Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: Oncology reports

Article Title: Downregulation of PDCD4 induced by progesterone is mediated by the PI3K/AKT signaling pathway in human endometrial cancer cells.

doi: 10.3892/or.2019.7202

Figure Lengend Snippet: Figure 1. Effect of estrogen on the expression of PDCD4 in human endometrial cancer cell lines. The results of qPCR showed that 17β‑estradiol (E2) had no effect on the expression of PDCD4 mRNA in (A) Ishikawa (P>0.05) and (B) HEC‑1‑A cells (P>0.05). (C and D) The level of PDCD4 protein was detected by western blot analysis after treatment with 17β‑estradiol (E2) in Ishikawa cells and no significant difference in the expression of PDCD4 protein was noted (P>0.05). (E and F) The level of PDCD4 protein was detected by western blot analysis after treated with 17β‑estradiol (E2) in HEC‑1‑A cells and no significant difference in the expression of PDCD4 protein was noted (P>0.05). PDCD4, programmed cell death 4.

Article Snippet: In addition, the cells were respectively pretreated with dimethyl sulfoxide (DMSO) and 5 μM (18) of PI3K inhibitor LY294002 (Selleckchem) for 1 h or 100 nM (19) of AKT inhibitor MK2206 (MedChem Express) for 2 h, and then the cells were treated with 10 μM of progesterone for 4 h. All treated cells were collected to detect the expression of PDCD4 protein and signaling molecules.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Downregulation of PDCD4 induced by progesterone is mediated by the PI3K/AKT signaling pathway in human endometrial cancer cells.

doi: 10.3892/or.2019.7202

Figure Lengend Snippet: Figure 2. Effect of progesterone on the expression of PDCD4 in human endometrial cancer cell lines. The results of qPCR showed that progesterone had no any effect on the expression of PDCD4 mRNA in (A) Ishikawa (P>0.05) and (B) HEC‑1‑A cells (P>0.05). (C) The expression of PDCD4 protein was detected by western blot analysis after treatment with progesterone (P4) in Ishikawa cells and (D) expression of PDCD4 protein was significantly lower after treatment with different concentrations (0, 0.01, 0.1, 1, 10 and 20 µM) of progesterone in Ishikawa cells (****P<0.0001 vs. 0 µM). (E) The expression of PDCD4 protein was detected by western blot analysis after treatment with progesterone (P4) in HEC‑1‑A cells and (F) expression of PDCD4 protein was significantly lower after treatment with 10 and 20 µM of progesterone in HEC‑1‑A cells (*P<0.05 vs. 0 µM). PDCD4, programmed cell death 4.

Article Snippet: In addition, the cells were respectively pretreated with dimethyl sulfoxide (DMSO) and 5 μM (18) of PI3K inhibitor LY294002 (Selleckchem) for 1 h or 100 nM (19) of AKT inhibitor MK2206 (MedChem Express) for 2 h, and then the cells were treated with 10 μM of progesterone for 4 h. All treated cells were collected to detect the expression of PDCD4 protein and signaling molecules.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Downregulation of PDCD4 induced by progesterone is mediated by the PI3K/AKT signaling pathway in human endometrial cancer cells.

doi: 10.3892/or.2019.7202

Figure Lengend Snippet: Figure 3. Effect of progesterone on the expression of PDCD4 protein at different time points. (A) The expression of PDCD4 protein was detected by western blot analysis in Ishikawa cells after treatment with 10 µM of progesterone at different time points (0, 6, 12, 24, 36 and 48 h). (B) The expression of PDCD4 protein began to decrease after progesterone treatment for 6 h in Ishikawa cells (*P<0.05 vs. 0 h; **P<0.01 vs. 0 h; ***P<0.01 vs. 0 h). (C) The expression of PDCD4 protein was detected by western blot analysis in HEC‑1‑A cells after treatment with 10 µM of progesterone at different time points (0, 6, 12, 24, 36 and 48 h). (D) The expression of PDCD4 protein started to decrease after progesterone treatment for 12 h in HEC‑1‑A cells (*P<0.05 vs. 0 h; **P<0.01 vs. 0 h; ns, not significant). PDCD4, programmed cell death 4.

Article Snippet: In addition, the cells were respectively pretreated with dimethyl sulfoxide (DMSO) and 5 μM (18) of PI3K inhibitor LY294002 (Selleckchem) for 1 h or 100 nM (19) of AKT inhibitor MK2206 (MedChem Express) for 2 h, and then the cells were treated with 10 μM of progesterone for 4 h. All treated cells were collected to detect the expression of PDCD4 protein and signaling molecules.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Downregulation of PDCD4 induced by progesterone is mediated by the PI3K/AKT signaling pathway in human endometrial cancer cells.

doi: 10.3892/or.2019.7202

Figure Lengend Snippet: Figure 5. The PI3K/AKT pathway participates in the downregulation of PDCD4 protein expression induced by progesterone. (A) The effect of LY294002 (PI3K inhibitor) on the expression of phosphorylated (p)‑mTOR, mTOR, p‑AKT, AKT, PDCD4 and β‑actin in Ishikawa and HEC‑1‑A cells. Cells were pre‑treated with 5 µM of LY294002 for 2 h, and then treated with 10 µM of progesterone (P4) for 4 h, and the expression of p‑mTOR, mTOR, p‑AKT, AKT, PDCD4 and β‑actin was detected by western blot analysis. (B) Quantitative analysis of the ratios of p‑mTOR/mTOR, p‑AKT/AKT and PDCD4/β‑actin by measuring the relative band density in (B) Ishikawa and (C) HEC‑1‑A cells. (D) The effect of MK2206 (AKT inhibitor) on the expression of p‑mTOR, mTOR, p‑AKT, AKT, PDCD4 and β‑actin in Ishikawa and HEC‑1‑A cells. Cells were pre‑treated with 100 nM of MK2206 for 2 h, and then treated with 10 µM of progesterone (P4) for 4 h, and the expression of p‑mTOR, mTOR, p‑AKT, AKT, PDCD4 and β‑actin was detected by western blot analysis. (E) Quantitative analysis of the ratios of p‑mTOR/mTOR, p‑AKT/AKT and PDCD4/β‑actin by measuring the relative band density in (E) Ishikawa and (F) HEC‑1‑A cells. *P<0.05 vs. the control group (P4 only); **P<0.01 vs. the control group (P4 only); ns, not significant; P4, progesterone.

Article Snippet: In addition, the cells were respectively pretreated with dimethyl sulfoxide (DMSO) and 5 μM (18) of PI3K inhibitor LY294002 (Selleckchem) for 1 h or 100 nM (19) of AKT inhibitor MK2206 (MedChem Express) for 2 h, and then the cells were treated with 10 μM of progesterone for 4 h. All treated cells were collected to detect the expression of PDCD4 protein and signaling molecules.

Techniques: Expressing, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control