Journal: International journal of oncology

Article Title: H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

doi: 10.3892/ijo.2020.4967

Figure Lengend Snippet: Figure 1. Upregulation of H19 and Lin28 indicates poor survival in BC. BC samples were divided into two groups based on clinical progression. (A) H19 and (B) Lin28 levels in the LN metastasis group (n=23) were significantly higher than those in the non‑metastasis group (n=20). (C) Linear regression analysis indicated an in vivo positive relationship between the expression levels of H19 and Lin28 in a statistically significant manner. Pearson's correlation coefficient, P‑value and sample size are indicated in the top left of the plot. Total RNA was subjected to RT‑qPCR analysis; the Cq values were normalized to Tubulin in each sample. Kaplan‑Meier survival curves of (D) H19 (P=0.0317) and (E) Lin28 (P=0.0128) were plotted using log‑rank tests for patients with BC in the bc‑GenExMiner v4.2 database. (F) Correlation map corresponding to the association between H19 and Lin28 expression was plotted for patients with BC in the bc‑GenExMiner v4.2 database. The numbers represent Pearson's correlation coefficient. (G) H19 and (H) Lin28 levels were evaluated via RT‑qPCR in four BC cell lines, and normal human MCF‑10A breast cells were used as a control. *P<0.05 vs. MCF‑10A. (I) Western blot analysis of Lin28 expression in four breast cancer cell lines. (J) RT‑qPCR analysis of the RNA expression levels of Lin28 and H19 in T‑47D cells transfected with siLin28. (K) Western blot analysis of Lin28 expression in SK‑BR‑3 cells S1 and S24. (L) RT‑qPCR analysis of the RNA expression levels of Lin28 and H19 in SK‑BR‑3 cells S1 and S24; mock was used as a control. (M) Western blot analysis of Lin28 expression in T‑47D cells transfected with siH19. (N) RT‑qPCR analysis of the RNA expres sion levels of H19 and Lin28 in T‑47D cells transfected with siH19. (O) Western blot analysis of Lin28 expression in MDA‑231 cells transfected with pH19. (P) RT‑qPCR analysis of the RNA expression levels of H19 and Lin28 in MDA‑231 cells transfected with pH19. Results represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. Control or Mock. BC, breast cancer; LN, lymph node; RT‑qPCR, reverse transcription‑quantitative PCR; Cq, quantification cycle; HR, hazard ratio; CI, confidence interval; si, small interfering RNA; NC, negative control; S1/S24, SK‑BR‑3 clones infected with a Lin28 overexpression lentivirus; pH19, pFlag‑CMV‑H19.

Article Snippet: Third-generation Lin28 lentiviral vectors were generated with the LV-LIN28A plasmid (Addgene, Inc.) and vesicular stomatitis virus G (Addgene, Inc.), and then transiently transfected into 2.5x105 293T (ATCC) cells at a ratio of 6:1 using Lipofectamine® 3000 transfection reagent (cat. no. L3000001; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol; viral supernatants were collected 36 h later and filtered through a 0.45‐μm filter.

Techniques: In Vivo, Expressing, Control, Western Blot, RNA Expression, Transfection, Small Interfering RNA, Negative Control, Clone Assay, Infection, Over Expression

Journal: International journal of oncology

Article Title: H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

doi: 10.3892/ijo.2020.4967

Figure Lengend Snippet: Figure 2. Reciprocal inhibition of the H19/let‑7/Lin28 loop and autophagy in breast cancer cells. (A) Western blot analysis of autophagy marker expression in MDA‑231 cells transfected with pH19 and T‑47D cells transfected with siH19. (B) Measurement of autophagy flux in MDA‑231 cells co‑transfected with p‑mCherry‑C1‑EGFP‑hLC3B and pH19, and T‑47D cells co‑transfected with p‑mCherry‑C1‑EGFP‑hLC3B and siH19. Scale bar, 20 µm. (C) Western blot analysis of autophagy marker expression in MDA‑231 cells transfected with Lin28 and T‑47D cells transfected with siLin28. (D) RT‑qPCR analysis of the RNA expression levels of H19 and Lin28 in T‑47D cells transfected with siH19 and let‑7 inhibitor. *P<0.05, ***P<0.001 vs. siNC + let‑7 inhibitor NC unless otherwise indicated. (E) Western blot analysis of Lin28 expression in T‑47D cells transfected with siH19 and let‑7 inhibitor. (F) Western blot analysis of autophagy markers expression in T‑47D cells transfected with siH19 and let‑7 inhibitor. (G) Western blot analysis of autophagy markers expression in T‑47D cells transfected with siLin28 and let‑7 inhibitor. RT‑qPCR analysis of the RNA expression levels of H19, let‑7 and Lin28 in MDA‑231 cells incubated with (H) 50 µM CQ or (I) 200 nM Rapa. RT‑qPCR analysis of the RNA expression levels of H19, let‑7 and Lin28 in T‑47D cells incubated with (J) 50 µM CQ or (K) 200 nM Rapa. Western blot analysis of Lin28 in T‑47D cells incubated with (L) 50 µM CQ or (M) 200 nM Rapa. (N) RT‑qPCR analysis of the RNA expression levels of H19 and Lin28 in T‑47D cells incubated with 200 nM Rapa and let‑7 inhibitor. (O) Western blot analysis of Lin28 in T‑47D cells incubated with 200 nM Rapa and let‑7 inhibitor. Results represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01. si, small interfering RNA; pH19, pFlag‑CMV‑H19; RT‑qPCR, reverse transcription‑quantitative PCR; LC3, light chain 3; CQ, chloroquine; Rapa, rapamycin; NC, negative control; n.s., not significant.

Article Snippet: Third-generation Lin28 lentiviral vectors were generated with the LV-LIN28A plasmid (Addgene, Inc.) and vesicular stomatitis virus G (Addgene, Inc.), and then transiently transfected into 2.5x105 293T (ATCC) cells at a ratio of 6:1 using Lipofectamine® 3000 transfection reagent (cat. no. L3000001; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol; viral supernatants were collected 36 h later and filtered through a 0.45‐μm filter.

Techniques: Inhibition, Western Blot, Marker, Expressing, Transfection, RNA Expression, Incubation, Small Interfering RNA, Negative Control

Journal: International journal of oncology

Article Title: H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

doi: 10.3892/ijo.2020.4967

Figure Lengend Snippet: Figure 3. H19 and Lin28 promote migration and invasion of breast cancer cells. (A) H19 overexpression was detected via RT‑qPCR. (B) MDA‑231 cells were transfected with pH19, and cell migration and invasion were evaluated by Transwell assays. Quantification of (C) migration and (D) invasion rates of the control and pH19 groups. (E) H19 knockdown was detected by RT‑qPCR. (F) T‑47D cells were transfected with siH19, and cell migration and invasion were evaluated by Transwell assays. Quantification of (G) migration and (H) invasion rates of siNC and siH19 groups. (I) Western blot analysis of Lin28 expression in S24 SK‑BR‑3 cells. Mock was used as a control. (J) Transwell assays were performed to investigate changes in cell migration and invasion in Mock and S24 cells. Quantification of (K) migration and (L) invasion rates of mock and S24 groups. (M) Lin28 knockdown was detected by western blot analysis. (N) T‑47D cells were transfected with siLin28, and cell migration and invasion were evaluated by Transwell assays. Quantification of (O) migration and (P) invasion rates of siCon and siLin28 groups. Scale bar=50 µm. Results represent the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. the corresponding control group. si, small interfering RNA; pH19, pFlag‑CMV‑H19; RT‑qPCR, reverse transcription‑quantitative PCR; NC, negative control; S24, SK‑BR‑3 clone infected with a Lin28 overexpression lentivirus.

Article Snippet: Third-generation Lin28 lentiviral vectors were generated with the LV-LIN28A plasmid (Addgene, Inc.) and vesicular stomatitis virus G (Addgene, Inc.), and then transiently transfected into 2.5x105 293T (ATCC) cells at a ratio of 6:1 using Lipofectamine® 3000 transfection reagent (cat. no. L3000001; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol; viral supernatants were collected 36 h later and filtered through a 0.45‐μm filter.

Techniques: Migration, Over Expression, Transfection, Control, Knockdown, Western Blot, Expressing, Small Interfering RNA, Negative Control, Infection

Journal: International journal of oncology

Article Title: H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

doi: 10.3892/ijo.2020.4967

Figure Lengend Snippet: Figure 4. H19/let‑7/Lin28 loop regulates the inhibitory effects of autophagy on the EMT of breast cancer cells. (A) T‑47D cells were incubated with 200 nM Rapa and 50 µM CQ, and cell migration and invasion were evaluated by Transwell assays. Scale bar=50 µm. (B) Quantification of migration and invasion of T‑47D cells incubated with 200 nM Rapa and 50 µM CQ. (C) Western blot analysis of EMT markers. (D) Reverse transcription‑quantitative PCR analysis of EMT‑inducing transcription factors in T‑47D cells incubated with 200 nM Rapa and 50 µM CQ. (E) Western blot analysis of autophagy and EMT markers in T‑47D cells treated with 200 nM Rapa and pH19. (F) T‑47D cells were treated with 200 nM Rapa and pH19, and cell migration and invasion were evaluated by Transwell assays. Scale bar=50 µm. Quantification of (G) migration and (H) invasion rates of T‑47D cells incubated with 200 nM Rapa and pH19. Results represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. Con unless otherwise indicated. pH19, pFlag‑CMV‑H19; Con, control; CQ, chloroquine; Rapa, rapamycin; EMT, epithelial‑mesenchymal transition; ZEB1, zinc finger E‑box binding homeobox 1; HMGA2, high‑mobility group AT‑hook 2; LC3, light chain 3.

Article Snippet: Third-generation Lin28 lentiviral vectors were generated with the LV-LIN28A plasmid (Addgene, Inc.) and vesicular stomatitis virus G (Addgene, Inc.), and then transiently transfected into 2.5x105 293T (ATCC) cells at a ratio of 6:1 using Lipofectamine® 3000 transfection reagent (cat. no. L3000001; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol; viral supernatants were collected 36 h later and filtered through a 0.45‐μm filter.

Techniques: Incubation, Migration, Western Blot, Control, Binding Assay

Journal: International journal of oncology

Article Title: H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

doi: 10.3892/ijo.2020.4967

Figure Lengend Snippet: Figure 5. Model of H19/let‑7/Lin28 loop inhibiting autophagy and promoting EMT in breast cancer. A proposed regulatory mechanism of the H19/let‑7/Lin28 loop inhibiting autophagy and promoting EMT. Black arrows indicate promoting effects; black hammerheads indicate inhibitory effects. Solid lines indicate effects reported in the present study; dotted lines indicate effects reported in previous studies. Red arrows pointing upwards indicate upregulation; red arrows pointing downwards indicate downregulation. LncRNA, long non‑coding RNA; EMT, epithelial‑mesenchymal transition; TF, transcription factor; ZEB1, zinc finger E‑box binding homeobox 1; HMGA2, high‑mobility group AT‑hook 2.

Article Snippet: Third-generation Lin28 lentiviral vectors were generated with the LV-LIN28A plasmid (Addgene, Inc.) and vesicular stomatitis virus G (Addgene, Inc.), and then transiently transfected into 2.5x105 293T (ATCC) cells at a ratio of 6:1 using Lipofectamine® 3000 transfection reagent (cat. no. L3000001; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol; viral supernatants were collected 36 h later and filtered through a 0.45‐μm filter.

Techniques: Binding Assay

Journal: Journal of Cardiothoracic Surgery

Article Title: Pulsatile arterial blood pressure mimicking aortic valve opening during continuous-flow LVAD support: a case report

doi: 10.1186/s13019-019-1039-z

Figure Lengend Snippet: Invasive arterial blood pressure tracing and LVAD flushing cycle. Timely coincidence of a transient arterial blood pressure reduction, followed by a brief increase, as assessed by invasive radial blood pressure monitoring ( a ), with the LVAD flushing cycle (scheme, rpm = rotations per minute) ( b ). Importantly, the flushing cycle transforms into a detectable change in peripheral blood pressure in presence of pulsatile blood flow, indicating left ventricular ejection through the apical inflow cannula and, thus, closure of the aortic valve during the complete cardiac cycle. LVAD = left ventricular assist device

Article Snippet: For pre-existent mild aortic regurgitation a bioprosthetic AV (Edwards Perimount Magna 3000) was implanted along with a third-generation, continuous-flow LVAD (HeartWare, Framingham, MA).

Techniques:

Journal: Cell Death & Disease

Article Title: Androgen receptor-dependent DRAM1 activation drives autophagic resistance to BRAF inhibitors in BRAFV600-mutant melanoma

doi: 10.1038/s41419-026-08547-x

Figure Lengend Snippet: A AR, LC3II and p62 protein level in A375 and WM793 cells with or without AR overexpression. B TEM analysis of autophagosomes and autolysosomes in A375 EV and A375 AR-OE cells (scale bars: 0.5 μm). A autophagosomes, AL autolysosomes, M: mitochondria and N nucleus. Manually counted. Mean ± SEM, n = 30, two-tailed Student’s t test, ** p < 0.01. C A375 EV and A375 AR-OE cells stably expressing TF-LC3 were imaged using fluorescence microscopy (Scale bars: 10 μm). White boxed regions in the panels are enlarged. Representative images of autolysosomes (red dots) and autophagosomes (yellow dots) are shown. Manually counted. n = 30, two tailed Student’s t test, ** p < 0.01, *** p < 0.001. Following treatment with DAB (0.5 μM) and/or 3-MA (2.5 mM), A375 EV and A375 AR OE cells were analyzed for D protein expression, E cell proliferation and F colony formation assays as indicated. WM793R cells transfected with si-AR or si-scramble were treated with DAB (0.5 μM) and/or Rapa (5 nM), G protein expression, H cell proliferation and I colony formation assays were measured as indicated. Mean ± SEM, n = 3, one-way ANOVA and Tukey’s post-hoc test. ** p < 0.01. For E , H EV(DAB) vs AR OE(DAB) ** p = 0.0018, AR OE(DAB) vs AR OE(DAB + 3MA) ** p = 0.0052, si-scramble (DAB) vs si-AR(DAB) ** p = 0.0037, si-AR(DAB) vs si-AR(DAB+Rapa) ** p = 0.0014.

Article Snippet: AR overexpression (Addgene # 213819) and TF-LC3 (EGFP-RFP-LC3, Addgene #22418) lentiviral vectors were packaged in HEK293T cells using third-generation plasmids (psPAX2/pMD2.G; Addgene #12260, #12259) via Lipofectamine 3000 (Invitrogen)-mediated transfection [ ].

Techniques: Over Expression, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Microscopy, Transfection

Journal: Cell Death & Disease

Article Title: Androgen receptor-dependent DRAM1 activation drives autophagic resistance to BRAF inhibitors in BRAFV600-mutant melanoma

doi: 10.1038/s41419-026-08547-x

Figure Lengend Snippet: DRAM1, LC3 and p62 protein expression were measured in A A375 and WM793 cells transfected with Flag-DRAM1, B A375R and WM793R cells transfected with si-DRAM1 or si-scramble C A375 and WM793 AR-OE cells transfected with si-DRAM1. D A375 cells expressing TF-LC3, AR-OE-TF-LC3 and AR-OE-TF-LC3 cells transfected with si-DRAM1 were imaged by fluorescence microscopy (Scale bars: 10 μm). White boxed regions in the panels are enlarged. Representative images of autolysosomes (red dots) and autophagosomes (yellow dots) are shown. Manually counted. Mean ± SEM, n = 30, two tailed Student’s t test, ** p < 0.01, *** p < 0.001. E , F cell proliferation and colony formation assays were measured in cells from ( D ) treated with DAB as indicated. Mean ± SEM, n = 3, one-way ANOVA and Tukey’s post-hoc test. ** p < 0.01. G , H WM793R cells transfected with/without si-DRAM1 were incubated with DAB (0.5 μM), and cell proliferation and colony formation assays were measured. Mean ± SEM, n = 3, two-tailed Student’s t test, ** p < 0.01. E EV vs AR-OE ** p = 0.0017, AR-OE + si-scramble vs AR-OE+si-DRAM1 ** p = 0.0032, G DAB + si-scramble vs DAB + si-DRAM1 ** p = 0.0047.

Article Snippet: AR overexpression (Addgene # 213819) and TF-LC3 (EGFP-RFP-LC3, Addgene #22418) lentiviral vectors were packaged in HEK293T cells using third-generation plasmids (psPAX2/pMD2.G; Addgene #12260, #12259) via Lipofectamine 3000 (Invitrogen)-mediated transfection [ ].

Techniques: Expressing, Transfection, Fluorescence, Microscopy, Two Tailed Test, Incubation