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: 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: PLoS ONE

Article Title: Analysis of Intracellular State Based on Controlled 3D Nanostructures Mediated Surface Enhanced Raman Scattering

doi: 10.1371/journal.pone.0015836

Figure Lengend Snippet: ( A ) SERS spectra of ( 1 ) HeLa, ( 2 ) HepG2, and ( 3 ) MCF-7 cells. ( B ) SERS spectra of (blue) MCF-7 and (black) HMEC cells. ( C ) SERS spectra of living HeLa cells (black curve) and dead HeLa cells (blue curve). Inset contains the confocal microscopic images of living and dead HeLa cells. ( D ) SERS spectra of living HepG2 cells (black curve) and dead HepG2 cells (blue curve). Inset contains the confocal microscopic images of living and dead HepG2 cells. ( E ) SERS spectra of living HMEC cells (black curve) and dead HMEC cells (blue curve). Inset contains the confocal microscopic images of living and dead HMEC cells. Circles in Figs. C, D, and E indicate the primary differences between the SERS spectra of living and dead cells.

Article Snippet: Human liver carcinoma cell line (HepG2), human epithelial carcinoma cell line (HeLa) and human breast adenocarcinoma cell line (MCF-7) were obtained from the Korean Cell Line Bank (Seoul, Korea).

Techniques:

Journal: Cancer Cell International

Article Title: Overexpression of trefoil factor 3 (TFF3) contributes to the malignant progression in cervical cancer cells

doi: 10.1186/s12935-016-0379-1

Figure Lengend Snippet: Forced expression of TFF3 in cervical cancer cells modulates the expression of malignant progression-related gene markers. a Determination of endogenous expression of TFF3 protein by Western blot in the cervical cancer cells lines CaSki, SiHa, Me180 and Hela and human non-tumor keratinocyte line HaCaT. b Semi-quantitative RT-PCR analysis was used to assess the mRNA levels of TFF3 in SiHa and Hela cells with either forced or depleted expression of TFF3 as described in “ ” section. c Western blot analysis was used to assess the protein levels of TFF3 in SiHa and Hela cells with either forced or depleted expression of TFF3. d , e Quantitative PCR analysis quantifying the change in expression of various genes associated with malignant progression in SiHa-TFF3/Hela cells. Change in gene expression is expressed as fold difference, respectively. Fold change values are representative of three independent experiments

Article Snippet: Human cervical cancer cell lines SiHa, CaSki, Hela, Me180 and human non-tumor keratinocyte line HaCaT were obtained from Nanjing KeyGen Biotech Co, Ltd (Nanjing,China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Gene Expression