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 ) .

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

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 ) .

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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 ) .

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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 ) .

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

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

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).


Proliferation ( 48 h ) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3 .

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Proliferation (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 ) .

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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 .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 .

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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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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.


Kaplan - 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 .

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Kaplan-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 ) .

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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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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.


E . 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 .

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

E. 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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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.


E . 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 ) .

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E. 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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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.


A . 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 .

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A. 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 .

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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.


B . 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 ) .

O O O O O O O O O O O O O O B-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-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O 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. 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 ) .

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

D. 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 O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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 O O O O O O 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 O O O O O O O O O O O

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).


D . 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 O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O 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 O O O O O O O

D. 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].


A . 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 O O O O O O O O O O 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 O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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).


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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 .

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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.


A . 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 .

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A. 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 .

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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 .

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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 ) .

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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 ) .

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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 .

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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.


Denervated 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 .

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Denervated 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 .

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. 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 .

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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 .

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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 .

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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.


A . 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 .

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A. 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 . ) .

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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.).


A . 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 .

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A. 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 .

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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.


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D . Age of disease onset as measured by a decrease of 5 % or more in forelimb grip strength ( not significant by log rank analysis ) .

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D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).


Tfeb 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 -

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Tfeb 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-GFP


Tfeb 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 -

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Tfeb 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-GFP


Alterations 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 ) .

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Alterations 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 ) .

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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 .

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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.


Reduced 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 +

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Reduced 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+ vessels


Reduced 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 .

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Reduced 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 ) .

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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 ) .

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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 ) .

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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).


Heatmap 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 .

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Heatmap 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.


Heatmap , Red : up - regulated genes ; green : down - regulated genes .

Heatmap, 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

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qPCR Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBP


immunoblots 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 -

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immunoblots 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-tubulin


Modulation 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

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Modulation 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 TBP


Analysis 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 ) .

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Analysis 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

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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 site


Modulation 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

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Modulation 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


Increase 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 ) .

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Increase 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

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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 areas


Increase 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

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Increase 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 areas


VEGFR2 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

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VEGFR2 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 TBP


VEGFR2 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

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VEGFR2 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 TBP


VEGFR2 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 -

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VEGFR2 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-tubulin


Analysis 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 .

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Analysis 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.


Silenced 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

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Silenced 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 VEGFR2


Silenced 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

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Silenced 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 TIRF


Silenced 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

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Silenced 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 total


Regulaton 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

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Regulaton 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 TBP


Regulaton 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

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Regulaton 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 ECs


TFEB silencing inhibits VEGFR2 internalization . Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF - A

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TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation


Analysis of TFEB binding and modulation of the MYO1C 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 MYO1C . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) .

Analysis of TFEB binding and modulation of the MYO1C 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 MYO1C. Bar graph shows the percent enrichment (n=3, mean±SD).


Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs . qPCR of MYO1C expression in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C ECs . Data are expressed as relative fold change compared with the expression in scr - shRNA ECs after normalization to the housekeeping gene

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Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. qPCR of MYO1C expression in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C ECs. Data are expressed as relative fold change compared with the expression in scr-shRNA ECs after normalization to the housekeeping gene TBP


MYO1C silencing reverses the effect of TFEB silencing on the up - regulation of PM VEGFR2 . Analyses were performed on human ECs carrying appropriate scr - shRNA or sh - TFEB in the presence or absence of sh - MYO1C . Representative western blot of MYOC1 , total and PM biotinylated VEGFR2 ( representative experiment out of 4 with similar results ) .

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MYO1C silencing reverses the effect of TFEB silencing on the up-regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr-shRNA or sh-TFEB in the presence or absence of sh-MYO1C. Representative western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results).


Representative immunostaining images of the vascular plexus of the retina ( p5 ) ( E ) and glomerulus ( p17 ) ( F ) of control and TfebiEC - / - mice with anti - CD31 and anti - MYO1C Abs ( scale bars : 50 µm ) . Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31 +

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Representative immunostaining images of the vascular plexus of the retina (p5) (E) and glomerulus (p17)(F) of control and TfebiEC-/- mice with anti-CD31 and anti-MYO1C Abs (scale bars: 50 µm). Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31+


Representative TIRF and epifluorescence images of VEGFR2 in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C human ECs ( scale bars : 10 µm ) . Bar graph shows the ratio between PM and total

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Representative TIRF and epifluorescence images of VEGFR2 in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C human ECs (scale bars: 10 µm). Bar graph shows the ratio between PM and total VEGFR2


Analysis of TFEB binding to DLEU2 and SMC4 promoters 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 DLEU2 and SMC4 .

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Analysis of TFEB binding to DLEU2 and SMC4 promoters 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 DLEU2 and SMC4.


DLEU2 expression is regulated by TFEB . qPCR of DLEU2 in human scr - shRNA , sh - TFEB or control and TFEBS142A ECs ( left panel ) and lung ECs obtained from control and TfebiEC - / - mice ( right panel ) . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene

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DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr-shRNA, sh-TFEB or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and TfebiEC-/- mice (right panel). Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene TBP


Human miR - 15a - 5p and miR - 16 - 5p are regulated by TFEB . qPCR of miR - 15a - 5p ( left panel ) and miR - 16 - 5p ( right panel ) in sh - TFEB or TFEBS142A ECs . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene

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

Human miR-15a-5p and miR-16-5p are regulated by TFEB. qPCR of miR-15a-5p (left panel) and miR-16-5p (right panel) in sh-TFEB or TFEBS142A ECs. Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene RNU44


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


( B ) Growth kinetics of PiCoVacc ( CN2 ) P5 stock in Vero cells .

(B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells.


( D ) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4 - 12 % Bis - Tris Gel .

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

(D) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12% Bis-Tris Gel.


( E ) Representative electron micrograph of PiCoVacc .

(E) Representative electron micrograph of PiCoVacc.


isolates . BALB / c mice and Wistar rats were immunized with various doses of PiCoVacc or control ( adjuvant only as the sham group ) ( n = 10 ) . Serums from recovered COVID19 patients ( RECOV ) and non - infected people ( NI ) act as positive and negative controls , respectively . The antibody responses were analyzed in mice ( A ) , humans ( B ) and rats ( C ) . Top : SARS - CoV - 2 - specific IgG response measured by

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

isolates.BALB/c mice and Wistar rats were immunized with various doses of PiCoVacc or control (adjuvant only as the sham group) (n=10). Serums from recovered COVID19 patients (RECOV) and non-infected people (NI) act as positive and negative controls, respectively. The antibody responses were analyzed in mice (A), humans (B) and rats (C). Top: SARS-CoV-2-specific IgG response measured by ELISA


The spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice ( D ) and rats ( E ) . Neutralization assays against the other 9 isolated SARS - CoV - 2 strains were performed using mouse and rat serums collected 3 weeks post vaccination . Points represent individual animals and humans ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group .

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

The spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice (D) and rats (E). Neutralization assays against the other 9 isolated SARS-CoV-2 strains were performed using mouse and rat serums collected 3 weeks post vaccination. Points represent individual animals and humans; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group.


primates . Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only ( sham ) or placebo ( n = 4 ) . SARS - CoV - 2 - specific IgG response ( A ) and neutralizing antibody titer ( B ) were measured . Points represent individual macaques ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group . Protective efficacy of PiCoVacc against SARS - CoV - 2 challenge at week 3 after immunization was evaluated in

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

primates.Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only (sham) or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titer (B) were measured. Points represent individual macaques; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group. Protective efficacy of PiCoVacc against SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques


( C - F ) . Viral loads of throat ( C ) and anal ( D ) swab specimens collected from the inoculated macaques at day 3 , 5 and 7 pi were monitored . Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured ( E ) . RNA was extracted and viral load was determined by qRT - PCR . All data are presented as mean ± SEM .

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

(C-F). Viral loads of throat (C) and anal (D) swab specimens collected from the inoculated macaques at day 3, 5 and 7 pi were monitored. Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured (E). RNA was extracted and viral load was determined by qRT-PCR. All data are presented as mean ± SEM.


Histopathological examinations ( F ) in lungs from all the inoculated macaques at day 7 post infection . Lung tissue was collected and stained with hematoxylin and eosin .

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

Histopathological examinations (F) in lungs from all the inoculated macaques at day 7 post infection. Lung tissue was collected and stained with hematoxylin and eosin.


primates . Macaques were immunized three times at day 0 , 7 and 14 through the intramuscular route with low dose ( 1 . 5 μg per dose ) or high dose ( 6 μg per dose ) of PiCoVacc or adjuvant only ( sham ) or placebo . ( A and B ) Hematological analysis in all four groups of macaques ( n = 4 ) . Lymphocyte subset percents ( A ) , including CD3 + , CD4 + and CD8 + were monitored at day − 1 ( 1 day before vaccination ) , 18 ( 3 day after the second vaccination ) and 29 ( 7 day after the third vaccination ) . Key cytokines ( B ) , containing TNF - α , IFN - γ and IL - 2 were examined at day − 1 , 1 ( the day for the first vaccination ) , 4 , 18 and 29 after vaccination . Values are mean ± s . d .

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

primates.Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μg per dose) or high dose (6 μg per dose) of PiCoVacc or adjuvant only (sham) or placebo. (A and B) Hematological analysis in all four groups of macaques (n=4). Lymphocyte subset percents (A), including CD3+, CD4+ and CD8+ were monitored at day −1 (1 day before vaccination), 18 (3 day after the second vaccination) and 29 (7 day after the third vaccination). Key cytokines (B), containing TNF-α, IFN-γ and IL-2 were examined at day −1, 1 (the day for the first vaccination), 4, 18 and 29 after vaccination. Values are mean ± s.d.


( C ) Histopathological evaluations in lungs from four groups of macaques at day 29 . Lung tissue was collected and stained with hematoxylin and eosin .

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

(C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.


B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co - transfected into HEK293 cells with HA - tagged Pellino - 1N plasmid . Cell lysates were immunoprecipitated with anti - HA antibody and immunoblotted with anti - Myc antibody . Data are representative of at least three independent experiments . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates .

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

B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.


C , D The SBE - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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

C, D The SBE-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


C , D The 5xNF - κB - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with LPS for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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

C, D The 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


E CMT - 93 cell lines stably expressing Smad6 amino acids 400 - 441 were treated with LPS for the indicated time and expression of IκBα , IKKα , and phospho - IKKα / β was monitored by immunoblotting . As a positive control , CMT - 93 cells were pre - treated with TGF - β1 for 2 h . CMT - 93 cells stably expressing the empty vector pMSCV - puro were used as a negative control . β - actin was used as a loading control . All data are representative of three independent experiments .

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

E CMT-93 cell lines stably expressing Smad6 amino acids 400-441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.


B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) was co - transfected with the HA - tagged full - length Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated ( IP ) with anti - Myc or anti - HA antibody , and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , CS3MTBXA - 6xMyc , was transfected as a negative control . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates . Data are representative of at least three independent experiments .

O O O O O O O O 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 B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O 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 A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.


C The Myc - Smad6 ( 422 - 441 ) plasmid was co - transfected with full - length Flag - IRAK1 , Flag - TRAF6 , Flag - MyD88 , or HA - Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti - Myc antibody . IgG was added as a negative control for IP . Data are representative of at least three independent experiments .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_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 O O O O O

C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments.


D , E The SBE - Luc or 5xNF - κB - Luc reporter plasmids were co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 6 h or LPS for 2 h , and luciferase activities were measured and normalized .

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

D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.


F The BRE - Luc reporter plasmid was co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid or full - length Smad6 into RAW264 . 7 cells , respectively . After 24 h , cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized .

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

F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized.


G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) or full - length Smad6 ( Myc - Smad6 ) was co - transfected with HA - tagged full - length Smad4 or HA - tagged full - length Pellino - 1 plasmid into HEK293 cells , respectively . Cell lysates were immunoprecipitated ( IP ) with anti - Myc and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , pCS3MTBXA - 6xMyc , was co - transfected with HA - Smad4 or HA - Pellino - 1 as a negative control . Data are representative of at least three independent experiments .

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

G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.


A Pre - treatment of Smaducin - 6 reduces LPS - induced interleukin - 6 ( Il6 ) gene expression in a dose - dependent manner in RAW264 . 7 cells . Il6 gene expression was analyzed by qRT - PCR .

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

A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by qRT-PCR.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( B ) NF - κB - mediated luciferase gene expression when RAW264 . 7 cells are treated with LPS for 2 h . Luciferase activity in ( B ) was normalized to β - galactosidase activity .

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

B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( C ) IκBα degradation and IKKα / β phosphorylation when RAW264 . 7 cells are treated with LPS for 2 h .

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

B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h.


D Peli1 knockdown or wild - type humanTHP1 cells were pre - treated with 100 nM Pal - Scram peptide and Smaducin - 6 for 30 min and subsequently treated with LPS for 2 h . Il6 and Peli1 gene expression were analyzed by qRT - PCR . Data show the mean ± SD of three independent experiments .

O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O 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

D Peli1 knockdown or wild-type humanTHP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by qRT-PCR. Data show the mean ± SD of three independent experiments.


E RAW264 . 7 cells were treated with 100 nM FITC - conjugated Smaducin - 6 and TAT - S6 ( 422 - 441 ) , and localization was observed by confocal microscopy . Scale bar , 10 μm ( magnification , 200 × ) . Nuclei were stained with DAPI .

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

E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.