Journal: EBioMedicine

Article Title: A truncating MEIOB mutation responsible for familial primary ovarian insufficiency abolishes its interaction with its partner SPATA22 and their recruitment to DNA double-strand breaks

doi: 10.1016/j.ebiom.2019.03.075

Figure Lengend Snippet: The MEIOB-truncating mutation disrupts MEIOB-SPATA22 recruitment to DNA damage foci. a-b- The truncated form of MEIOB is stable when expressed in HeLa cells. a- Western blot showing the expression of the wild-type (MEIOB MF ) and the truncated forms (MEIOB MF-mut ) of MEIOB transfected in HeLa cells. The ProSieve QuadColor protein marker 4,6 kDa–300 kDa (Lonza) was run on the left (Ladder). b- MEIOB MF or MEIOB MF-mut and SPATA22 were co-expressed from a single plasmid MEIOB∧SPATA22 in HeLa cells before exposure to etoposide (25 μM). Cells were fixed in PFA without pre-permeabilisation to retain both cytoplasmic and nuclear fractions. Antibodies were used to immunostain MEIOB (red) and SPATA22 (white). Yellow arrows highlight transfected cells expressing both MEIOB and SPATA22. c-d- The truncation prevents MEIOB recruitment with SPATA22 to DSBs. MEIOB MF or MEIOB MF-mut and SPATA22 HA were expressed in HeLa cells before exposure to etoposide. Cells were permeabilised before fixation in PFA to analyse the chromatin-associated fraction of proteins. RPA foci are formed at DNA damage sites. c- MEIOB or SPATA22 alone are not recruited to DNA damage sites. d- When co-expressed, MEIOB MF and SPATA22 are recruited to DNA-damage sites and colocalise with RPA. This colocalisation is not observed for the truncated MEIOB MF-mut . Antibodies are used to immunostain MEIOB (red), SPATA22 (white), and RPA (green). Scale bar = 10 μm in all panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: HeLa cells were used for minigene assays (CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030).

Techniques: Mutagenesis, Western Blot, Expressing, Transfection, Marker, Plasmid Preparation

Journal: iScience

Article Title: Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks

doi: 10.1016/j.isci.2022.104142

Figure Lengend Snippet:

Article Snippet: Hela human cell line , ATCC , CRL-12401; RRID: CVCL_W341.

Techniques: Virus, Generated, Control, Recombinant, Protease Inhibitor, Extraction, cDNA Synthesis, Plasmid Preparation, Software

Journal: Virology

Article Title: Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

doi: 10.1016/j.virol.2005.01.008

Figure Lengend Snippet: Fig. 1. Detection of NS3, NS3-4A complex, and NS4A expression in HepG2 and HeLa cells. HepG2 and HeLa cells (HepG2-NS3, HeLa-NS3, HepG2-NS3-4A, HeLa-NS3-4A, HepG2-NS4A, and HeLa-NS4A) cul- tured in the presence (+Tc) or in the absence (Tc) of 4 Ag/ml tetracycline for 48 h. 100 Ag protein was separated by 12% SDS–PAGE and analyzed by immunoblotting. (A) Expression of HCV NS3 in HepG2-NS3 and HeLa-NS3 cells. (B) Expression of HCV NS3-4A complex in HepG2- NS3-4A and HeLa-NS3-4A cells. (C) Expression of HCV NS4A in HepG2-NS4A and HeLa-NS4A cells. The same blots were reprobed with an anti-actin antibody to compare loading and transfer.

Article Snippet: HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator and the RetroPack pT67 cells, were purchased from Clontech (California, USA).

Techniques: Expressing, SDS Page, Western Blot

Journal: Virology

Article Title: Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

doi: 10.1016/j.virol.2005.01.008

Figure Lengend Snippet: Fig. 2. Effect of HCV NS3 protein on cell growth. HepG2-NS3 and HeLa-NS3 transfectants were cultured in the presence or in the absence of tetracycline for the indicated time points. The proliferation rate was assessed by [3H] thymidine uptake assay (A) and MTT assay (B). The enhancement of cell proliferation by NS3 is reversible up on re-addition of tetracycline to the culture medium. The growth rate of both HepG2-NS3 and HeLa-NS3 transfectants was assessed at the indicated time points using [3H] thymidine uptake assay (C) or MTT assay (D). The results are means F SE of three independent experiments.

Article Snippet: HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator and the RetroPack pT67 cells, were purchased from Clontech (California, USA).

Techniques: Cell Culture, MTT Assay

Journal: Virology

Article Title: Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

doi: 10.1016/j.virol.2005.01.008

Figure Lengend Snippet: Fig. 4. Inhibition of HCV NS3 protein-induced AP-1 and ATF-2 activation, and c-jun expression, and cell growth by JNK inhibitor SP600125. HepG2-NS3 and HeLa-NS3 transfectants as well as HepG2-Luc and HeLa-Luc transfectants were pre-treated with SP600125 (20 AM/ml) 1 h prior to the cultivation in medium with (+Tc) or without (Tc) 4 Ag/ml tetracycline for the indicted time points. (A–C) The nuclear extracts were prepared and equal amounts of nuclear proteins (4 Ag) were analyzed for the activation of either AP-1 (A), ATF-2 (B), and Oct-1 (A and B). The specificity of the DNA binding of AP-1, ATF-2, and Oct-1 was analyzed by both competition with unlabelled oligonucleotides (data not shown) and supershift assay (C) in HepG2-NS3 transfectants. The same analysis was performed also in HeLa-NS3 transfectants and the obtained results were comparable (data not shown). (D) The total RNAs were prepared and the expression of c-jun was analyzed by Northern blot. Membranes were then rehybridized with a GAPDH to compare loading and transfer. (E and F) Measurement of cell growth in HepG2-NS3 and HeLa-NS3 transfectants as well as in control cells HepG2-Luc and HeLa-Luc transfectants in the presence or in the absence of JNK inhibitor by either [3H] thymidine uptake assay (E) or by MTT assay (F). Results are representative of three separate experiments.

Article Snippet: HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator and the RetroPack pT67 cells, were purchased from Clontech (California, USA).

Techniques: Inhibition, Activation Assay, Expressing, Binding Assay, Northern Blot, Control, MTT Assay

Journal: Virology

Article Title: Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

doi: 10.1016/j.virol.2005.01.008

Figure Lengend Snippet: Fig. 5. Inhibition of HCV NS3-mediated cell growth by cofactor NS4A. HepG2 and HeLa cells (HepG2-NS3, HeLa-NS3, HepG2-NS3-4A, HeLa-NS3-4A, HepG2-NS4A, and HeLa-NS4A) were cultured in the presence or in the absence of tetracycline for 48 h, and [3H] thymidine uptake assay (A) and MTT assay (B) were performed. The results are means F SE of three independent experiments.

Article Snippet: HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator and the RetroPack pT67 cells, were purchased from Clontech (California, USA).

Techniques: Inhibition, Cell Culture, MTT Assay

Journal: Virology

Article Title: Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

doi: 10.1016/j.virol.2005.01.008

Figure Lengend Snippet: Fig. 6. Effect of NS4A co-expression on JNK, p38, and ERK MAP kinase activation. HepG2 and HeLa transfectants (HepG2-NS3, HeLa-NS3, HepG2-NS3- 4A, HeLa-NS3-4A, HepG2-NS4A, and HeLa-NS4A) were cultured in the presence or in the absence of tetracycline for 48 h. Cells were washed, lysed, and the JNK, p38, and ERK were immunoprecipitated from extracts using anti-JNK, p38, or ERK antibodies, respectively. JNK activity was measured by using the immune complex in a kinase assay with GST–c-jun as the substrate, whereas the activities of p38 or ERK were measured by using the immune complex in a kinase assay with MBP as substrate. These results are representative of three independent experiments.

Article Snippet: HeLa Tet-Off cells, which constitutively express the tetracycline-controlled transactivator and the RetroPack pT67 cells, were purchased from Clontech (California, USA).

Techniques: Expressing, Activation Assay, Cell Culture, Immunoprecipitation, Activity Assay, Kinase Assay

Journal: bioRxiv

Article Title: VASH1 phosphorylation regulates tubulin detyrosination via CNP/PRKG1α signaling in DRG neurons

doi: 10.64898/2026.10.14.759141

Figure Lengend Snippet: Comparison of the activity of sfGFP-VASH1-SVBP (WT) and the mutant (S337E) enzyme complexes (50 pM) on taxol-stabilized HeLa tyrosinated (Tyr) microtubules measured by immunofluorescence in BRB40 buffer supplemented with 50 mM KCl as in . ( A ) Representative images of tyrosinated (Tyr, red) and detyrosinated (dTyr, turquoise) α-tubulin pools of microtubules after 30 min incubation in the absence (control) or presence of the indicated enzyme complexes. Scale bar, 10 μm. ( B ) Quantification of tyrosinated (Tyr) and detyrosinated (dTyr) tubulin signals. Each point represents a microtubule, at least 270 microtubules across 3 experiments done with 3 independent sets of recombinant enzymes were analyzed. Data are represented as mean ± SD. Statistical significance was determined using Kruskal-Wallis test, ****P < 0.0001.

Article Snippet: HeLa S3 cells (CLS Cell Lines Service GmbH) were first cultivated ∼5 days in adherent cultures, and then used to inoculate 2 liters of suspension culture that were cultured during ∼14 days at 65 rpm, 37°C, 5% CO 2 in a New Brunswick S41i incubator (Eppendorf).

Techniques: Comparison, Activity Assay, Mutagenesis, Immunofluorescence, Incubation, Control, Recombinant

Journal: Nature Communications

Article Title: Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin

doi: 10.1038/s41467-023-42714-4

Figure Lengend Snippet: a Activity of 3033 on JAK family enzymes. Human HEL 92.1.7 cells were treated with siRNA at 1.5 μM for 72 h, and mRNA levels of JAK1, JAK2, JAK3, and TYK2 were measured using the QuantiGene 2.0 assay ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, **** P < 0.0001, ns: not significant). b Ruxolitinib inhibits JAK1 and JAK2 to prevent the pathogenesis of vitiligo driven by IFN-γ signaling. c mRNA expression of IFN-γ-inducible chemokines CXCL9, 10, and 11. Cells were first treated with siRNA or ruxolitinib at 1.5 μM for 72 h, and then stimulated with recombinant human IFN-γ for 24 h ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). d , e Chemical engineering of 3033 for skin delivery. Terminal nucleotides were stabilized with phosphorothioate backbone modifications for nuclease stability ( d ). Human HeLa cells were transfected with DCA-siRNAs for 72 h through lipofectamine RNAiMax-mediated uptake and mRNA levels were measured using the QuantiGene 2.0 assay ( n = 3 biologically independent samples, mean ± s.d.; two-way ANOVA multiple comparison, * P < 0.05). f Molecular modeling (PyMOL 2) of DCA-siRNA 3033 in scaffold 2, si3033. g – i ruxolitinib and si3033 both reduce luciferase activity in IFN-γ-JAK1/2-STAT1 HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). Cells were treated with ruxolitinib or siRNA for 72 h and then stimulated with IFN-γ for 18 h. si3033 was transfected to the HeLa reporter cells using lipofectamine RNAiMax. j JAK1 and JAK2 mRNA expression in si3033-treated and untreated HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; two-sided unpaired t test, **** P < 0.0001, ns: not significant). Source data are provided as a Source Data file.

Article Snippet: GAS reporter (Luc)-HeLa cell line (IFN-γ/JAK/STAT1 pathway) was purchased from BPS Bioscience (#79041).

Techniques: Activity Assay, Expressing, Recombinant, Transfection, Comparison, Luciferase

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