a301 096a  (Bethyl)

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: A Construction of platinum-resistant OC cell lines A2780-DDP and OVCAR3-DDP cells. Western blot ( B ) and RT‒qPCR ( C ) were performed to detect the expression of CSTF3 in A2780-DDP, OVCAR3-DDP and corresponding parental cells. D CSTF3 protein was measured to confirm the knockdown efficiency in A2780-DDP and OVCAR3-DDP cells. E Cell viability was evaluated to measure the IC50 of platinum after knockdown of CSTF3 and treatment with gradient concentrations of platinum in A2780-DDP and OVCAR3-DDP cells. F A2780-DDP and OVCAR3-DDP cells were treated with 100 μM and 40 μM platinum for 6 h respectively. Colony formation assay was performed with control and CSTF3 knockdown A2780-DDP and OVCAR3-DDP cells treated with or without platinum. G Western blot was performed to verify CSTF3 knockdown efficiency in WT A2780 and OVCAR3 cells. H A2780 and OVCAR3 cells with CSTF3 knockdown were treated with different concentrations of platinum, and cell survival was determined. I A2780 and OVCAR3 cells were treated with 20 μM and 5 μM platinum for 6 h respectively. Colony formation assays were performed in A2780 and OVCAR3 cells treated with or without platinum after CSTF3 knockdown. J FLAG-NC and FLAG-CSTF3 were stably transfected into A2780 and OVCAR3 cells. Then, cell viability curves ( K ) and colony formation assays ( L ) were performed to measure the sensitivity of cells to platinum after overexpression of CSTF3. *P < 0.05, **P < 0.01, ***P < 0.001 vs. shNC.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Western Blot, Expressing, Knockdown, Colony Assay, Control, Stable Transfection, Transfection, Over Expression

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: OVCAR3 cells transfected with CSTF3 knockdown or negative control constructs were injected subcutaneously into BALB/c nude mice. The xenografted mice were treated with or without DDP (4 mg/kg) through intraperitoneal injection once every 6 days. A Photo of representative tumors from six groups of xenografted nude mice on the 22nd day. B The growth of tumor volumes was monitored every 3 days beginning on the 7th day. C Tumor weights are presented as the mean ± SD from five mice per group. D , E HE staining was performed to evaluate tissue morphology, and IHC was performed to visualize Ki-67- and Caspase-3-positive staining in xenografted tumors. *P < 0.05, **P < 0.01, ***P < 0.001 vs. shNC.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Transfection, Knockdown, Negative Control, Construct, Injection, Staining

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: A CSTF3 knockdown induced an APA shift of target genes in A2780 cells, left: scatterplot of the 3’UTR alteration in CSTF3 knockdown cells when compared with the control cells, right: the usage of proximal and distal PAS in CSTF3 knockdown cells. B Detection of APA events in CSTF3 knockdown OVCAR3 cells, combining 3’UTR alteration and usage of PAS. C Filtering out the directly bound target genes of CSTF3 by eCLIP-seq. D The consensus sequences of CSTF3 binding sites detected by HOMER motif analysis with eCLIP-seq data. E Distribution of CSTF3-targeted transcript types (left) and CSTF3 binding sites within different regions (right) as identified through eCLIP-seq. F The Venn diagram screened the candidate target genes using CSTF3 eCLIP-seq, A2780 and OVCAR3 PAS-seq data. G IGV genome browser showing the PAS usage of the NEAT1 3′UTR by eCLIP-seq. H Schematic diagram for primer design of short and long NEAT1 transcripts to validate the NEAT1 APA regulation by CSTF3. I Histogram showing the relative expression of the NEAT1 isoform with the distal PAS (dPAS) relative to that with the proximal PAS (pPAS) when CSTF3 was downregulated in A2780, A2780-DDP, OVCAR3 and OVCAR3-DDP cells compared with the negative control. J CLIP-qPCR detected the interaction between CSTF3 and NEAT1. K RT-qPCR was used to detect the relative expression of NEAT1 in A2780-DDP, OVCAR3-DDP and corresponding parental cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Knockdown, Control, Binding Assay, Expressing, Negative Control, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: A The IC50 values were evaluated when silencing the expression of NEAT1 and NEAT1_2 in A2780, OVCAR3 and corresponding platinum-resistant cells compared with the negative control. B A2780, OVCAR3, A2780-DDP and OVCAR3-DDP cells were treated with 20 μM, 5 μM, 100 μM and 40 μM platinum for 6 h respectively. Colony formation assay was performed to measure the sensitivity of the drug when silencing the expression of NEAT1 and NEAT1_2 in A2780, OVCAR3 and corresponding platinum-resistant cells treated with or without platinum. C , D The IC50 values and colony formation were evaluated when NEAT1_1 was overexpressed in A2780, OVCAR3 and platinum-resistant cells with CSTF3 knockdown treated with platinum or not. *P < 0.05, **P < 0.01, ***P < 0.001. ns not significant.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Expressing, Negative Control, Colony Assay, Knockdown

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: Immunofluorescence assays were performed with CSTF3 knockdown in A2780 ( A ) and OVCAR3 ( B ) cells, and images of DAPI (blue), CSTF3 (green), PSPC1 (red, left) and SFPQ (red, right) were obtained. C Immunofluorescence of A2780 and OVCAR3 cells transfected with si-NEAT1, si-NEAT1_2 and the negative control. DAPI (blue), PSPC1 (green) and SFPQ (red) were detected.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Immunofluorescence, Knockdown, Transfection, Negative Control

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: A Overlap of upregulated and downregulated genes in A2780 and OVCAR3 cells when CSTF3 was knocked down. B KEGG enrichment analysis for overlap of upregulated and downregulated genes. C Overlap of differentially expressed genes (DEGs) in OVCAR3 cells when NEAT1 and NEAT1_2 were silenced. D KEGG analysis of DEGs for silencing NEAT1. E KEGG analysis of DEGs for silencing NEAT1_2. F Histogram of KEGG analysis for the independent DEGs list with NEAT1 and NEAT1_2 silenced. G KEGG enrichment analysis of the individual DEGs list with NEAT1 silenced. H KEGG enrichment analysis of the individual DEGs list with NEAT1_2 silenced. I The levels of PI3K/AKT/mTOR pathway-related proteins were detected by western blot when CSTF3 was downregulated in A2780 and OVCAR3 cells. J Western blot was used to measure the levels of PI3K/AKT/mTOR pathway-related proteins with NEAT1 or NEAT1_2 siRNA transfected into A2780 and OVCAR3 cells.

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

doi: 10.1038/s41419-024-06816-1

Figure Lengend Snippet: Diagram of the regulatory mechanism and function underlying CSTF3 mediating the platinum resistance of OC cells .

Article Snippet: The primary antibodies were as follows: CSTF3 (Bethyl, A301-096A), PI3K (Abcam, ab151549), AKT (Proteintech, 60203-2-Ig), mTOR (CST, 2983 T), phospho-PI3K (CST, 4228 S), phospho-AKT (CST, 4060 S), phospho-mTOR (CST, 5536 S), FLAG-tag (MBL, M185-3L, PM020), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: