Fig 1: BRD4 inhibition restores impaired autophagic influx in vivo via promoting autophagosome-lysosome fusion and lysosomal degradation. (A) Immunoblot analysis for LC3 and p62 in the pancreas from CER (upper) or ethanol plus POA (lower). (B) Immunoblot analysis for ATG14 and STX17 in the pancreas from CER (upper) or ethanol plus POA (lower). (C) Immunoblot analysis for LAMP2 in the pancreas from CER (upper) or ethanol plus POA (lower). Data represent the mean values ± SEM (n = 3). Statistical analysis was performed by Student’s un-paired, two-tailed t-test between two groups, *P < 0.05, compared to the control; #P < 0.05, compared to AP group.
Fig 2: Co-localization changes of LC3, LAMP1, and AD-like pathological proteins after the Stx17 overexpression. (a) Co-localization of LC3 and Bace-1 after the Stx17 overexpression. (b) Co-localization of LC3 and Aß1-42 after the Stx17 overexpression. (c) Co-localization of LAMP1 and Bace-1 after the Stx17 overexpression. (d) Co-localization of LAMP1 and Aß1-42 after the Stx17 overexpression. (e) Co-localization of LC3 and p-S396tau after the Stx17 overexpression. (f) Co-localization of LAMP1 and pS396-tau after the Stx17 overexpression. Scale bar: 5 µm.(g) Co-localization of LC3-Bace-1, LC3-Aß1-42, LAMP1-Bace-1, LAMP1-Aß1-42, LC3-pS396-tau, and LAMP1-pS396-tau was quantified and expressed as Pearson coefficient value (*p < 0.05,**p < 0.01, and ***p < 0.001 compared with the control group; #p < 0.05 and ##p < 0.01 compared to the Meth group).
Fig 3: Stx17 overexpression recovered the Meth-induced autophagosome-late endosome/lysosome fusion deficiency. (a) Co-localization of LC3 and Stx17. (b) Co-localization of LC3 and Rab5. (c) pHBAd-EF1-Stx17-3 flag (MOI = 40, 36 h) or empty adenovirus vector (Ad) was overexpressed in primary hippocampal neurons. (d) Co-localization of LC3 and Rab7 after the Stx17 overexpression. (e) Co-localization of LAMP1 and Rab7 after the Stx17 overexpression. (f) Co-localization of LC3 and LAMP1 after the Stx17 overexpression. Scale bar: 5 μm. (g) Co-localization of LC3-Rab7, LAMP1-Rab7, and LC3-LAMP1 was quantified and expressed as Pearson coefficient value (∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the control group and ###p < 0.001 compared to the Meth group).
Fig 4: BRD4 inhibition upregulated SIRT1 and inhibition of SIRT1 reversed the effects of BRD4 inhibition on autophagic flux. (A) mRNA level of Sirt1 at 4 h after CCK stimulation in isolated pancreatic acinar cells (n = 3). (B) Immunoblot analysis of SIRT1 level at 4 h after CCK stimulation (n = 5). (C) Immunoblot analysis for LC3B and p62 expression in isolated pancreatic acinar cells pretreated with 500 nmol·L-1 JQ1 or 10 μmol·L-1 EX527 followed by stimulation with 200 nmol·L-1 CCK for 4 h (n = 4). (D) Immunoblot analysis for ATG14, STX17, and LAMP2 expression (n = 5). (E) Immunoblot analysis for cathepsin B (n = 3) and cathepsin L expression (n = 5). (F) The activities of cathepsin B (n = 3) and cathepsin L (n = 3). Data represent the mean values ± SEM. Statistical analysis was performed by Student’s un-paired, two-tailed t-test between two groups, *P < 0.05, compared to the control; #P < 0.05, compared to CCK-stimulated group; +P < 0.05, compared to JQ1-treated group.
Fig 5: Effect of Meth on LAMP1, CTSL, CTSD, Stx17, Rab5, and Rab7 expression. (a) Western blot analysis of the LAMP1, CTSL, CTSD, and Stx17 expression in primary hippocampal neurons. (c) Western blot analysis of the Rab5 and Rab7 expression in primary hippocampal neurons. (b, d) β-actin levels were assessed and compared with the control group (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 compared with the control group). (e) Immunofluorescence analysis of Rab5 expression. (f) Immunofluorescence analysis of Rab7 expression. Scale bar: 5 μm. (g, h) Quantification of the mean fluorescence intensity (MFI) in the confocal images showed the Rab5 and Rab7 levels (∗p < 0.05 compared with the control group).
Supplier Page from Abcam for Anti-STX17 antibody