F . Scanning electron microscopy of Fn 12230 , US1 ( ∆ fadA ) lam in log stationary phase at 10 , 000x magnification . Note fibrous structure coating Fn in stationary phase , pointed by 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 in presence of 0 . 1 % SDS . FadA ( 0 . 8 mg / ml ) were incubated with 10 μM Thioflavin - T at room for 10 min . was measured at excitation wavelength of 440 nm emission wavelength of 500 nm . experiment was performed in duplicate repeated three times . error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 ( compared to BSA , t - ) .

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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 . of amyloid - like FadA produced by Fn 23726 its ∆ fap2 mutant . An aliquot of 50 μg detergent - resistant pellets was loaded onto each lane , followed by SDD - Western blot as described above . mutant produced significantly less amyloid - like polymers than 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 . Three 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 ) . was performed as described using mAb 7H7 at 1 : 800 dilution . Anti - mouse IgG isotype control of diseased site is shown ( right panels ) . 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 of paired normal carcinoma tissues from two CRC . frozen tissue sections were incubated with mAb 7H7 at 1 : 50 dilution anti - amyloid antibody A11 at 1 : 25 dilution , or mouse rabbit IgG control , followed by incubation with Alexa Fluor 555 - conjugated goat anti - mouse Alexa Fluor 680 - conjugated donkey anti - rabbit , both at 1 : 1000 dilution . Co - of FadA ( red ) amyloid oligomers ( green ) was observed in carcinoma but not normal tissues . 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 presence of 0 , 10 or 50 µg / ml Congo Red ( CR ) , followed by addition of Fn 12230 incubated as described above . results shown are average of four experiments each performed in duplicate . error bars indicate SD . A representative image following 72 hours of incubation is shown in top panel . * * p < 0 . 01 , * * * p < 0 . 001 ( t - ) .

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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 incubated in PBS at pH4 in presence of absence of 50 μg / ml Congo Red ( CR ) for 1 or 2 hours . live at time 0 were designated as 100 % , those after 1 or 2 hrs of incubation were expressed relative to time 0 . results shown are average of 5 independent experiments , each performed in duplicate . error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( One - way ANOVA ) .

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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 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 attachment performed as above . attachment value by Fn alone was designated as 100 % , with other values expressed relative to it . FadA mutant L14A was included as a negative control . Data shown are mean values ± . experiment was performed in duplicate repeated three times . * * p < 0 . 01 ( One - way ANOVA ) .

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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 ) US1 ( ∆ fadA ) on growth . HCT116 were inoculated into nude mice as described above . In one group of mice ( n = 3 ) , sarkosyl - resistant pellets prepared from Fn 12230 US1 ( ∆ fadA ) were injected into 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 tumors on opposite sides . volumes were measured described above . vertical lines represent standard deviations . pairs are shown ( right panel ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ compared to US1 ( ∆ fadA ) , t - ] .

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

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

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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 ( bars , n = 3 ) controls ( black bars , n = 3 ) ( mean + / − ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t .

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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 ) spliced ( XBP1s ) transcripts were resolved on a nondenaturing polyacrylamide gel 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 + / − ) . Mice evaluated were littermate WT ( n = 6 ) , AR113Q ( n = 6 ) , castrated WT ( C - WT , n = 6 ) castrated AR113Q ( 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 PDI expression in proximal hind limb muscle . Right panels show of signal relative to loading control ( mean + / − ) . * p < 0 . 05 by Student ' s t .

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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 ) AR113Q ( n = 3 ) backcrossed to C57BL / 6J . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t . F . Relative mRNA expression in spinal cord of AR21Q ( n = 5 ) AR113Q ( n = 3 ) ( mean + / − ) . n . s . = not significant by Student ' s t .

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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 ( 100 / mouse ) was quantified from proximal hind limb muscle of AR113Q ( black ) or AR113Q , CHOP − / − mice ( ) at 12 wks . Left panel shows fiber distribution , middle panel shows cumulative of as a function of fiber , right panel shows relative fiber cross sectional ( mean + / − ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 001 by Student ' s t .

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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 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 from wt ( black ) CHOP − / − ( ) mice at 12 wks . Difference not significant by Mann - Whitney .

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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 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 p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot . Right panels show of signal relative to GAPDH . * * p < 0 . 01 by Student ' s t .

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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 .

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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 expression in skeletal muscle of 12 wk mice . Hsp90 serves as a loading control . Right panel shows of relative signal ( 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 stained for 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 P - eIF2 alpha expression ( top , middle ) XBP1 mRNA splicing ( bottom ) in proximal hind limbmuscle of 12 wk mice . Right panels show of signal relative to loading control . * * p < 0 . 01 by Student ' s t . 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 or intact controls were harvested at indicated times following unilateral sciatic nerve transection in 6 wk mice . A . Western blot shows enhanced eIF2 alpha phosphorylation ( top ) RT - PCR demonstrates increased XBP1 mRNA splicing ( bottom ) in denervated muscle . Right panels show relative of signal . * p < 0 . 05 by Student ' s t .

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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 CHOP mRNA ( n = 3 ) . * p < 0 . 05 by Student ' s t .

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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 p62 expression was assessed by western blot . Right panels show 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 ( 100 / mouse ) was quantified from wild type ( black , n = 5 ) , CHOP − / − ( , n = 3 ) Beclin - 1 + / − ( grey , n = 3 ) mice 7 days post sciatic nerve transection . Shown is relative fiber cross sectional ( mean + / − ) . * * * 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 ( 100 / mouse ) was quantified from CHOP − / − ( black , n = 6 ) or CHOP − / − , Beclin - 1 + / − mice ( , n = 6 ) 7 days post sciatic nerve transection . Left panel shows fiber distribution , middle panel shows cumulative of as a function of fiber , right panel shows relative fiber cross sectional ( mean + / − ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 001 by Student ' s t .

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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 ( 100 / 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 distribution , right panel shows relative fiber cross sectional ( mean + / − ) . Left panel , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 0001 by Student ' s t .

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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 of signal relative to GAPDH ( mean + / − ) . Differences not significant ( n . s . ) .

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 ( red , n = 12 ) AR113Q , Beclin - 1 + / − ( blue , n = 15 ) . * p < 0 . 05 by log - rank . Right panel , mean survival + / − . * p < 0 . 05 by Student ' s t - .

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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 . ( panel B ) grip strength ( panel C ) at different ages for wild type ( wt , green , n = 7 ) , Beclin - 1 + / − ( yellow n = 9 ) , AR113Q ( red , n = 12 ) , AR113Q , Beclin - 1 + / − ( blue , n = 15 ) 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.


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

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 vasculature ( mice n = 10 ) . ( A ) Representative of retinal vessels ( p5 ) ( A ) ( scale bar : 50 µm ) of Tfeb - EGFP mice stained with anti - iB4 ( A ) 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 vasculature ( mice n = 10 ) . Representative of glomerular , arterial ( scale bars : 10 µm ) interstitial vessels ( scale bar : 50 µm ) of kidney ( p17 ) of Tfeb - EGFP mice stained with anti - CD31 ( B ) 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 embryonic vasculature in TfebEC - / - mice at E10 . 5 ( mice n = 25 ) . Representative of whole - mount yolk sacs ( i , ii ) ( scale bars : 0 . 5 mm ) . Vessels of head ( iii ) , ocular ( iv ) 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 of whole mounts of retina immunostaining of vascular front vascular plexus with an anti - iB4 Ab ( scale bars : 50 µm ) . Bar graphs indicate of vascular versus total of retina , of vascular density , number of branches per field , vessel diameter , number of filopodia per field of filopodia ( mice n = 10 , mean ± ; * * * p < 0 . 0001 versus control mice by Student ' s t - ) .

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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 glomerular ultrastructure in TfebiEC - / - mice at p17 ( mice n = 5 ) . Representative transmission electron of renal tissue . Magnification : i ) x6000 , ii ) x20000 ) , iii - iv ) x10000 , v - vi ) x25000 ; scale bars : 1µm ) . Asterisks indicate accumulation of extracellular matrix in mesangium ; black triangles indicate fusion of podocyte foot processes lack of endothelial fenestrae , respectively .

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

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 retina ( p5 ) of in TfebiEC - / - mice . Representative of vessels of vascular front vascular plexus in retina of control TfebiEC - / - mice stained with anti - iB4 Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates 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 kidney ( p17 ) of in TfebiEC - / - mice . ( B ) Representative of vessels of kidney in control TfebiEC - / - mice stained with anti - CD31 Ki - 67 antibodies ( scale bars : 50 µm ) . Bar graph indicates of EC Ki - 67 + nuclei versus total nuclei co - localized with CD31 + vessels ( mice n = 6 , mean ± ; * * p < 0 . 001 versus control mice by Student ' s t - ) . Podocin 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 sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . ( C ) incorporation of EdU was detected by flow cytometry . of proliferating is indicated ( n = 4 , mean ± ; * * * p < 0 . 0001 versus scr - shRNA ECs by Student ' s t - ) .

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

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 sh - TFEB ECs treated for 24 hours with FCS ( 20 % ) or VEGF - A ( 30 ng / ml ) . content was determined by propidium iodide assessed by fluorescences - activated sorter ( representative experiment out of 4 with similar results ) .

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

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 of tube - like - structure of scr - shRNA sh - TFEB human ECs stained with phalloidin - 555 . Bar graph indicates of phalloidin + in sh - TFEB scr - shRNA ECs ( scale bars : 0 . 25 mm ; n = 6 , mean ± ; * * * p < 0 . 0001 versus scr - shRNA by Student ' s t - ) .

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).


showing unsupervised hierarchical clustering of human differentially expressed genes between human scr - shRNA 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.


, 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 expression in scr - shRNA after normalization to 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 differentially expressed cycle related genes between scr - shRNA sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA sh - TFEB ECs probed with specific Abs . bar graph shows densitometric expressed as between cycle genes 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 cycle genes expression in lung ECs derived from control TfebiEC - / - mice . Data are expressed as relative fold - change compared with expression in ECs of control mice after normalization to 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


of TFEB binding modulation of CDK4 promoter in human ECs . ChIP was performed using digested from control ECs TFEBS142A ECs incubated with IgG ( indicated in bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in bar graph as " + Ab anti - TFEB " ) , followed by qPCR for CDK4 . Bar graph shows 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).


of TFEB binding modulation of CDK4 promoter in human ECs . of TFEB modulation of CDK4 promoter in human ECs . Bar graph shows relative luciferase activity % evaluated in control TFEBS142A human ECs after transfection of CDK4 full promoter CDK4 promoter deleted of TFEB binding

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 in sh - TFEB ECs . Immunoblots of total lysates from scr - shRNA sh - TFEB ECs probed with phospho - Rb total Rb Abs . bar graph shows densitometric of immunoblotting expressed as % of sh - TFEB vs scr - shRNA between phospho - Rb total Rb in different

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

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 vasculature of TfebEC - / - mice . Representative immunostaining of TfebEC - / - embryonic vessels ( E10 . 5 ) stained with anti - endomucin anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates Vegfr2 mean only in endomucin + vessel areas ( n = 6 , mean ± ; * * * p < 0 . 0001 versus control by Student ' s t - ) .

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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 vasculature of TfebiEC - / - mice . Representative immunostaining ( i ) detail ( ii ) of vascular front vascular plexus of retina ( p5 ) of control TfebiEC - / - mice with anti - iB4 anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graph indicates Vegfr2 mean 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 vasculature of TfebiEC - / - mice . Representative immunostaining of glomerulus ( p17 ) of control TfebiEC - / - mice with anti - podocin , anti - CD31 anti - Vegfr2 Abs ( scale bars : 50 µm ) . Bar graphs indicate Vegfr2 mean 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 TfebiEC - / - mice ( D ) Data are expressed as relative fold - change compared with expression in control after normalization to 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 in human sh - TFEB Data are expressed as relative fold - change compared with expression in control after normalization to housekeeping gene

O O O O O O O O O O O O O O O O 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. 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 sh - TFEB ECs probed with anti - VEGFR2 α - tubulin Abs . bar graph shows densitometric expressed as between VEGFR2 α -

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


of TFEB binding to VEGFR2 promoter in human ECs . ChIP was performed using digested from human control ECs TFEBS142A ECs incubated with IgG ( indicated in bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in 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 localization of VEGFR2 . FACS of surface VEGFR2 expression on human scr - shRNA sh - TFEB ECs . Bar graph shows between total 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 localization of VEGFR2 . Representative TIRF epifluorescence of human scr - shRNA sh - TFEB ECs after with anti - VEGFR2 Ab ( scale bars : 10 µm ) . Bar graphs show of VEGFR2 analyzed in epifluorescence 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 localization phosphorylation state of VEGFR2 its signal transduction . Representative immunoblot of PM biotinylated portion total lysates of scr - shRNA sh - TFEB ECs after VEGF - A stimulation ( 30 ng / ml ) . Blots of total or PM lysates were probed with anti - VEGFR2 . Blots of total 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 α - tubulin Abs . bar graphs ( i , ii ) show densitometric of stimulated versus unstimulated scr - shRNA sh - TFEB ECs expressed as : ( i ) % of VEGFR2 on PM fraction ( n = 3 , mean ± ; ANOVA p < 0 . 02 ; * * p < 0 . 001 versus scr - shRNA by Bonferroni post - ) , ( ii ) % of VEGFR2

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

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 lysosome pathway by TFEB silencing . qPCR showing differentially expressed autophagy lysosome related genes between human scr - shRNA sh - TFEB ECs . Data are expressed as relative fold - change compared with expression in control after normalization to housekeeping gene

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 lysosome pathway by TFEB silencing . immunoblots showing differentially expressed autophagy lysosome related genes between human scr - shRNA sh - TFEB ECs . Immunoblots of total lysates from human scr - shRNA sh - TFEB ECs probed with anti - ULK - 1 , anti - ATG9 , anti - LC3 - I / II , anti - LAMP - 1 α - tubulin Abs . bar graph shows densitometric expressed as between scr - shRNA 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 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


of TFEB binding modulation of MYO1C promoter in human ECs . ChIP was performed using digested from control ECs TFEBS142A ECs incubated with IgG ( indicated in bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in bar graph as " + Ab anti - TFEB " ) , followed by qPCR for MYO1C . Bar graph shows 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).


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

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 effect of TFEB silencing on up - regulation of PM VEGFR2 . Analyses were performed on human ECs carrying appropriate scr - shRNA or sh - TFEB in presence or absence of sh - MYO1C . Representative western blot of MYOC1 , total 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 of vascular plexus of retina ( p5 ) ( E ) glomerulus ( p17 ) ( F ) of control TfebiEC - / - mice with anti - CD31 anti - MYO1C Abs ( scale bars : 50 µm ) . Bar graphs indicate Myo1C mean 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 epifluorescence of VEGFR2 in scr - shRNA , sh - TFEB , sh - MYO1C sh - TFEB + sh - MYO1C human ECs ( scale bars : 10 µm ) . Bar graph shows between PM 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


of TFEB binding to DLEU2 SMC4 promoters in human ECs . ChIP was performed using digested from control ECs TFEBS142A ECs incubated with IgG ( indicated in bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in bar graph as " + Ab anti - TFEB " ) , followed by qPCR for DLEU2 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 TFEBS142A ECs ( left panel ) lung ECs obtained from control TfebiEC - / - mice ( right panel ) . Data are expressed as relative fold - change compared with expression in scr - shRNA control after normalization to housekeeping gene

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

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 sh - TFEB ECs in control treated with a specific miR - control , miR - 15a - 5p miR - 16 - 5p mimics Data are expressed as relative fold - change compared with expression in control after normalization to housekeeping gene

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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 sh - TFEB ECs in control treated with a specific miR - control , miR - 15a - 5p miR - 16 - 5p mimics Representative western blot of VEGFR2 expression under same experimental conditions previously reported . bar graph shows densitometric expressed as between VEGFR2 α -

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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 TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p miR - 16 - 5p inhibitors . Data are expressed as relative fold - change compared with expression in control after normalization to housekeeping gene

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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 TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p miR - 16 - 5p inhibitors . Representative western blot of VEGFR2 expression under same experimental conditions previously reported . bar graph shows densitometric expressed as between VEGFR2 α -

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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 .

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


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

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

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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


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

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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 intramuscular route with various doses of PiCoVacc or adjuvant only ( sham ) or placebo ( n = 4 ) . SARS - CoV - 2 - specific IgG response ( A ) neutralizing antibody titer ( B ) were measured . Points represent macaques ; dotted lines indicate limit of detection ; horizontal lines indicate 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

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

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(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 inoculated macaques at day 7 post infection . Lung tissue was collected stained with hematoxylin eosin .

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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 14 through 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 B ) Hematological in four groups of macaques ( n = 4 ) . Lymphocyte subset percents ( A ) , including CD3 + , CD4 + CD8 + were monitored at day − 1 ( 1 day before vaccination ) , 18 ( 3 day after second vaccination ) 29 ( 7 day after third vaccination ) . Key cytokines ( B ) , containing TNF - α , IFN - γ IL - 2 were examined at day − 1 , 1 ( day for first vaccination ) , 4 , 18 29 after vaccination . Values are mean ± s . d .

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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 stained with hematoxylin eosin .

(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 Smad6 MH2 domain were co - transfected into HEK293 with HA - tagged Pellino - 1N plasmid . lysates were immunoprecipitated with anti - HA antibody immunoblotted with anti - Myc antibody . Data are representative of at least three independent experiments . IP , immunoprecipitation ; IB , immunoblot ; TCL , total lysates .

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

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

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

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

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

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

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

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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 truncated mutant composed of Smad6 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) or full - Smad6 ( Myc - Smad6 ) was co - transfected with HA - tagged full - Smad4 or HA - tagged full - Pellino - 1 plasmid into HEK293 , respectively . lysates were immunoprecipitated ( IP ) with anti - Myc immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . 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 .

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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 . Il6 gene expression was analyzed by qRT - PCR .

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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 are treated with LPS for 2 h . Luciferase activity in ( B ) was normalized to β - galactosidase activity .

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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 IKKα / β phosphorylation when RAW264 . 7 are treated with LPS for 2 h .

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

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

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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.


F After pre - treating RAW264 . 7 with biotin - conjugated Smaducin - 6 ( 100 nM ) for 30 min , were treated with LPS for 2 h . Subsequent precipitation by streptavidin - agarose showed that endogenous Pellino - 1 binds to Smaducin - 6 peptide . A biotin - conjugated scrambled peptide was used as a negative control .

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F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin-agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.


G - I Immunoprecipitation assays in peritoneal macrophages show that Smaducin - 6 inhibits formation of endogenous ( G ) IRAK1 - , ( H ) RIP1 - , or ( I ) IKKε - mediated signaling complexes induced by 2 h LPS treatment , compared to a scrambled peptide . IB , immunoblot ; TCL , total lysates . Data shown are representative of three independent experiments .

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G-I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.


Subcutaneous injection of Smaducin - 6 increases survival rate of severe CLP - induced sepsis mice ( BALB / c mice ) . Different amounts of Smaducin - 6 were subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group per experiment .

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Subcutaneous injection of Smaducin-6 increases the survival rate of severe CLP-induced sepsis mice (BALB/c mice). Different amounts of Smaducin-6 were subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group per experiment.


Smaducin - 6 treatment reduces abnormal nuclei observed in tissues of CLP - induced BALB / c mice , as observed by hematoxylin eosin ( H / E ) . Data shown are representative of five independent experiments . Scale bar , 40 μm ( magnification , 400 × ) .

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Smaducin-6 treatment reduces abnormal nuclei and cell morphology observed in tissues of CLP-induced BALB/c mice, as observed by hematoxylin and eosin (H/E) staining. Data shown are representative of five independent experiments. Scale bar, 40 μm (magnification, 400×).


survival rates of severe CLP - induced sepsis mice by subcutaneously injected Smaducin - 6 were compared with those by intraperitoneally injected ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) . were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . n = 10 mice per group per experiment .

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The survival rates of severe CLP-induced sepsis mice by subcutaneously injected Smaducin-6 were compared with those by intraperitoneally injected antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin). Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. n = 10 mice per group per experiment.


Therapeutic effects of Smaducin - 6 alone , or ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) , or different amounts of Smaducin - 6 plus were observed in CLP mice . were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . Smaducin - 6 was subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group .

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Therapeutic effects of Smaducin-6 alone, or antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin), or different amounts of Smaducin-6 plus antibiotics were observed in CLP mice. Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. Smaducin-6 was subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group.


Comparison of mortality of severe CLP - induced sepsis mice upon subcutaneous injections of a total of 16 mg / kg Smaducin - 6 or TAT - S6 ( 422 - 441 ) peptide . n = 10 mice per group .

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Comparison of mortality of severe CLP-induced sepsis mice upon subcutaneous injections of a total of 16 mg/kg Smaducin-6 or TAT-S6(422-441) peptide. n = 10 mice per group.


A - I Smaducin - 6 treatment decreased of IL - 6 , TNF - α , IFN - γ , IL - 1β in blood of CLP mice ( A - D ) , but not IL - 4 , IL - 10 , TGF - β1 , IL - 12 , or IL - 17A ( E - I ) . Cytokine concentrations were analyzed by ELISA . n = 10 mice per group per experiment . Data were statistically analyzed by Dunnett ' s multiple comparison ( one - way ANOVA ) . * * * P < 0 . 001 , * * P < 0 . 005 , * P < 0 . 05 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A-I Smaducin-6 treatment decreased the concentration of IL-6, TNF-α, IFN-γ, and IL-1β in the blood of CLP mice (A-D), but not IL-4, IL-10, TGF-β1, IL-12, or IL-17A (E-I). Cytokine concentrations were analyzed by ELISA. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001, **P < 0.005, *P < 0.05 compared to sham or vehicle control (CLP + Pal-Scram #1).


J Expression of IL - 6 is reduced in spleen liver of Smaducin - 6 - treated CLP mice . Each lane represents independent mice .

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J Expression of the IL-6 protein is reduced in the spleen and liver of Smaducin-6-treated CLP mice. Each lane represents independent mice.


Subcutaneous injection of Smaducin - 6 into CLP mice reduced loads in peritoneal fluid . n = 10 mice per group per experiment . Data were statistically analyzed by Mann - Whitney U - . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Subcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann-Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


Smaducin - 6 increases neutrophil numbers in peritoneal lavage fluids from LPS - induced endotoxemia BALB / c mice . n = 5 mice per group per experiment . Data were statistically analyzed by a t - . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


CXCR2 expression in human neutrophils , measured by FACS , was decreased by LPS treatment restored by Smaducin - 6 treatment .

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CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.


Treatment with Smaducin - 6 downregulates LPS - induced GRK2 expression in RAW264 . 7 human neutrophils .

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Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.


CXCR2 - expressing neutrophils isolated from CLP mice in presence of Smaducin - 6 or Pal - Scram peptide were analyzed by FACS . n = 5 mice per group were used . Data were statistically analyzed by a t - show mean ± SD of at least three independent experiments . * * P < 0 . 005 compared to sham or vehicle controls ( CLP + PBS CLP + Pal - Scram # 1 ) .

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CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).


GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in presence of Smaducin - 6 or Pal - Scram # 1 peptide .

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GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.


A , B Quantitative of ( A ) TUNEL - positive ( B ) caspase - 3 - positive in in spleens of CLP + PBS , CLP + Pal - Scram # 1 , CLP + Smaducin - 6 mice . Three independent experiments ( n = 3 mice per group per experiments ) were performed . At least five hot spots in a section of TUNEL per experiment were selected , average was determined . data were expressed as a mean of total numbers statistically analyzed by a t - show mean ± SD of three independent experiments . * * P < 0 . 005 , * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A, B Quantitative analysis of (A) TUNEL-positive cells and (B) caspase-3-positive cells in IHC in the spleens of CLP + PBS, CLP + Pal-Scram #1, and CLP + Smaducin-6 mice. Three independent experiments (n = 3 mice per group per experiments) were performed. At least five hot spots in a section of TUNEL and IHC per experiment were selected, and average count was determined. The data were expressed as a mean percentage of total cell numbers and statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.005, ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


C ELISA showing that Smaducin - 6 treatment reduced IFN - β1 in peritoneal lavage fluids compared to Pal - Scram # 1 - treated CLP mice . n = 10 mice per group per experiment . Data were statistically analyzed by Dunnett ' s multiple comparison ( one - way ANOVA ) . * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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C ELISA showing that Smaducin-6 treatment reduced IFN-β1 levels in peritoneal lavage fluids compared to Pal-Scram #1-treated CLP mice. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


D , E IFN - β1 ( D ) TRAIL ( E ) expression detected by FACS in splenocytes is decreased in Smaducin - 6 - treated CLP mice compared to scrambled peptide - treated CLP mice . Data are representative of three independent experiments .

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D, E IFN-β1 (D) and TRAIL (E) expression detected by FACS in splenocytes is decreased in Smaducin-6-treated CLP mice compared to scrambled peptide-treated CLP mice. Data are representative of three independent experiments.


F Decreased expression of TRAIL was detected by in spleen of CLP - induced mice treated with Smaducin - 6 compared to mice treated with Pal - Scram . Scale bar , 100 μm ( magnification , 200 × ) . Data are representative of three independent experiments .

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F Decreased expression of TRAIL was detected by IHC in the spleen of CLP-induced mice treated with Smaducin-6 compared to mice treated with Pal-Scram. Scale bar, 100 μm (magnification, 200×). Data are representative of three independent experiments.


B . truncated receptor encoded by RNA1 is endogenously expressed . HEK293 were transfected with a cDNA encoding human truncated 5HT2C ( RNA1 ) . SDS - lysates from these transfected from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 ( Figure EV1 ) . Mousehypothalamus was exposed 10 times longer . In brain , a band around 75kDa of unknown origin is detected by antiserum ( indicated by a star ) .

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B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).


D . RT - PCR of serotonin 2C receptor minigene after oligonucleotide addition . Below is a of three independent experiments . A statistical evaluation showed significant differences between oligo # 5 ( p = 0 . 00008 ) oligo # 5 - 3 ( p = 0 . 0019 ) control .

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D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.


A . Titration of oligo # 5 . An increasing amount of oligo # 5 was added to HEK293 stably expressing serotonin 2C receptor reporter minigene was analyzed by RT - PCR . - RT : negative control without reverse transcriptase . B . of three independent experiments . half - maximal effect was seen at 13 . 1 nM .

A. Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT-PCR. -RT: negative control without reverse transcriptase.B. Quantification of three independent experiments. The half-maximal effect was seen at 13.1 nM concentration.


C . Influence of oligo # 5 shortening on alternative splicing of serotonin 2C receptor . Oligo # 5 : 5 ' AGU AUU GAG CAU AGC CGC 3 ' ( 18mer ) ; Oligo # 5 - 3 : 5 ' GAG CAU AGC CGC 3 ' ( 12mer ) ; Oligo # 5 - 10 : 5 ' G CAU AGC CGC 3 ' ( 10mer ) were used in transfection assays analyzed by RT - PCR . D . Statistical of effect of oligonucleotide shortening . effect of oligos testes is significant , oligo # 5 : p = 0 . 0015 ; oligo # 5 - 3 : p = 0 . 031 ; oligo # 5 - 10 : p = 0 . 048 , n = 6 .

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C. Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5' AGU AUU GAG CAU AGC CGC 3' (18mer); Oligo#5-3: 5' GAG CAU AGC CGC 3' (12mer); Oligo#5-10: 5' G CAU AGC CGC 3' (10mer) were used in transfection assays analyzed by RT-PCR.D. Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: p= 0.0015; oligo#5-3: p=0.031; oligo#5-10: p= 0.048, n=6.


E . Influence of cy3 label on 5 ' 3 ' end of oligo # 5 . Using a phosphate linker , Cy3 was added to 5 ' or 3 ' phosphate , respectively oligonucleotides assayed by RT - PCR . Oligo # 5 with cy3 at 5 ' end showed 86 % inclusion , with Cy3 at 3 ' end 17 % exon inclusion . F . Statistical of effect of oligonucleotide modification . effect is significant , p = 0 . 009 , n = 4 .

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E. Influence of cy3 label on the 5' and 3' end of oligo#5. Using a phosphate linker, Cy3 was added to the 5' or 3' phosphate, respectively and the oligonucleotides assayed by RT-PCR. Oligo#5 with cy3 at the 5' end showed 86% inclusion, with Cy3 at the 3' end 17% exon inclusion.F. Statistical analysis of the effect of oligonucleotide modification. The effect is significant, p=0.009, n=4.


D . In vitro splicing : Uniformly labeled in vitro transcribed was incubated with HeLanuclear extract ( NE ) under splicing conditions for two hours at 30 ºC . structures of in vitro splicing products are shown on right . LI LII are two lariats formed . Numbers indicate expected product lengths . E . of selected bands from four independent experiments . of each form in total products is shown . differences of splice products ( Va + b - MINX ) is significant , p = 0 . 0055 ; n = 3 .

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D. In vitro splicing assay: Uniformly labeled in vitro transcribed RNA was incubated with HeLanuclear extract (NE) under splicing conditions for two hours at 30 ºC. The structures of the in vitro splicing products are shown on the right. LI and LII are two lariats formed. Numbers indicate the expected product lengths.E. Quantification of selected bands from four independent experiments. The percentage of each form in the total products is shown. The differences of splice products (Va+b-MINX) is significant, p=0.0055; n=3.


B . Effect of oligo # 5 on pre - mRNA splicing of reporter gene , stably transfected into HeLa .

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B. Effect of oligo#5 on pre-mRNA splicing of the reporter gene, stably transfected into HeLa cells.


C . Effect of oligo # 5 on truncated 5HT2C . Stable lines expressing construct shown in panel ( A ) were treated with oligo # 3 oligo # 5 . After 48 hrs were separated in containing fractions soluble supernatant . fractions were analyzed by Western Blot using anti - RNA1 antiserum ( Figure EV1 ) . samples were on same , but supernatant fractions were exposed about ten times longer . D . Effect of oligo # 5 on full - 5HT2C . was prepared as in panel ( C ) analyzed using a polyclonal anti - GFP antiserum . E . of panels C D from three independent experiments . of signal after oligo # 3 oligo # 5 treatment is shown for RNA1 ( p = 0 . 002 ) RNA2 ( p = 0 . 0003 ) , n = 3 , respectively .

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C. Effect of oligo#5 on the truncated 5HT2C protein. Stable cell lines expressing the construct shown in panel (A) were treated with oligo#3 and oligo#5. After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant. The fractions were analyzed by Western Blot using the anti-RNA1 antiserum (Figure EV1). The protein samples were on the same membrane, but the supernatant fractions were exposed about ten times longer.D. Effect of oligo#5 on the full-length 5HT2C protein. The protein was prepared as in panel (C) and analyzed using a polyclonal anti-GFP antiserum.E. Quantification of panels C and D from three independent experiments. The ratio of protein signal after oligo#3 and oligo#5 treatment is shown for RNA1 (p=0.002) and RNA2 (p=0.0003), n=3, respectively.


F , G . Effect of oligo # 5 on localization of EGFP - tagged serotonin 2C receptor . Confocal microscopy of three representative pictures are shown . H . Quantitification of GFP signal found on after oligo # 5 addition . signal around peripheral region was determined by visual inspection calculated as a fraction of total signal using NIH image . 200 from three experiments were analyzed , p < 0 . 008 , n = 200 .

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F, G. Effect of oligo#5 on localization of the EGFP-tagged serotonin 2C receptor. Confocal microscopy images of three representative pictures are shown.H. Quantitification of GFP signal found on the membrane after oligo#5 addition. The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image. 200 cells from three individual experiments were analyzed, p<0.008, n=200.


A . Accumulation of Cy3 - labeled oligo # 5 in around 3rd ventricle one hour post injection , shown is ventral part of a coronal section - 1 . 34 mm from Bregma . injection coordinates were AP : - 1 . 82 mm , DV : - 4 . 5 mm , ML : 0 . B . Uptake of oligo # 5 in arcuate nucleus region ( arc ) , enlarged from panel A .

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A. Accumulation of Cy3-labeled oligo#5 in the area around the 3rd ventricle one hour post injection, shown is the ventral part of a coronal section -1.34 mm from Bregma. The injection coordinates were AP: -1.82 mm, DV: -4.5 mm, ML: 0.B. Uptake of oligo#5 in the arcuate nucleus region (arc), enlarged from panel A.


C . Oligo # 5 enters neurons . Enlargement of hypothalamus region taking up oligo # 5 ( red ) , stained for neuronal marker NeuN ( green ) . Most of oligo # 5 signal is in neurons , but some is also in NeuN negative ( arrow ) , likely glia . D . Oligo # 5 enters some glia . Enlargement of a hypothalamic region adjacent to 3rd ventricle one hour after oligo # 5 injection . oligo ( red ) can be found in some GFAP - positive glia ( arrow ) , but numerous GFAP - positive ( green ) show no oligo # 5 uptake .

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C. Oligo#5 enters neurons. Enlargement of hypothalamus region taking up oligo#5 (red), stained for the neuronal marker NeuN (green). Most of the oligo#5 signal is in neurons, but some is also in NeuN negative cells (arrow), likely glia.D. Oligo#5 enters some glia cells. Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo#5 injection. The oligo (red) can be found in some GFAP-positive glia cells (arrow), but numerous GFAP-positive cells (green) show no oligo#5 uptake.