Fig 1: Maf1 suppressed inflammation in the LPS-induced in vitro BBB model through inactivation of the NLRP3 inflammasome. BMECs were transfected with the control vector (NC), Maf1 or a combination of Maf1and NLRP3, followed by LPS stimulation. The BMECs were then co-cultured with astrocytes to establish an in vitro BBB model. (A) The TEER values of monolayer cells at 0, 6, 12, and 24 h were measured using a bioelectric impedance analyzer. (B) Trans-endothelial permeability assays were performed to assess in vitro BBB integrity by using the diffusion of 10 kDa FITC-dextran method. The relative permeability coefficient was determined after the Transwell experiment. (C) Cell viability was analyzed using the CCK-8 assay. (D) Quantification of cell apoptosis. (E) Western blot results for the levels of Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and GSDMD. (F) The cell-free conditioned culture medium was collected and analyzed IL-1ß and IL-18 levels via ELISA (G) Western blot analyses for pro-inflammatory cytokine (ASC, Caspase-1, IL-1ß, and IL-18) and NLRP3 expression levels. Results are expressed as the mean value ± SD of data obtained from three separate experiments. ***p < 0.001, compared with Control; # p < 0.05, ## p < 0.01 and ### p < 0.001, compared with LPS + NC; $ p < 0.05 and $$ p < 0.01 compared with LPS + Maf-1.
Fig 2: Effects of inhibiting miR-503 on protein levels involved in the PI3K/Akt and STAT3 pathways under H/R condition. (a–c)Western blotting analysis of Bcl-2, Bax, PI3K p85, p-Akt (T450), and p-Stat3 (Y705) and densitometry analysis of proteins. Data are expressed as mean ± SEM, n = 6/group. (a–c) *P < 0.05 vs. Con; $P < 0.05 vs. H/R; #P < 0.05 vs. H/R+NC. One-way ANOVA followed by Newman-Keuls multiple comparison test was applied.
Fig 3: Inhibition of miR-503 in H9c2 cells under normoxia. (a) miR-503 expression level, quantified by RT-qPCR. (b–g) Western blotting analysis of PI3K p85, p-Stat3 (Y705), p-Akt (T450), Bcl-2, and Bax and densitometry analysis of proteins. Data are expressed as mean ± SEM, n = 6/group. *P < 0.05 vs. Antagomir-Co. Student's t-test was applied. Antgomir-Co: negative control of Antagomir-503.
Fig 4: Severe mitochondrial damage and autophagy in Calr+/- mouse kidneys. (A): Representative electron micrographs for ultrastructural morphology of mitochondria from Calr+/- mouse kidney—(a,b): distal convoluted tubule cells swelling mitochondria enclosed in membrane structures, some of the mitochondria are in advanced stages of autophagy; (c–e): damaged mitochondrial enclosed in multi-membrane structure undergoing autophagy, also shown are advanced stages where the mitochondria are almost completely eliminated; (f): a podocyte with damaged vacuolated mitochondria highlighted with red asterisks in Calr+/- mouse kidneys. (B): Western blot analysis of protein extract from Calr+/+ and Calr+/- kidney tissue showed down-regulation of Bcl-2 and up-regulation of Becn-1, indicating an activation of the autophagy. (C): Immunofluorescence staining of LC3 confirmed the initiation and formation of autophagosomes. (D): Western blot analysis with antibody against LC3 confirmed the shift toward LC3-II in Calr+/- kidney, as evidenced by the ratio LC3-II/LC3-I calculation. (E): Proteomic analysis revealed an up-regulation of the Atg3, an important player in autophagy in Calr+/- kidneys. Results are given as means ± SD of the relative intensity in the case of Western blot analysis, or of the normalized spectral accounts in the case of proteomic data **: p<0.01, ***: p<0.001).
Fig 5: Restoration of PARP1 attenuates the inhibitive effect of NEAT1 knockdown on cisplatin resistance in cisplatin-resistant ovarian cancer cells. (A-D) The expression levels of PARP1 mRNA and protein were detected in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by qRT-PCR and Western blot. (E and F) Cell viability and IC50 of cisplatin (DDP) were measured in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 after treatment of cisplatin for 48 h by MTT. (G and H) Cell viability, (I and J) apoptosis and (K and L) protein levels of Bcl-2, Bax and Cleaved-casp-3 were examined in A2780/DDP and SKOV3/DDP cells transfected with si-NC, si-NEAT1 #2, si-NEAT1 #2 and vector or PARP1 by MTT, flow cytometry and Western blot, respectively. The difference was compared with the indicated control group and analyzed via ANOVA followed via Tukey post hoc test. *P<0.05.
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