Journal: Cancer Research

Article Title: Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

doi: 10.1158/0008-5472.can-08-4689

Figure Lengend Snippet: Figure 2. ACSVL3 expression in cultured human glioma cells and xenografts correlates with RTK activation. A, human U87, U373, and Mayo 22 glioma cells were grown to ∼80% confluence. Cells were serum starved overnight and were incubated for 16 h in the absence or presence of HGF (20 ng/mL; a gift from Genentech) or EGF (30 ng/mL; Calbiochem/EMD) before harvesting and Western blot analysis for ACSVL3 expression. Each lane contained 30 μg of total cellular protein. B, U87 cells stably expressing constitutively active EGFR (EGFRvIII) or corresponding control U87 cells were harvested and analyzed by Western blot. C, tumors were produced in the flanks of athymic nude mice (n = 12) by s.c. injection (two sites per mouse) of 4 × 106 U87 cells. When tumor size reached ∼300 mm3 (day 14), animals were randomly divided into two groups of six mice. One group received i.p. injection (100 μg/20 g body weight) of anti-HGF mAb (L2G7) and the other group received isotype control mAb (5G8) on days 14, 16, and 18 (17). Mice were sacrificed on day 20. Tumor homogenates (50 μg of protein) were subjected to Western blot analysis for ACSVL3 expression. Actin expression was used as a loading control for all blots.

Article Snippet: Human U87 MG (ATCC #HTB-14) and U373 MG (ATCC #HTB-17) glioblastoma cell lines have been maintained in our laboratory for >10 y and were cultured as previously described (10).

Techniques: Expressing, Cell Culture, Activation Assay, Incubation, Western Blot, Stable Transfection, Control, Produced, Injection

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) RT-qPCR for the expression of CCHCR1 and TCF19 in HeLa cells transfected with E2F activators (E2F1, E2F2 or E2F3). pEGFP-N1 was the empty vector control. Gene expression level was normalized to the expression level of RPL13A . Results were mean ± SD of three replicates. Statistically significant differences of the gene expression levels were compared to empty vector control and were analyzed by paired t-test (** = P ≤ 0.01). ( b ) Down-regulation of CCHCR1 and TCF19 mRNA by E2F1 knockdown. Cells were transfected with siE2F1 and were harvested for the time indicated (time post-transfection). mRNA expressions (normalized to RPL13A gene expression) were analyzed by RT-qPCR. Results were mean ± SD of three replicates. Statistically significant differences of the gene expression levels were compared to 0 h post-siE2F1 transfection and were analyzed by paired t-test (*** = P ≤ 0.001).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Gene Expression, Knockdown

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were synchronized and arrested in G1/S transition by a double thymidine block. The cells were released and collected at different time points, stained with propidium iodide (PI). The percentage of cells in different cell cycle phases in each time point (G1/S, S, G2/M, and G1) were analyzed by flow cytometry. ( b ) Percentage of cells in different cell cycles after double thymidine block and release. (C) Relative expression of CCHCR1 and TCF19 in different cell cycle phases. CCHCR1 and TCF19 gene expression were normalized to RPL13A gene expression. The time points after release from the double thymidine block were indicated for different cell cycle phases; i.e. 0 h: G1/S, 2 h: S, 8 h: G2/M and 14 h: G1. Results were mean ± SD of three replicates. Statistically significant differences of the gene expressions were compared to 0 h after double thymidine release (G1/S transition) and were analyzed by paired t-test (** = P ≤ 0.01 and *** = P ≤ 0.001)

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Blocking Assay, Staining, Flow Cytometry, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were transfected with the corresponding siRNAs. Non-viable cells were counted using trypan blue exclusion assay. siCTL was Silencer select negative control #1 (Ambion, Life Technologies, USA). Results are mean ± SD of five replicates. Statistically significant differences of the cell counts were compared to the untransfected samples in the corresponding time point, and were analyzed by paired t-test (* = ≤0.05 and ** = P ≤ 0.01). ( b ) Up-regulation of p21 mRNA was observed after knockdown of CCHCR1 . siRNAs were transfected into HeLa cells and were collected for analysis 4 days post-transfection. Gene expressions (normalized to RPL13A gene expression) were analyzed by RT-qPCR. Results are mean ± SD of three replicates. Statistically significant differences of the gene expressions were compared to untransfected sample and were analyzed by paired t-test (* = ≤0.05, ** = P ≤ 0.01 and *** = P ≤ 0.001).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Transfection, Trypan Blue Exclusion Assay, Negative Control, Knockdown, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

doi: 10.1101/2023.04.08.535951

Figure Lengend Snippet: ( a ) HeLa cells were treated with 0.1% DMSO (vehicle control) or 2 μM CPT for 24 hours. The percentage of cells in different cell cycle phases were analyzed by flow cytometry. ( b ) Relative expression of CCHCR1 and TCF19 (normalized to RPL13A gene expression) in HeLa cells after the treatment with CPT for 24 hours. Results are mean ± SD of three replicates. Statistically significant differences of the gene expression level were compared to 0.1% DMSO treatment and were analyzed by paired t-test (* = ≤0.05 and ** = P ≤ 0.01).

Article Snippet: Human cervical cancer HeLa cells (ATCC ® CRM-CCL-2 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Control, Flow Cytometry, Expressing, Gene Expression

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Knockdown of FLNB promotes proliferation and inhibits apoptosis of HeLa cells. (A) Relative mRNA expression of FLNB in HeLa cells after it was knocked down using FLNB -specific shRNA was determined by reverse transcription-qPCR. shFLNB_1 and shFLNB_2 indicate two biological repeats of HeLa cells transfected with FLNB-specific shRNA. Ctrl_1 and Ctrl_2 indicate two biological repeats of HeLa cells transfected with empty vector as controls. (B) Cell proliferation of shFLNB was measured by an MTT assay in HeLa cells. Cell apoptosis of cells transfected with shFLNB was measured by (C) flow cytometry and (D) subsequent analysis by a 7-ADD and Annexin V assay. *P<0.05 and **P<0.01 vs. respective Ctrl. FLNB , filamin B; sh, short hairpin; qPCR, quantitative PCR; Ctrl, control; 7-AAD, 7-amino actinomycin D; OD, optical density; PE, phycoerythrin.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Knockdown, Expressing, shRNA, Reverse Transcription, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Annexin V Assay, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: GO and KEGG analysis of differentially expressed genes between short hairpin FLNB-transfected and control HeLa cells. Top 10 most enriched GO terms (biological process) of (A) upregulated and (B) downregulated genes upon FLNB knockdown. Rectangles around GO terms indicate notable cancer-related and cartilage development terms. Top 10 most enriched KEGG pathways of (C) upregulated and (D) downregulated genes upon FLNB knockdown. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FLNB , filamin B.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Transfection, Control, Knockdown

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Validation of FLNB -regulated genes (DEGs). (A) Relative expression level (FPKM, up) and RT-qPCR measurement (down) of cartilage development-related DEGs. (B) Related expression level (FPKM, up) and RT-qPCR measurement (down) of apoptotic-related DEGs. (C) Western blot analysis of two apoptotic-related proteins in shFLNB and Ctrl HeLa cells. FLNB , filamin B; DEGs, differentially expressed genes; FPKM, fragments per kilobase of transcript per million fragments mapped; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; Ctrl, control; ATP7A, ATPase copper transporting α; BMP7, bone morphogenetic protein 7; COL2A1, collagen type II α 1 chain; MMP13, matrix metallopeptidase 13; IL23A, interleukin 23 subunit α; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NAIP, NLR family apoptosis inhibitory protein; SLC25A36, solute carrier family 25 member 36; MAP2K7, mitogen-activated protein kinase kinase 7.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Validation of FLNB -affected ASEs. Genome visualization (left panel) shows FLNB -regulated ASEs in shFLNB and control. (A) Validation of an ASE of VDR in HeLa cells. (B) Validation of an ASE of PACS2 in HeLa cells. (C) Validation of an ASE of MAP2K7 in HeLa cells. (D) Validation of an ASE of MALAT1 in HeLa cells. The number of junction reads were marked on the line representing splice junction composing ASE. The structures of ASEs are depicted in the top-right panel. The altered ratio of ASEs in RNA-sequencing and in reverse transcription-quantitative PCR were calculated and plotted (right panel, bottom). FLNB , filamin B; ASEs, alternative splicing events; sh/SH, short hairpin; NC, negative control; VDR, vitamin D receptor; PACS2, phosphofurin acidic cluster sorting protein 2; MAP2K7, mitogen-activated protein kinase kinase 7; MALAT1, metastasis associated lung adenocarcinoma transcript 1. *P<0.05 and ***P<0.001 vs. respectively NC.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Biomarker Discovery, Control, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Alternative Splicing, Negative Control