Journal: Molecular human reproduction

Article Title: Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis.

doi: 10.1093/molehr/gah016

Figure Lengend Snippet: Figure 2. Identi®cation of TRAIL ligand and receptor proteins in the adult human testis. The four samples from left to right are: (1) 40 mg and (2) 50 mg of whole HeLa cell lysate (positive control): (3) 60 mg and (4) 100 mg of proteins from adult human testicular tissue were used to perform western blotting analysis. Individual membranes were incubated with primary antibody raised against TRAIL, DR4, DR5, DcR1 or with antibody raised against DcR2.

Article Snippet: Human HeLa cell total RNA, used as positive control, was obtained from Clontech Laboratories, USA. cDNA were obtained from reverse transcription (RT) of 5 mg total RNA using random hexanucleotides as primers (50 mmol/l) in the presence of dNTP (250 mmol/l) (Gibco BRL), dithiothreitol (10 mmol/l), and Moloney murine Figure 1.

Techniques: Positive Control, Western Blot, Incubation

Journal: Molecular human reproduction

Article Title: Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis.

doi: 10.1093/molehr/gah016

Figure Lengend Snippet: Figure 3. Identi®cation of TRAIL ligand and receptor mRNA in the adult human testis. cDNA obtained from reverse transcription of total RNA from adult human testicular tissue and total RNA of HeLa cells used as positive control were ampli®ed by PCR with corresponding primers (see Table I and Materials and methods).

Article Snippet: Human HeLa cell total RNA, used as positive control, was obtained from Clontech Laboratories, USA. cDNA were obtained from reverse transcription (RT) of 5 mg total RNA using random hexanucleotides as primers (50 mmol/l) in the presence of dNTP (250 mmol/l) (Gibco BRL), dithiothreitol (10 mmol/l), and Moloney murine Figure 1.

Techniques: Reverse Transcription, Positive Control

Journal: PLoS ONE

Article Title: APOL1 renal risk variants promote cholesterol accumulation in tissues and cultured macrophages from APOL1 transgenic mice

doi: 10.1371/journal.pone.0211559

Figure Lengend Snippet: HeLa cells were infected with different APOL1 alleles using a lentiviral system. (A) HeLa cells were fixed and stained with BODIPY 493/503 and cell-mask blue. The number of lipid droplets per cell was determined by high throughput Perkin Elmer OPERA analysis and Acapella Image Analysis software. Dot blot analysis indicating a significantly increased number of Bodipy493/503-positive lipid droplets per cell in HeLa cells infected with APOL1 G1 or APOL1 G2 risk variants when compared to APOL1 G0 transfected HeLa cells (left). Representative images of Bodipy493/503 staining (right, upper panel) to identify lipid droplets and cell-mask staining (right, lower panel) to identify individual cells. *p<0.05, **p<0.01. (B,C) HeLa cells expressing the different APOL1 alleles were labeled with 1 μCi/ml 3 H-cholesterol in medium with 1% FBS for 24 hours, then washed with PBS, incubated in RPMI containing 2% human serum for 6 hours. Dot plot analysis indicating no changes in cholesterol efflux to APOA1 (B) or human serum (C) in APOL1 G1 and APOL1 G2 risk variant expressing HeLa compared to APOL1 G0 expressing cells. Both APOL1-G1 and APOL1-G2 HeLa cells showed increased lipid droplets compard to APOL1-G0 HeLa cells. There were no statistically significant differences in cholesterol efflux that were detected in this system.

Article Snippet: HeLa cells were purchased from the American Type Collection, Manassas, VA. Pooled normal human serum used for cholesterol efflux assays in HeLa cells was purchased from Innovative Research, Novi, MI (Cat. No. IPLA-SER).

Techniques: Infection, Staining, High Throughput Screening Assay, Software, Dot Blot, Transfection, Expressing, Labeling, Incubation, Variant Assay

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