Fig 2: JUP is colocalized with Bcl-XL in cells after exposure to TcdB. (A and B) Caco-2 cells were either untreated (w/o toxin) or treated with 1 nM TcdB for 4 h and 8 h. Cells were costained for JUP and Bcl-XL (A) or Bax (B). Representative images are from one of three independent experiments. All scale bars, 10 µm.

Fig 3: Knockdown of JUP and HMGB1 rescues TcdB-induced cell death. (A and B) Caco-2 cells were transfected with siNC or siRNAs targeting JUP (A) or HMGB1 (B) for 48 h and then treated with a serial concentration of TcdB for 18 h. Caspase 3/7 activation and cell viability were assayed. JUP and HMGB1 mRNA or protein levels were determined by quantitative RT-PCR (qPCR) or Western blotting (WB), respectively. GAPDH served as a loading control for WB. (C and D) The JUP-/- (C) and HMGB1-/- (D) knockout Caco-2 cells generated by CRISPR technology were treated with a serial concentration of TcdB for 18 h. Caspase 3/7 activation and cell viability were assayed. JUP and HMGB1 protein levels were determined by WB. All results are representative of at least three independent experiments performed in triplicate. Error bars represent the standard error of the mean (SEM). Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4: Generation of Cdk13tm1a mice. (A) Scheme of Cdk13tm1a allele. The Cdk13tm1a allele consists of strong splicing acceptor (SA), ß-galactosidase gene (lacZ) and neomycine resistance gene (neo) both with poly A sites (pA), being surrounded by two FRT sites within intron 2, having two and one lox P sites within intron 2 and 4. (B) PCR genotyping from E12.5 embryos using specific primers distinguishing Cdk13tm1a, mutant (KO, upper bands) and Cdk13+, wild-type, allele (WT, lower bands). (C) Brain extracts were prepared from E14.5 embryos of Cdk13+/+, Cdk13tm1a/+ and Cdk13tm1a/tm1a mice and protein levels of Ser2 and Ser5 of RNAPII, CDK13 and ?-catenin (CTNNG) were evaluated by Western blotting. The short and long expositions for CDK13 are presented to demonstrate residual expression of CDK13 in Cdk13tm1a/tm1a embryos.

Fig 5: JUP silencing protects cells against TcdB-induced cell damage. (A) Caco-2 cells were transfected with two siRNAs targeting JUP (siJUP-1 and siJUP-2) or siNC for 48 h and then were treated with a serial concentration of TcdB for 18 h. Representative images from one of two independent experiments performed in triplicate show the cell morphology. Scale bar, 50 µm. (B) Cells were treated the same as in panel A but were stained with propidium iodide (PI) after 18 h of incubation with TcdB. Representative images from one of two independent experiments performed in triplicate show PI-stained dead cells. Scale bar, 50 µm. The percentage of PI-stained cells was analyzed with the HCS platform. Data are presented as the mean ± SEM. Asterisks indicate statistically significant differences between the experimental sample and the control group (siNC) at the same concentration of TcdB. Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) The expression levels of JUP and HMGB1 were examined by immunoblot analysis of cell lysates from three different cell lines. GAPDH was used as the loading control.