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

Journal: bioRxiv

Article Title: Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability

doi: 10.1101/2021.05.27.445952

Figure Lengend Snippet: A. Representative images of HeLa and HEK293T cells showing localization of BG4, the G4 binding antibody to mitochondria following immunofluorescence study. Nucleus is stained with DAPI (blue color), mitochondria with MitoTracker DR (red) and BG4 with Alexa-Fluor 488 (green). A merged image is shown with merge of red and green as depicted by Coste’s mask (colocalization is represented as a white dot). B, C. Quantitation showing colocalization of BG4 with MitoTracker indicated as dot plots. The colocalization was quantified using Mander’s colocalization coefficient (ImageJ software) analyzing a minimum of 100 cells as red over green (B) and green over red (C). D. Representative image of rho(0) cell showing localization of BG4 to mitochondria following immunofluorescence as investigated in panel A. E. Quantitation showing comparison of colocalization of BG4 between HeLa cells and rho(0) cells is shown as dot plots. The colocalization was quantified using Mander’s colocalization coefficient analyzing a minimum of 50 cells as red over green. F. BG4 bound mtDNA was purified after reverse crosslinking and used for real-time PCR using primers derived from different regions of the mitochondrial genome, which include 5 G-quadruplex forming regions and 10 random regions. Input DNA served as template control. No antibody control was also used. Bars in blue (first 5) are for G-quadruplex forming regions, while in green (last 10) are for random regions. Y-axis depicts threshold Ct value obtained following real time PCR for each primer. Error bar represents mean ± SEM. G. Agarose gel profile showing the amplification of Input DNA (left panel) and BG4 pull down DNA (right panel). “M” denotes 100 bp ladder.

Article Snippet: HeLa (human cervical cancer), MEF (mouse embryonic fibroblast) and HEK293T (human embryonic kidney epithelial cell line) were purchased from National Centre for Cell Science, Pune, India.

Techniques: Binding Assay, Immunofluorescence, Staining, Quantitation Assay, Software, Comparison, Purification, Real-time Polymerase Chain Reaction, Derivative Assay, Control, Agarose Gel Electrophoresis, Amplification

Journal: Molecular Medicine Reports

Article Title: hsa_circ_0101119 facilitates the progression of cervical cancer via an interaction with EIF4A3 to inhibit TCEAL6 expression

doi: 10.3892/mmr.2021.12293

Figure Lengend Snippet: hsa_circ_0101119 is highly expressed in CC tissues and cells. (A) Heatmap of differentially expressed circRNAs in CC tissues and normal tissues according to the online data set (GSE102686). (B) Expression level of hsa_circ_0101119 was detected via reverse transcription-quantitative PCR in CC cell lines (C-33A, SiHa, CaSki and HeLa) and normal human cervical epithelial cell line, HcerEpic. **P<0.01 vs. HcerEpic cells. (C) Expression level of hsa_circ_0101119 in CC tissues and normal tissues, according to the online data set (GSE102686). circRNA/circ, circular RNA.

Article Snippet: The four human CC cell lines (C-33A, SiHa, CaSki and HeLa) and the normal human cervical epithelial cell line, HcerEpic, were supplied by VCANBIO Cell & Gene Engineering Co., Ltd. All the cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured at 37°C in a humidified 5% CO 2 incubator.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: hsa_circ_0101119 facilitates the progression of cervical cancer via an interaction with EIF4A3 to inhibit TCEAL6 expression

doi: 10.3892/mmr.2021.12293

Figure Lengend Snippet: hsa_circ_0101119 recruits EIF4A3 to inhibit TCEAL6 expression in CC. (A) Bioinformatics was used to predict the interaction probabilities of the RNA-binding protein EIF4A3 with hsa_circ_0101119. Predictions with probabilities >0.5 were considered ‘positive’, suggesting that the corresponding RNA and protein are likely to interact. (B) RIP assay using anti-EIF4A3 showed that EIF4A3 precipitated hsa_circ_0101119 in SiHa and HeLa cell lysates. (C) Pull down assay indicated that biotin-labeled hsa_circ_0101119 interacted with EIF4A3. (D) Bioinformatics was used to predict the interaction probabilities of EIF4A3 with TCEAL6. (E) RIP assay using anti-EIF4A3 showed that EIF4A3 precipitated TCEAL6 in SiHa and HeLa cell lysates. (F) Expression levels of EIF4A3 and TCEAL6 were detected via RT-qPCR in CC cell lines (C-33A, SiHa, CaSki and HeLa) and a normal human cervical epithelial cell line, HcerEpic. (G) Expression levels of EIF4A3 and TCEAL6 in CC tissues and normal tissues, according to the analysis of TCGA. (H) Correlation between EIF4A3 and TCEAL6 in CC samples from TCGA. (I) After transfection with sh-EIF4A3, RT-qPCR was used to detect EIF4A3 expression in SiHa and HeLa cells. (J) After transfection with sh-EIF4A3, western blotting was performed to detect the expression level of TCEAL6 in SiHa and HeLa cells. (K) After co-transfection with si-hsa_circ_0101119 and sh-EIF4A3, western blotting was performed to measure the expression level of TCEAL6 in SiHa and HeLa cells. (L) A proposed model whereby hsa_circ_0101119 sequesters EIF4A3 away from TCEAL6 mRNA, in turn suppressing TCEAL6 mRNA translation. **P<0.01 vs. IgG group (B and E); *P<0.05, **P<0.01 vs. HcerEpic cells group (F); *P<0.05 vs. normal tissues group (G); **P<0.01, vs. sh-NC group (I and J); **P<0.01 vs. sh-NC group, ## P<0.01, vs. si-hsa_circ group. (K) RIP, RNA immunoprecipitation; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; sh, short hairpin RNA; NC, negative control; si, small interfering RNA; circ, circular RNA; EIF4A3, eukaryotic initiation factor 4A-3; TCEAL6, transcription elongation factor A-like 6; T, tumor; N, normal; CC, cervical cancer.

Article Snippet: The four human CC cell lines (C-33A, SiHa, CaSki and HeLa) and the normal human cervical epithelial cell line, HcerEpic, were supplied by VCANBIO Cell & Gene Engineering Co., Ltd. All the cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured at 37°C in a humidified 5% CO 2 incubator.

Techniques: Expressing, RNA Binding Assay, Pull Down Assay, Labeling, Quantitative RT-PCR, Transfection, Western Blot, Cotransfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA, Negative Control, Small Interfering RNA