .

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(B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody.


( C ) Flag immunoprecipitation . 293T were co - transfected with Flag - Cyclin B2 - WT or Flag - Cyclin B2 - 4A together with GFP ( negative control ) GFP - CDK1 . After 36 h , were collected lysed , immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitation samples were resolved by SDS - PAGE analyzed by Western blotting using anti - GFP antibody anti - Flag antibody .

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(C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( D ) LCL1 . 11 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 .

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


( B ) LCL1 . 11 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 .

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


( D ) HE 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 ) .

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


( I ) HE 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 .

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


( A ) 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 .

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


( A ) 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 ± .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

(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 .

(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 .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

(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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

(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 ) .

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(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 ) .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 + ) .

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(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 .

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


( E ) Dots indicate frequency of HBHA - reactive memory B 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 .

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


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

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


( G ) Anti - HBHA serum IgG ELISA reactivity for TB 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 .

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


( H ) Absolute number of somatic hypermutations ( SHM ) in 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 ) .

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


( I ) Number 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 .

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