Fig 1: miR-100 increases IL-1ra secretion through stat5a-mediated transcriptional regulation.a Supernatants of RAW264.7 cells transfected with miR-100 mimic and nc mimic were collected and secreted cytokines were measured by the mouse cytokine array panel. b IL-1ra mRNA expression was measured by qPCR in miR-100 mimic transfected RAW264.7 cells. c IL-1ra+ cells percentage in PMs transfected with miR-100 mimic were measured by flow cytometry. d IL-1ra expression in miR-100 antagomir-treated 4T1 mouse breast cancer tissues was detected by ELISA. e, f qPCR and Western blot to detect IL-1ra expression in RAW264.7 cells transfected with siRNAs targeting several candidate TFs. g Western blot to detect Stat5a and Stat5b expression in RAW264.7 cells transfected with miR-100 mimic or treated with rapamycin. h Stat5a binding sites were predicted by ALLGEN database in IL-1ra promoter region from −3k to +1k. i Three stat5a binding sites were validated by qPCR in genomic DNA extracted from RAW264.7 cells. Data were shown as mean ± s.e.m. from three independent experiments; **P < 0.01, *P < 0.05
Fig 2: Pimozide sensitizes DOX-resistant cells to DOX by suppressing STAT5a. (A) Survival rate of MCF/DOX cells after treatment with pimozide for 48 h, with the IC50 calculated to be 14.79 μM. (B, C) Expression of p-STAT5 (Try 694), STAT5a and ABCB1 in MCF7/DOX cells after treatment with 0, 1, 2, or 5 μM DOX for 48 h (B) or with 5 μM DOX for 0, 12, 24, 48 or 72 h (C). (D) Survival rate of MCF/DOX cells after treatment with a combination of 0, 1, 2 or 5 μM pimozide and the indicated concentration of DOX for 48 h. (E, F) Apoptosis rate of MCF7/DOX cells after the indicated treatment assessed by flow cytometry (E); bar graphs showing the percentage of apoptotic cells (F). (G) Expression of apoptosis markers in MCF7/DOX cells given the indicated treatments determined by Western blotting. (H, I) Accumulation of DOX in MCF7/DOX cells after treatment with DOX or a combination of DOX and pimozide (H) and quantification (I). (J, K) Western blotting was performed to determine the expression of STAT5a, ABCB1, cleaved PARP, cleaved caspase 7 and cleaved caspase 3 in MCF7/DOX cells treated with DOX and transfected with the indicated vectors. ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: The lncRNA KLF3-AS1 segregates miR-23c to promote STAT5B expression. The putative miR-23c binding sites in the 3′UTR of (A) KLF3-AS1 or (B) STAT5B. A luciferase reporter assay was employed to analyze the binding relationship between miR-23c and (C) KLF3-AS1 or (D) STAT5B. (E) The binding relationship between KLF3-AS1 and mmiR-23c was validated with a RIP assay. (F,G) The expression of the STAT5B protein in MSCs was determined using Western blotting. (H) RT-qPCR analysis was used to examine the expression of the STAT5B mRNA in MSCs. **P < 0.01 and ***P < 0.001.
Fig 4: The exosomal lncRNA KLF3-AS1 induces IGF-1 secretion from MSCs to inhibit myocardial injury in vivo. (A) Echocardiography was used to assess the cardiac function, such as LVEF, LVFS, LVSP, and LVEDP. (B) The infarct area of I/R-injured rats was detected by performing TTC staining. (C) The expression of STAT5B in I/R-injured rats was measured using Western blotting. (D) RT-qPCR analysis was implemented to measure the expression level of the IGF-1 mRNA in I/R-injured rats. (E) HE staining and TUNEL assays were performed to estimate the degree of myocardial tissue damage. The apoptosis cells were indicated by red arrows (Scale bar = 100 μm). For the TUNEL assay, apoptotic cells are indicated by red arrows. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: MKL-1 and STAT5b are high expressed in Treg cells. a QPCR analysis of MKL-1 and STAT5b mRNA level in PBMC, CD3+T cells and Treg cells. GAPDH is the loading control. **p < 0.01, *p < 0.05. n = 3; b Western blot analysis of MKL-1 and STAT5b expression in PBMC, CD3+T cells and Treg cells. Data were quantified using Quantity One software. GAPDH is the loading control. **p < 0.01, *p < 0.05. n = 3
Supplier Page from Abcam for Anti-STAT5b antibody [EPR16671]