Fig 2: Expression of APRIL, MCL-1 and COX-2 proteins in HT-29 and LoVo as detected by western blot analysis. APRIL, A proliferating-inducing ligand; MCL-1, myeloid cell leukemia-1; COX-2, cyclooxygenase-2; NC, negative control.

Fig 3: Protein expression from four different constitutive promoters driving long mRNA.Transfected HEK293T cells were lysed with RIPA buffer and processed for immunoblotting using antibodies to detect (a) GFP (b) c-Myc tag for Her2 CAR and (c) Mcl-1 expression with β-actin used as a loading control for the Western blots. All representative blots above are repeated three times and quantified and presented in the bar graph (right) using Image Studio Lite. Bar graph values represent the mean values ± SD from three independent repeats.

Fig 4: miR-340-5p directly targets the Mcl-1 apoptotic regulator. (a) Strategy to identify nuclear targets for miR-340-5p in diabetic cardiomyopathy. (b) Time course analysis of miR-340-5p and Mcl-1 expression in HL-1 cells cultured with high glucose (30 mM) and palmitate (0.5 mM) at each time point (0, 3, 6, and 12 hours after treatment), n = 4 at each time point. (c) Pearson correlation analyses between miR-340-5p and Mcl-1 expression were performed in heart tissues of db/db mice. Total 54 samples were measured. (d, e). Bioinformatics analysis showed that Mcl-1 was a target of miR-340-5p (d), and the luciferase activity was analyzed (e) using AC-16, HL-1, and H9c2 cells treated with either miR-340 mimic. n = 4 independent experiments. (f) AC-16, HL-1, and H9c2 cells were treated with miR-340 mimic for 24 hours, and western blot was used to measure Mcl-1 expression. n = 4 independent experiments. (g) HL-1 cells were treated with miR-340 mimic or miR-340-5p inhibitor, and qPCR was performed to detected mcl-1 mRNA expression. n = 4 independent experiments. (h, i) RNA pulldown (h) and RIP RT-PCR assay (i) were performed to confirm whether miR-340-5p directly target mcl-1 in HL-1 cells. n = 3 independent experiments. (j) Western blot analysis of Mcl-1 expression in wt and db/db mice under rAAV-random or rAAV-miR-340-5p treatment. n = 6 in each group of mice. Data were represented as the mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01. n.s.: nonsignificant.

Fig 5: (A) MCL-1 immunohistochemistry in canine normal mammary gland. (B) Benign mammary gland tumor showing low MCL-1 expression. (C, D) Malignant mammary gland tumor showing high MCL-1 expression. (E) In the positive control, MCL-1 expression was abundantly observed at Leydig cells in the mouse testis. (F) No specific staining was observed in the negative control (A, B, C, E, F original magnification ×400; D, original magnification ×1000).