Figure 5 . ( A ) The C106 mutation ( C106S ) of HMGB1 impairs its nuclear localization . Hmgb1 − / − MEFs were transfected with wild - type and cysteine mutant HMGB1 - GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved ( HBSS ) for 3 h . The mean nuclear ( Nuc ) and cytosolic ( Cyt ) HMGB1 intensity per cell was analyzed by imaging cytometric analysis . * , P < 0 . 05 versus HMGB1 + / + group ; n = 3 . Representative images of HMGB1 location are shown on the left ( green , HMGB1 ; blue , nucleus ) . The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C - terminal domain , with the cysteine mutation locations identified . ( B and C ) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and then were starved ( HBSS ) for the indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h and then starved as indicated . LC3 punctae formation was assayed by imaging cytometric analysis ( B ) ( B and C ) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and then were starved ( HBSS ) for the indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h and then starved as indicated . Apoptosis was assayed by FACS ( C ) ( C ) as described in Materials and methods . * , P < 0 . 05 and * * , P < 0 . 005 ; n = 3 . UT , untreated . Non , nontransfection . ( D ) C23 / C45 is required for the binding of HMGB1 to Beclin1 . Hmgb1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and stimulated with starvation ( HBSS ) for 3 h . These cells were then assayed for protein expression levels as indicated by IP or Western blotting . Blots are representative of three independent experiments with similar results . ( E ) Reducing reagents disrupt the interaction between wild - type / C106 HMGB1 and Beclin1 . As a control , before IP , samples were incubated with 50 mM DTT ( + DTT ) and assayed for protein expression levels as indicated by IP or Western blotting . Blots are representative of two independent experiments with similar results . ( F ) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol . Cells were stimulated with HBSS , rapamycin ( Rap ) , rotenone ( Rot ) , or thenoyltrifluoroacetone ( TTFA ) for 3 h or 12 h , and LC3 punctae formation was assayed as indicated . * * , P < 0 . 005 versus Beclin1 shRNA group ; n = 3 . Ctrl , control . Data are means ± SEM .

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Figure 5.(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified.(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B)(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection.(D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of three independent experiments with similar results.(E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results.(F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.


Figure 6 . ( A and B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . Cells as indicated were stimulated with 1 µg / ml HMGB1 protein ( rHMGB1 ) for 24 h , and LC3 expression was detected by Western blotting ( A ) . ( A and B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . Cells as indicated were stimulated with 1 µg / ml HMGB1 protein ( rHMGB1 ) for 24 h , LC3 punctae formation ( arrows ) was assayed by immunofluorescence ( B ; n = 3 ; * , P < 0 . 05 ) . ( C and D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with and without pUNO1 - HMGB1 transfection . Cells as indicated were stimulated with Earle ' s balanced salt solution for 3 h , and LC3 expression was detected by Western blotting ( C ) . ( C and D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with and without pUNO1 - HMGB1 transfection . Cells as indicated were stimulated with Earle ' s balanced salt solution for 3 h , and LC3 punctae formation ( arrows ) was assayed by immunofluorescence ( D ; n = 3 ; * , P < 0 . 05 ) . Data are means ± SEM .

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Figure 6.(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting (A).(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05).(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 expression was detected by Western blotting (C).(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3; *, P < 0.05). Data are means ± SEM.


Figure 7 . Expression of HMGB1 or the C106S mutant , but not C23S and C45S mutants , restore autophagic flux in Hmgb1 − / − MEFs . ( A ) HMGB1 − / − MEFs were transfected with wild - type ( WT ) or the cysteine mutant HMGB1 - GFP plasmids as indicated , starved ( HBSS ) for 3 h in the presence or absence of 100 nM bafilomycin A1 , and then immunostained with LAMP2 - specific antibody / Alexa Fluor 594 secondary antibody ( shown in red ) , LC3 - specific antibody / Alexa Fluor 647 secondary antibody ( shown in green ) , and Hoechst 33342 ( shown in blue ) . Images were acquired digitally from a randomly selected pool of 10 - 15 fields under each condition . ( B ) Quantitative analysis of the percentage of LAMP2 / LC3 colocalization was detected by Image - Pro Plus 5 . 1 software . Data are means ± SEM .

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Figure 7.Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1−/− MEFs. (A) HMGB1−/− MEFs were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of 100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro Plus 5.1 software. Data are means ± SEM.


f1a , b , LDH release , IL - 1β secretion ( a ) and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b ) and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b ) from unprimed BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) . c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S . typhimuriumd , e , LDH release and IL - 1β secretion from primed BMDMs infected with SPI - 1 - expressing logarithmic phase S . typhimurium , treated with monosodium urate , alum and nigericin or transfected with poly ( dA : dT ) and LPS . LDH release and IL - 1β secretion from unprimed wild - type and Gbp2 − / − BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( b ) and three ( a , c - f ) independent experiments . * Crossreactive band ; * * P < 0 . 01 ; NS , not significant ( two - tailed t - test ) .

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f1a, b, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b)and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase).c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimuriumd, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).


f2a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow - cytometrya , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow - cytometry ( b ) in unprimed BMDMs at 16 h post - infection . c , Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post - infection . Arrowheads , bacteria shown in insets . d , Quantification of GBP - positive bacteria , LDH release and IL - 1β secretion at indicated time points from BMDMs infected with Salmonella , live or killed by different means . e , Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella ( mCherry - positive ) - infected mice ( representative of n = 3 per group ) . S . tm . , S . typhimurium . f , g , Quantification of GBP - positive Salmonella in anti - IFN - γ - treated or control animals ( f ) and live and dead bacteria among GBP2 - negative / - positive Salmonella ( g ) ( n = 3 per group ) . Scale bars , 10 µm ( c ) , 1 µm ( e ) . Graphs show mean and 5 - 95 percentile ( box plots ) or s . d . of technical triplicates , and data are representative of three independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed t - test ) .

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f2a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometrya, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection.c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets.d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means.e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium.f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


f3a , b , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for LC3 and GBP2 . Arrowheads , bacteria shown in insets . Scale bars , 10 & amp ; amp ; mgr ; m . . c , Quantification of results from b . b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) . b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) . b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) . b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) . h , i , LDH release and IL - 1b secretion from BMDMs infected for 16 h or transfected with LPS . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( e , i ) and three ( a - d , f - h ) independent experiments . * P 0 . 05 , * * P 0 . 01 ; NS , not significant ( two - tailed t - test ) .

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f3a, b, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &amp;mgr;m.. c, Quantification of results from b.b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).


f4a , Quantification of galectin - 8 - positive Salmonella in unprimed BMDMs at 4 h post - infection . bc , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for galectin - 8 , GBP2 and LC3 . Arrowheads , bacteria shown in insets . Scale bars , 10 µm . d , Quantification of galectin - 8 / LC3 - double - positive Salmonella at indicated time points post - infection . e - h , Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry - positive S . typhimurium ( e - g ) or S . flexneri ( h , wild - type or ΔT3SS ) for 4 h . Graphs show mean and s . d . or 5 - 95 percentile ( Box plots ) of technical triplicates . Data are representative of 2 ( g , h ) , 3 ( a - d ) and 4 ( e , f ) independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed t - test ) .

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f4a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. bc, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection.e-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


Figure 1mRNA levels of TMX1 ( A ) and NFAT1 ( B ) in primary melanocytes , keratinocytes and melanoma cell lines . data are are normalized to the expression of TBP and are presented as mean ± SEM ( n ≥ 3 ) . Representative immunoblot ( from 3 ) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines ; actin was used as a loading control . Indicated band intensities were normalized to actin and background signal was subtracted . Melan - A ( brown ) , TMX1 ( red - brown ) and NFAT1 ( deep red ) staining ( IHC ) in a primary stage 4 high risk nodular melanoma patient I ( out of four ) , the enlarged regions show healthy tissue ( panels 2 , 5 and 8 ) and tumor tissue ( panels 3 , 6 and 9 ) . Arrows indicate Melan - A positive melanocytes . TMX1 ( red - brown ) and NFAT1 ( deep red ) staining of healthy human skin and increasing melanoma stages ; P1 - P13 refer to the donor patient number .

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Figure 1mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines. data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3).Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.


Figure 2Insulin ( 50 µg / mL ) induced the translocation of NFAT1 - GFP to the nucleus of WM3734 cells . ( A ) Ratio images ( F / F0 ) , ( B ) corresponding endpoint quantification ( n = 5 ) . Reduced nuclear translocation of NFAT1 - GFP in TMX1 or TMX3 silenced ( siRNA ) melanoma cells . ( C ) Images show NFAT1 - GFP fluorescence intensity before and after stimulation with thapsigargin ( Tg ; 1 µM ) in WM3734 cells . ( D ) Corresponding time - dependent nuclear import of NFAT1 as a change of F / F0 . ( E ) Normalized endpoint quantification . Same analysis was performed with Mel Juso cells with ( F ) images , ( G ) time - dependent nuclear import and ( H ) normalized endpoint quantification . data are presented as mean ± SEM ( n values : WM3734 , control = 142 , TMX1 kd = 116 , TMX3 kd = 148 ; Mel Juso , control = 75 , TMX1 kd = 47 , TMX3 kd = 67 ) .

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Figure 2Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).


Figure 3Thapsigargin ( Tg ) induced Fura - 2 based cytosolic Ca2 + imaging in Ringer ' s buffer containing 0 . 25 mM Ca2 + . ( B ) Quantification of basal cytosolic calcium levels and ( C ) SOCE quantification ( plateau - basal ) for WM3734 after stable silencing of TMX1 ( two clones ) . : In ( B - C ) , data are presented as mean ± SEM ( n values : WM3734 , control = 939 , TMX1 kds 1 = 988 , TMX1 kds 2 = 508 ) . Cytosolic Ca2 + imaging ( Fura - 2 ) and ( E ) SOCE quantification ( plateau - basal ) for WM3734 after transient silencing of TMX1 or TMX3 . In ( E ) , data are presented as mean ± SEM ( n values : WM3734 , control = 30 , TMX1 kd = 49 , TMX3 kd = 52 ) . Cellular H2O2 ( HyPer ) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd . ( F ) Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for WM3734 and Mel Juso . Quantification of basal cellular H2O2 in WM3734 ( G ) and Mel Juso ( H ) cells . In ( G - H ) , data are presented as mean ± SEM ( n values : WM3734 : control = 168 , TMX1 kd = 209 , TMX3 kd = 192 ; Mel Juso : control = 297 , TMX1 kd = 343 , TMX3 kd = 440 ) . In ( F ) scale bars : 10 µm ; color code : WM3734 : color code : blue = 0 , red = 3 ; Mel Juso : color code : blue = 0 , red = 1 . 5 .

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Figure 3Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.


Figure 4WM3734 cells were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP nuclear translocation was measured as F / F0 . The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13 . 2 µM ( red mark ) . Tg - induced NFAT1 - GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre - incubation with 100 µM N - acetylcysteine ( NAC ) for 48 h . Endpoint quantification of the data from ( C ) and upon treatment with NAC , PEG - catalase ( 50 U / mL ) , or DTT ( 1 mM ) . data are presented as mean ± SEM ( n values : control = 73 , control + NAC = 19 , TMX1 kd = 57 , TMX1 kd + NAC = 63 , TMX1 kd + catalase = 58 , TMX1 kd + DTT = 22 ) . Cytosolic calcineurin activity ( CaNAR2 ) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation . Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) . data are presented as mean ± SEM ( n values : control = 49 , TMX1 kd = 48 , TMX3 kd = 63 ) . Normalized Tg - induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre - incubation with extracellular H2O2 ( 0 - 200 µM ) . Quantification of the maximum cytosolic calcineurin activity ( plateau - basal ) . Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) 8 h after treatment with the antioxidant NAC ( 100 µM ) in WM3734 with transient knockdown of TMX1 . data are presented as mean ± SEM ( n values : control = 15 , TMX1 kd = 24 , TMX1 kd + NAC = 19 ) .

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Figure 4WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63).Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).


Figure 5Mitochondrial H2O2 ( mito - HyPer ) was measured in two cell lines upon transient TMX knockdown . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown ( A ) ; corresponding quantification for ( B ) WM3734 and ( C ) Mel Juso . , data are presented as mean ± SEM ( n values : WM3734 : control = 546 , TMX1 kd = 510 , TMX3 kd = 621 ; Mel Juso : control = 416 , TMX1 kd = 418 , TMX3 kd = 442 ) . Mitochondrial Ca2 + uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria - targeted calcium sensor ( 4mt - D3cpV ) ( D ) . Cells were exposed to 0 . 25 mM Ca2 + containing Ringer ' s buffer and Tg . ( E ) Quantification of basal mitochondrial Ca2 + levels and ( F ) mitochondrial Ca2 + uptake ( basal - plateau ) . , data are presented as mean ± SEM ( n values : control = 62 , TMX1 kds = 45 ) . Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1 . The mean mitochondrial volume was modeled based on microscopy , the quantification of mean volume ( µm3 ) and mean surface ( µm2 ) is depicted in ( G ) and ( H ) respectively . data are presented as boxplots ( n values : control = 101 , TMX1 kds = 90 ; center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ; outliers are shown as dots ) . Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1 . A peripheral mask was applied based on the membrane staining ( CellMask Green ) and mitochondria covered area ( MitoTracker DeepRed ) was evaluated ( see Fig . 9 ) . Representative images ( I ) and quantification ( J ) . In ( J ) , data are presented as mean ± SEM ( n values : control = 163 , TMX1 kds = 116 ) . Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown ( K ) . Corresponding quantification of the distance between ( L ) mitochondria and plasma membrane , ( M ) mitochondria and ER , and ( N ) the length of the MAM ( mitochondria - ER contact site ) . In ( L ) , data are presented as mean ± SEM ( n values : control = 100 , TMX1 kds = 100 ) . In ( M - N ) , data are presented as mean ± SEM ( n values : control = 60 , TMX1 kds = 60 ) . Cellular H2O2 concentration ( HyPer ) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 ( 140 nM ) ( O ) Representative ratiometric images ( F505 nm / F420 nm ) and ( P ) quantification . In ( P ) , data are presented as mean ± SEM ( n values : control = 837 , TMX1 kd = 888 , TMX1 kd + NOX4 kd = 793 , TMX1 kd + GKT = 844 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots).Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116).Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60).Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).


Figure 6Proliferation ( 48 h ) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3 . Proliferation ( 48 h ) of melanoma cell lines with stable TMX1 knockdown ( two clones ) . Proliferation ( 24 h ) of melanoma cell lines after transient knockdown of NFAT1 . Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 100 nM ) . Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 or TMX3 . Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 1 µM ) . Transwell migration ( 48 h ) of melanoma cell lines after preventing NFAT - calcineurin interaction with dipyridamole ( 40 µM ) or inhibition of calcineurin with cyclosporine A ( CsA , 2 µM ) . Invasion of WM3734 with stable TMX1 knockdown ( two clones ) through Matrigel for 96 h . Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown ( TMX1 kds ; two clones ) in immunodeficient mice . ( I ) Tumor growth in the first 19 days after injection ; ( J ) the corresponding tumor volume quantified after 19 days and ( K ) after 45 days .

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Figure 6Proliferation (48 h) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3.Proliferation (48 h) of melanoma cell lines with stable TMX1 knockdown (two clones).Proliferation (24 h) of melanoma cell lines after transient knockdown of NFAT1.Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 or TMX3.Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).Invasion of WM3734 with stable TMX1 knockdown (two clones) through Matrigel for 96 h.Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown (TMX1 kds; two clones) in immunodeficient mice. (I) Tumor growth in the first 19 days after injection; (J) the corresponding tumor volume quantified after 19 days and (K) after 45 days.


Figure 7Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with high or low expression of any of the three genes of interest ( GOI ) , TMX1 , TMX3 and NFAT1 . mRNA expression levels of NFAT1 , TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients . In ( B ) , expression levels are based on the FPKM values of the TCGA samples ( center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ) . Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with ( C ) BRAF WT and ( D ) BRAF V600E and with high or low expression of NFAT1 .

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Figure 7Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with high or low expression of any of the three genes of interest (GOI), TMX1, TMX3 and NFAT1.mRNA expression levels of NFAT1, TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients. In (B), expression levels are based on the FPKM values of the TCGA samples (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range).Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with (C) BRAF WT and (D) BRAF V600E and with high or low expression of NFAT1.


Figure 1E . Double immunofluorescent staining of Fn12230 , fadA - deletion mutant US1 ( ∆ fadA ) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1 : 800 dilution and anti - human β - amyloid polyclonal antibody A11 at 1 : 500 dilution , followed by incubation with Alexa Fluor 680 - conjugated donkey anti - rabbit and Alexa Fluor 555 - conjugated goat anti - mouse , both at 1 : 1 , 000 dilution . Co - staining of FadA and A11 was observed in Fn in stationary phase , not in log phase , or in US1 or lam . The imagines were taken under 60X objective . Scale bar equals 5 μm . F . Scanning electron microscopy of Fn 12230 , US1 ( ∆ fadA ) and lam in log and stationary phase at 10 , 000x magnification . Note the fibrous structure coating Fn in stationary phase , pointed by the clear arrows , but not in log phase . Scale bar equals 1 μm .

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Figure 1E. Double immunofluorescent staining of Fn12230, fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in stationary phase, not in log phase, or in US1 or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm.F. Scanning electron microscopy of Fn 12230, US1 (∆fadA) and lam in log and stationary phase at 10,000x magnification. Note the fibrous structure coating Fn in stationary phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm.


Figure 2E . Thioflavin - T binding assay in the presence of 0 . 1 % SDS . FadA proteins ( 0 . 8 mg / ml ) were incubated with 10 μM Thioflavin - T at room temperature for 10 min . The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm . The experiment was performed in duplicate and repeated three times . The error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 ( compared to BSA , t - test ) . G . Analysis of amyloid - like FadA produced by Fn 23726 and its ∆ fap2 mutant . An aliquot of 50 μg detergent - resistant pellets was loaded onto each lane , followed by SDD - AGE and Western blot analysis as described above . The mutant produced significantly less amyloid - like polymers than the wild type .

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Figure 2E. Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to BSA, t-test).G. Analysis of amyloid-like FadA produced by Fn 23726 and its ∆fap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type.


Figure 3A . Detection of FadA in subgingival plaque samples by IHC . Three patients with periodontitis are shown here who provided plaque samples from their healthy sites ( probing depth ≤ 3 mm , left panels ) as well as periodontal diseased sites ( probing depth ≥ 7 mm , middle panels ) . IHC was performed as described using mAb 7H7 at 1 : 800 dilution . Anti - mouse IgG isotype control of the diseased site is shown ( right panels ) . The images were taken using 100X objective . Scale bar equals 50 μm . B . Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients . The frozen tissue sections were incubated with mAb 7H7 at 1 : 50 dilution and anti - amyloid antibody A11 at 1 : 25 dilution , or mouse and rabbit IgG control , followed by incubation with Alexa Fluor 555 - conjugated goat anti - mouse and Alexa Fluor 680 - conjugated donkey anti - rabbit , both at 1 : 1000 dilution . Co - staining of FadA ( red ) and amyloid oligomers ( green ) was observed in the carcinoma but not normal tissues . The images were taken under 60X objective . Scale bar equals 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. Detection of FadA in subgingival plaque samples by IHC. Three patients with periodontitis are shown here who provided plaque samples from their healthy sites (probing depth ≤ 3 mm, left panels) as well as periodontal diseased sites (probing depth ≥ 7 mm, middle panels). IHC was performed as described using mAb 7H7 at 1:800 dilution. Anti-mouse IgG isotype control of the diseased site is shown (right panels). The images were taken using 100X objective. Scale bar equals 50 μm.B. Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients. The frozen tissue sections were incubated with mAb 7H7 at 1:50 dilution and anti-amyloid antibody A11 at 1:25 dilution, or mouse and rabbit IgG control, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse and Alexa Fluor 680-conjugated donkey anti-rabbit, both at 1:1000 dilution. Co-staining of FadA (red) and amyloid oligomers (green) was observed in the carcinoma but not normal tissues. The images were taken under 60X objective. Scale bar equals 10 μm.


Figure 4B . Inhibition of biofilm formation mediated by detergent - resistant Fn pellets by Congo Red . An aliquot of 10 μg detergent - resistant pellets prepared from Fn 12230 ( pellet ) was added in 96 - well plates in the presence of 0 , 10 or 50 µg / ml Congo Red ( CR ) , followed by addition of Fn 12230 and incubated as described above . The results shown are the average of four experiments each performed in duplicate . The error bars indicate SD . A representative image following 72 hours of incubation is shown in the top panel . * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) . D . Effects of Congo Red on Fn survival in acidic environment . Wild type Fn 12230 ( Fn ) or its fadA deletion mutant US1 ( ∆ fadA ) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg / ml Congo Red ( CR ) for 1 or 2 hours . The live bacterial counts at time 0 were designated as 100 % , and those after 1 or 2 hrs of incubation were expressed relative to time 0 . The results shown are the average of 5 independent experiments , each performed in duplicate . The error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( One - way ANOVA ) . F . Inhibition of Fn attachment by FadAc in the presence or absence of anti - amyloid antibody OC . An aliquot of 50 μg purified recombinant FadAc was pre - incubated with DLD1 for 45 min either alone , or mixed with 20 μl OC or rabbit IgG control , before Fn was added and the attachment assay performed as above . The attachment value by Fn alone was designated as 100 % , with all other values expressed relative to it . FadA mutant L14A was included as a negative control . Data shown are mean values ± SEM . The experiment was performed in duplicate and repeated three times . * * p < 0 . 01 ( One - way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B. Inhibition of biofilm formation mediated by detergent-resistant Fn pellets by Congo Red. An aliquot of 10 μg detergent-resistant pellets prepared from Fn 12230 (pellet) was added in 96-well plates in the presence of 0, 10 or 50 µg/ml Congo Red (CR), followed by addition of Fn 12230 and incubated as described above. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01, ***p<0.001 (t-test).D. Effects of Congo Red on Fn survival in acidic environment. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg/ml Congo Red (CR) for 1 or 2 hours. The live bacterial counts at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to time 0. The results shown are the average of 5 independent experiments, each performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).F. Inhibition of Fn attachment by FadAc in the presence or absence of anti-amyloid antibody OC. An aliquot of 50 μg purified recombinant FadAc was pre-incubated with DLD1 for 45 min either alone, or mixed with 20 μl OC or rabbit IgG control, before Fn was added and the attachment assay performed as above. The attachment value by Fn alone was designated as 100%, with all other values expressed relative to it. FadA mutant L14A was included as a negative control. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated three times. **p< 0.01 (One-way ANOVA).


Figure 5D . Effect of detergent - resistant pellets prepared from Fn 12230 ( Fn ) and US1 ( ∆ fadA ) on tumor growth . HCT116 cells were inoculated into the nude mice as described above . In one group of mice ( n = 3 ) , sarkosyl - resistant pellets prepared from Fn 12230 and US1 ( ∆ fadA ) were injected into the tumors on opposite sides . In another group ( n = 5 ) , sarkosyl - resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control ( rIgG ) before injecting into the tumors on opposite sides . The tumor volumes were measured described above . The vertical lines represent the standard deviations . The individual tumor pairs are shown ( right panel ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ compared to US1 ( ∆ fadA ) , t - test ] .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].


Figure 6A . Approximately 1x109 CFU of Fn 12230 , US1 ( ∆ fadA ) , and lam suspended in carboxymethylcellulose ( CMC ) were orally administered to C57BL / 6 mice three times a week for 10 weeks . CMC alone was administered as a control . Maxillae from mice inoculated with CMC alone ( n = 5 ) , Fn 12230 ( n = 5 ) , US1 ( ∆ fadA ; n = 5 ) , and lam ( n = 4 ) were harvested and fixed in 4 % paraformaldehyde and stored in 70 % ethanol , followed by microCT ( μCT ) scanning using a Scanco vivaCT 80 system at 55 kVp , 145 µA , and 250 ms integration time . Shown in the figure are reconstructed grayscale images . ImageJ was used to measure difference in bone height from the cementoenamel junction ( CEJ ) to the alveolar crest between palatal roots of first and second molars ( see arrows ) . B . The average bone loss of each group shown in ( A ) , with the lines above each bar representing standard deviations . * p < 0 . 05 . * * p < 0 . 01 ( t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. Approximately 1x109 CFU of Fn 12230, US1 (∆fadA), and lam suspended in carboxymethylcellulose (CMC) were orally administered to C57BL/6 mice three times a week for 10 weeks. CMC alone was administered as a control. Maxillae from mice inoculated with CMC alone (n=5), Fn 12230 (n=5), US1 (∆fadA; n=5), and lam (n=4) were harvested and fixed in 4% paraformaldehyde and stored in 70% ethanol, followed by microCT (μCT) scanning using a Scanco vivaCT 80 system at 55 kVp, 145 µA, and 250 ms integration time. Shown in the figure are reconstructed grayscale images. ImageJ was used to measure difference in bone height from the cementoenamel junction (CEJ) to the alveolar crest between palatal roots of first and second molars (see arrows). B. The average bone loss of each group shown in (A), with the lines above each bar representing standard deviations. *p< 0.05. **p< 0.01 (t-test).


Figure 1A . Relative mRNA expression in skeletal muscle from SBMA patients ( white bars , n = 3 ) and controls ( black bars , n = 3 ) ( mean + / − SEM ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . B . Splicing of XBP1 mRNA was assessed by RT - PCR . Products from unspliced ( XBP1u ) and spliced ( XBP1s ) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green . C . Relative mRNA expression in proximal hind limb muscle ( mean + / − SEM ) . Mice evaluated were littermate WT ( n = 6 ) , AR113Q ( n = 6 ) , castrated WT ( C - WT , n = 6 ) and castrated AR113Q males ( C - AR113Q , n = 5 ) on a mixed C57BL / 6J - 129 genetic background . * p < 0 . 05 by ANOVA . D . Western blot of BiP and PDI expression in proximal hind limb muscle . Right panels show quantification of signal relative to loading control ( mean + / − SEM ) . * p < 0 . 05 by Student ' s t test . E . Relative mRNA expression in proximal hind limb muscle of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males backcrossed to C57BL / 6J . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . F . Relative mRNA expression in spinal cord of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males ( mean + / − SEM ) . n . s . = not significant by Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A. Relative mRNA expression in skeletal muscle from SBMA patients (white bars, n = 3) and controls (black bars, n = 3) (mean +/− SEM). * p<0.05, ** p<0.01 by Student's t test.B. Splicing of XBP1 mRNA was assessed by RT-PCR. Products from unspliced (XBP1u) and spliced (XBP1s) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green.C. Relative mRNA expression in proximal hind limb muscle (mean +/− SEM). Mice evaluated were littermate WT (n = 6), AR113Q (n = 6), castrated WT (C-WT, n = 6) and castrated AR113Q males (C-AR113Q, n = 5) on a mixed C57BL/6J-129 genetic background. *p<0.05 by ANOVA.D. Western blot of BiP and PDI expression in proximal hind limb muscle. Right panels show quantification of signal relative to loading control (mean +/− SEM). * p<0.05 by Student's t test.E. Relative mRNA expression in proximal hind limb muscle of AR21Q (n = 5) and AR113Q (n = 3) males backcrossed to C57BL/6J. ** p<0.01, ***p<0.001 by Student's t test. F. Relative mRNA expression in spinal cord of AR21Q (n = 5) and AR113Q (n = 3) males (mean +/− SEM). n. s. = not significant by Student's t test.


Figure 2A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limb muscle of AR113Q ( black ) or AR113Q , CHOP − / − mice ( white ) at 12 wks . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test . B . Representative image of muscle fibers following NADH stain . Bar = 20 µM . C . Distribution of proximal hind limbmuscle fiber size from wt ( black ) and CHOP − / − ( white ) mice at 12 wks . Difference not significant by Mann - Whitney test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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 2A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.B. Representative image of muscle fibers following NADH stain. Bar = 20 µM.C. Distribution of proximal hind limbmuscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.


Figure 3A . Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice ( n = 5 - 6 / genotype ) . * p < 0 . 05 by ANOVA , n . s . = not significant . B . LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * p < 0 . 01 by Student ' s t test . C . Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle . * p < 0 . 05 by Student ' s t test . Androgen receptor protein expression in skeletal muscle of 12 wk mice . Hsp90 serves as a loading control . Right panel shows quantification of relative signal intensity ( n = 3 / genotype ) . D . Proximal hind limb muscles stained for the androgen receptor ( in red ) exhibit intranuclear inclusions . Nuclei are stained by DAPI ( in blue ) . E . P - JNK and P - eIF2 alpha expression ( top , middle ) and XBP1 mRNA splicing ( bottom ) in proximal hind limbmuscle of 12 wk mice . Right panels show quantification of signal relative to loading control . * * p < 0 . 01 by Student ' s t test . n . s . = not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice (n = 5-6/genotype). *p<0.05 by ANOVA, n. s. = not significant.B. LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot. Right panels show quantification of signal relative to GAPDH. **p<0.01 by Student's t test.C. Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle. *p<0.05 by Student's t test.Androgen receptor protein expression in skeletal muscle of 12 wk mice. Hsp90 serves as a loading control. Right panel shows quantification of relative signal intensity (n = 3/genotype).D. Proximal hind limb muscles stained for the androgen receptor (in red) exhibit intranuclear inclusions. Nuclei are stained by DAPI (in blue).E. P-JNK and P-eIF2 alpha expression (top, middle) and XBP1 mRNA splicing (bottom) in proximal hind limbmuscle of 12 wk mice. Right panels show quantification of signal relative to loading control. **p<0.01 by Student's t test. n. s. = not significant.


Figure 4Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice . A . Western blot shows enhanced eIF2 alpha phosphorylation ( top ) and RT - PCR demonstrates increased XBP1 mRNA splicing ( bottom ) in denervated muscle . Right panels show relative quantification of signal intensity . * p < 0 . 05 by Student ' s t test . . B . Relative expression of BiP , ATF4 and CHOP mRNA ( n = 3 ) . * p < 0 . 05 by Student ' s t test . C . Following surgical denervation of wild type or CHOP − / − mice , LC3 and p62 expression was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * * p < 0 . 001 by ANOVA , n . s . = not significant . D . Muscle fiber size ( 100 fibers / mouse ) was quantified from wild type ( black , n = 5 ) , CHOP − / − ( white , n = 3 ) and Beclin - 1 + / − ( grey , n = 3 ) mice 7 days post sciatic nerve transection . Shown is relative fiber cross sectional area ( mean + / − SEM ) . * * * p < 0 . 001 by ANOVA . E . Muscle fiber size ( 100 fibers / mouse ) was quantified from CHOP − / − ( black , n = 6 ) or CHOP − / − , Beclin - 1 + / − mice ( white , n = 6 ) 7 days post sciatic nerve transection . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test.. B. Relative expression of BiP, ATF4 and CHOP mRNA (n = 3). *p<0.05 by Student's t test.C. Following surgical denervation of wild type or CHOP−/− mice, LC3 and p62 expression was assessed by western blot. Right panels show quantification of signal relative to GAPDH. ***p<0.001 by ANOVA, n. s. = not significant.D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA.E. Muscle fiber size (100 fibers/mouse) was quantified from CHOP −/− (black, n = 6) or CHOP −/−, Beclin-1 +/− mice (white, n = 6) 7 days post sciatic nerve transection. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


Figure 5A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limbmuscle of AR113Q ( red , n = 6 ) or AR113Q , Beclin - 1 + / − ( blue , n = 6 ) mice at 16 wks . Left panel shows fiber size distribution , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left panel , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 0001 by Student ' s t test . B . AR expression in skeletal muscle of 16 wk mice was assessed by western blot . GAPDH controls for loading . Right panel shows quantification of signal relative to GAPDH ( mean + / − SEM ) . Differences not significant ( n . s . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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 5A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limbmuscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test.B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).


Figure 6A . Left panel , Kaplan - Meyer survival curve of AR113Q males ( red line , n = 12 ) and AR113Q , Beclin - 1 + / − males ( blue line , n = 15 ) . * p < 0 . 05 by log - rank analysis . Right panel , mean survival + / − SEM . * p < 0 . 05 by Student ' s t - test . B , C . Body weight ( panel B ) and grip strength ( panel C ) at different ages for wild type ( wt , green line , n = 7 ) , Beclin - 1 + / − ( yellow line n = 9 ) , AR113Q ( red line , n = 12 ) , and AR113Q , Beclin - 1 + / − ( blue line , n = 15 ) male mice . B , C . Body weight ( panel B ) and grip strength ( panel C ) at different ages for wild type ( wt , green line , n = 7 ) , Beclin - 1 + / − ( yellow line n = 9 ) , AR113Q ( red line , n = 12 ) , and AR113Q , Beclin - 1 + / − ( blue line , n = 15 ) male mice . D . Age of disease onset as measured by a decrease of 5 % or more in forelimb grip strength ( not significant by log rank analysis ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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 6A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test.B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).


Figure 1Tfeb expression in the vasculature ( mice n = 10 ) . ( A ) Representative images of retinal vessels ( p5 ) ( A ) ( scale bar : 50 µm ) of Tfeb - EGFP mice stained with anti - iB4 ( A ) and anti - GFPTfeb expression in the vasculature ( mice n = 10 ) . Representative images of glomerular , arterial ( scale bars : 10 µm ) and interstitial vessels ( scale bar : 50 µm ) of kidney ( p17 ) of Tfeb - EGFP mice stained with anti - CD31 ( B ) and anti - GFPAlterations in the embryonic vasculature in TfebEC - / - mice at E10 . 5 ( mice n = 25 ) . Representative images of whole - mount embryos and yolk sacs ( i , ii ) ( scale bars : 0 . 5 mm ) . Vessels of the head ( iii ) , ocular ( iv ) and intrasomitic regions ( v ) were stained with anti - endomucin Ab ( scale bars : 100 µm ) . Alteration of retinal vascular maturation in TfebiEC - / - at p5 . Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti - iB4 Ab ( scale bars : 50 µm ) . Bar graphs indicate the percent of vascular area versus total area of the retina , the percent of vascular density , the number of branches per field , vessel diameter , the number of filopodia per field and the length of filopodia ( mice n = 10 , mean ± SEM ; * * * p < 0 . 0001 versus control mice by Student ' s t - test ) . Alteration of the glomerular ultrastructure in TfebiEC - / - mice at p17 ( mice n = 5 ) . Representative transmission electron micrographs of renal tissue . Magnification : i ) x6000 , ii ) x20000 ) , iii - iv ) x10000 , v - vi ) x25000 ; scale bars : 1µm ) . Asterisks indicate the accumulation of extracellular matrix in mesangium ; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) of Tfeb-EGFP mice stained with anti-iB4 (A) and anti-GFPTfeb expression in the vasculature (mice n=10). Representative images of glomerular, arterial (scale bars: 10 µm) and interstitial vessels (scale bar: 50 µm) of kidney (p17) of Tfeb-EGFP mice stained with anti-CD31 (B) and anti-GFPAlterations in the embryonic vasculature in TfebEC-/- mice at E10.5 (mice n=25). Representative images of whole-mount embryos and yolk sacs (i, ii) (scale bars: 0.5 mm). Vessels of the head (iii), ocular (iv) and intrasomitic regions (v) were stained with anti-endomucin Ab (scale bars: 100 µm).Alteration of retinal vascular maturation in TfebiEC-/- at p5. Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti-iB4 Ab (scale bars: 50 µm). Bar graphs indicate the percent of vascular area versus total area of the retina, the percent of vascular density, the number of branches per field, vessel diameter, the number of filopodia per field and the length of filopodia (mice n=10, mean±SEM; ***p< 0.0001 versus control mice by Student's t-test).Alteration of the glomerular ultrastructure in TfebiEC-/- mice at p17 (mice n=5). Representative transmission electron micrographs of renal tissue. Magnification: i) x6000, ii) x20000), iii-iv) x10000, v-vi) x25000; scale bars: 1µm). Asterisks indicate the accumulation of extracellular matrix in mesangium; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae, respectively.


Figure 2Reduced EC proliferation in the retina ( p5 ) of in TfebiEC - / - mice . Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC - / - mice stained with anti - iB4 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percentage of EC Ki - 67 + nuclei versus total nuclei co - localized with iB4 + vesselsReduced EC proliferation in the kidney ( p17 ) of in TfebiEC - / - mice . ( B ) Representative images of vessels of the kidney in control and TfebiEC - / - mice stained with anti - CD31 and Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates the percent of EC Ki - 67 + nuclei versus total nuclei co - localized with CD31 + vessels ( mice n = 6 , mean ± SEM ; * * p < 0 . 001 versus control mice by Student ' s t - test ) . Podocin staining is shown to glomerular localization . TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . ( C ) DNA incorporation of EdU was detected by flow cytometry . The percentage of proliferating cells is indicated ( n = 4 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA ECs by Student ' s t - test ) . TFEB silencing reduced EC proliferation . Representative graph of scr - shRNA and sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . DNA content was determined by propidium iodide staining and assessed by fluorescences - activated cell sorter analysis ( representative experiment out of 4 with similar results ) . TFEB silencing reduced human EC morphogenesis . Representative images of tube - like - structure of scr - shRNA and sh - TFEB human ECs stained with phalloidin - 555 . Bar graph indicates the percentage of phalloidin + area in sh - TFEB and scr - shRNA ECs ( scale bars : 0 . 25 mm ; n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus scr - shRNA by Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Reduced EC proliferation in the retina (p5) of in TfebiEC-/- mice. Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC-/- mice stained with anti-iB4 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percentage of EC Ki-67+ nuclei versus total nuclei co-localized with iB4+ vesselsReduced EC proliferation in the kidney (p17) of in TfebiEC-/- mice. (B) Representative images of vessels of the kidney in control and TfebiEC-/- mice stained with anti-CD31 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percent of EC Ki-67+ nuclei versus total nuclei co-localized with CD31+ vessels (mice n=6, mean±SEM; **p<0.001 versus control mice by Student's t-test). Podocin staining is shown to glomerular localization.TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student's t-test).TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results).TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student's t-test).


Figure 3Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr - shRNA and sh - TFEB ECs . Red : up - regulated genes ; green : down - regulated genes .

O B-PANEL_START O O O O O O O O O O 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 3Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr-shRNA and sh-TFEB ECs. Red: up-regulated genes; green: down-regulated genes.


Figure 4Heatmap , Red : up - regulated genes ; green : down - regulated genes . qPCR Data are expressed as relative fold - change compared with the expression in scr - shRNA cells after normalization to the housekeeping gene TBPimmunoblots showing the differentially expressed cell cycle related genes between scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with specific Abs . The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α - tubulinModulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC - / - mice . Data are expressed as relative fold - change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBPAnalysis of TFEB binding and modulation of the CDK4 promoter in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for CDK4 . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) . Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs . Analysis of TFEB modulation of CDK4 promoter in human ECs . Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding siteModulation of Rb protein in sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with phospho - Rb and total Rb Abs . The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh - TFEB vs scr - shRNA and the ratio between phospho - Rb and total Rb in the different

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Heatmap, Red: up-regulated genes; green: down-regulated genes.qPCR Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBPimmunoblots showing the differentially expressed cell cycle related genes between scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with specific Abs. The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α-tubulinModulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC-/- mice. Data are expressed as relative fold-change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBPAnalysis of TFEB binding and modulation of the CDK4 promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD).Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. Analysis of TFEB modulation of CDK4 promoter in human ECs. Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding siteModulation of Rb protein in sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with phospho-Rb and total Rb Abs. The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh-TFEB vs scr-shRNA and the ratio between phospho-Rb and total Rb in the different conditions


Figure 5Increase of Vegfr2 expression in the vasculature of TfebEC - / - mice . Representative immunostaining images of TfebEC - / - embryonic vessels ( E10 . 5 ) stained with anti - endomucin and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in endomucin + vessel areas ( embryos n = 6 , mean ± SEM ; * * * p < 0 . 0001 versus control embryos by Student ' s t - test ) . Increase of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images ( i ) and detail ( ii ) of the vascular front and vascular plexus of the retina ( p5 ) of control and TfebiEC - / - mice with anti - iB4 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates the Vegfr2 mean intensity only in iB4 + vessel areasIncrease of Vegfr2 expression in the vasculature of TfebiEC - / - mice . Representative immunostaining images of the glomerulus ( p17 ) of control and TfebiEC - / - mice with anti - podocin , anti - CD31 and anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graphs indicate the Vegfr2 mean intensity in CD31 + vessel areasVEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC - / - mice ( D ) Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB . qPCR of VEGFR2 in lung ECs obtained from control and in human sh - TFEB Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB . Immunoblots of total lysates from scr - shRNA and sh - TFEB ECs probed with anti - VEGFR2 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α - tubulinAnalysis of TFEB binding to the VEGFR2 promoter in human ECs . ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for VEGFR2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Increase of Vegfr2 expression in the vasculature of TfebEC-/- mice. Representative immunostaining images of TfebEC-/- embryonic vessels (E10.5) stained with anti-endomucin and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in endomucin+ vessel areas (embryos n=6, mean±SEM; ***p< 0.0001 versus control embryos by Student's t-test).Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and TfebiEC-/- mice with anti-iB4 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in iB4+ vessel areasIncrease of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images of the glomerulus (p17) of control and TfebiEC-/- mice with anti-podocin, anti-CD31 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graphs indicate the Vegfr2 mean intensity in CD31+ vessel areasVEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC-/- mice (D) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and in human sh-TFEB Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPVEGFR2 expression is regulated by TFEB. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with anti-VEGFR2 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulinAnalysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for VEGFR2.


Figure 6Silenced TFEB alters the localization of VEGFR2 . FACS analysis of surface VEGFR2 expression on human scr - shRNA and sh - TFEB ECs . Bar graph shows the ratio between total and PM VEGFR2Silenced TFEB alters the localization of VEGFR2 . Representative TIRF and epifluorescence images of human scr - shRNA and sh - TFEB ECs after staining with anti - VEGFR2 Ab ( scale bars : 10 µm ) . Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRFSilenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction . Representative immunoblot of PM biotinylated portion and total cell lysates of scr - shRNA and sh - TFEB ECs after VEGF - A stimulation ( 30 ng / ml ) . Blots of total or PM cell lysates were probed with anti - VEGFR2 . Blots of total cell lysates were probed with anti - p - Y1175 - VEGFR2 , anti - PLCγ , p - PLCγ , anti - ERK - 1 / 2 , anti - pERK1 / 2 , anti - p - Src , anti - Src , anti CD31 and α - tubulin Abs . The bar graphs ( i , ii ) show densitometric analysis of stimulated versus unstimulated scr - shRNA and sh - TFEB ECs expressed as : ( i ) % of VEGFR2 on PM fraction ( n = 3 , mean ± SEM ; ANOVA p < 0 . 02 ; * * p < 0 . 001 versus scr - shRNA by Bonferroni post - test ) , ( ii ) % of VEGFR2 totalRegulaton of autophagy and lysosome pathway by TFEB silencing . qPCR showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene TBPRegulaton of autophagy and lysosome pathway by TFEB silencing . immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from human scr - shRNA and sh - TFEB ECs probed with anti - ULK - 1 , anti - ATG9 , anti - LC3 - I / II , anti - LAMP - 1 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between scr - shRNA and sh - TFEB ECsTFEB silencing inhibits VEGFR2 internalization . Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF - A

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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 6Silenced TFEB alters the localization of VEGFR2. FACS analysis of surface VEGFR2 expression on human scr-shRNA and sh-TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2Silenced TFEB alters the localization of VEGFR2. Representative TIRF and epifluorescence images of human scr-shRNA and sh-TFEB ECs after staining with anti-VEGFR2 Ab (scale bars: 10 µm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRFSilenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 totalRegulaton of autophagy and lysosome pathway by TFEB silencing. qPCR showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBPRegulaton of autophagy and lysosome pathway by TFEB silencing. immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from human scr-shRNA and sh-TFEB ECs probed with anti-ULK-1, anti-ATG9, anti-LC3-I/II, anti-LAMP-1 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr-shRNA and sh-TFEB ECsTFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation