Journal: bioRxiv
Article Title: Differential immune- and apoptosis-related gene signatures in pancreatic alpha and beta cells contribute to their fate in type 1 diabetes
doi: 10.1101/2025.07.26.666935
Figure Lengend Snippet: EndoC-βH1 cells were transfected with small interfering RNA (siCTRL or siMEG3). After recovery, cells were left untreated or exposed to either IL-1β (50U/ml) +IFN-γ (1000U/ml) for 48h, to IFN-α(1000U/ml) alone for 24h or to IFN-γ alone for 2h, 4h, 8h or 24h. (A) pSTAT1 and GAPDH protein expression were analyzed by immunoblot after silencing MEG3 and exposing to the cytokines detailed in the figure. (B) pSTAT1 bands were quantified by densitometry and normalized to GAPDH. (C) Representative blots of pSTAT1 and GAPDH protein expression after silencing MEG3 and exposing to the cytokines detailed in the figure. (D-E) pSTAT1, tSTAT1 and GAPDH protein expression were analyzed by immunoblot after silencing MEG3 and exposing to IFN-γ at different time points. P-STAT1 bands were quantified by densitometry and normalized to tSTAT1. (F) The AUC from the figure shown in (E) . (G) CXCL10 mRNA expression assessed by RT-qPCR, normalized to the geometric mean of ACTIN and VAPA and presented as fold change compared with siCTRL cytokine-treated cells. (H) CXCL10 secretion in the supernatant was measured by ELISA. Results are means ± SEM. Each point represents an independent experiment. *p<0.05, ****p<0.0001 vs siCTRL, ###p<0.001, ####p<0.0001 vs untreated one-way ANOVA, Bonferroni correction. P-STAT1: phosphorylated STAT1, tSTAT1: total STAT1, AUC: Area under the curve, SEM: Standard Error of the Mean, Unt.: untreated.
Article Snippet: The nitrocellulose membranes were probed using specific primary antibodies: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167, Stat1 (9H2) Mouse mAb #9176 or Human/Mouse/Rat GAPDH Antibody R&D Systems # 2275-PC-100 diluted 1:1000 in TBST (TBS, 0.1% Tween 20) with 5% BSA (GAPDH was diluted 1:10 000).
Techniques: Transfection, Small Interfering RNA, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay
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