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

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