hela frt cells  (ATCC)

Journal: eLife

Article Title: A specific role for importin-5 and NASP in the import and nuclear hand-off of monomeric H3

doi: 10.7554/eLife.81755

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Article Snippet: HeLa FRT cell line (T-REx-HeLa) was sourced from Invitrogen (R71407).

Techniques: Plasmid Preparation, Transfection, Construct, Transformation Assay, Expressing, Sequencing, Blocking Assay, Recombinant, Concentration Assay, Software, Staining

Journal: Nucleic Acids Research

Article Title: XAB2 depletion induces intron retention in POLR2A to impair global transcription and promote cellular senescence

doi: 10.1093/nar/gkz532

Figure Lengend Snippet: XAB2 depletion led to substantial loss of POLR2A and reduction of newly transcribed RNA. ( A ) RT-PCR showing decreased expression of POLR2A mRNA in HeLa cells treated with XAB2 siRNA. Cells were harvested after 48 hours of siRNA treatment. ( B ) Quantitation of relative mRNA expression compared to control sample in A ( n = 3, *** P < 0.001). ( C ) RT-PCR showing decreased expression of POLR2A mRNA in HeLa cells expressing XAB2 shRNA. Cells were harvested after 72 hours of Dox induction. ( D ) Quantitation of relative mRNA expression compared to control sample in C ( n = 3, *** P < 0.001). ( E ) Western blot showing reduction of POLR2A protein in HeLa cells treated with XAB2 siRNA. Cells were harvested after 48 h of siRNA treatment. ( F ). Quantitation of relative protein expression compared to control sample in E ( n = 3, ** P < 0.01, *** P < 0.001). ( G ) Western blot showing reduction of POLR2A protein in HeLa cells expressing XAB2 shRNA. Cells were harvested after 72 h of Dox induction. ( H ) Quantitation of relative protein expression compared to control sample in G ( n = 3, ** P < 0.01, *** P < 0.001). ( I ) Immunofluorescence (IF) staining to show substantial loss of POLR2A and altered SC35 pattern after XAB2 depletion. IF staining of POLR2A was performed after 48 h of siRNA treatment. ( J ) EU incorporation assay showing significant decrease of newly transcribed RNA after XAB2 depletion. Assay was performed after 48 h of siRNA treatment.

Article Snippet: HeLa and FRT HeLa cells were gifts from Reed Lab in Harvard Medical School, 293T, MDA-MB-231 and HFF1 cells were purchased from the American Type Culture Collection (USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitation Assay, Control, shRNA, Western Blot, Immunofluorescence, Staining, Depletion Assay

Journal: Nucleic Acids Research

Article Title: XAB2 depletion induces intron retention in POLR2A to impair global transcription and promote cellular senescence

doi: 10.1093/nar/gkz532

Figure Lengend Snippet: XAB2 depletion induced severe splicing defects in POLR2A. ( A ) Localization of primers using for assay in splicing efficiency. EE: both primers were in exons; EI: forward primer in exon, reverse primer in intron; IE: forward primer in intron, reverse primer in exon. ( B ) RT-PCR showing decrease of spliced POLR2A mRNA and increase of unspliced pre-mRNA after XAB2 depletion. Cells were harvested after 48 h of siRNA treatment. RT: reverse transcriptase. ( C ) Quantification showing increased ratio of unspliced vs spliced transcripts ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001). ( D ) RNA-seq revealed much more reads mapping to exon-intron junction regions after XAB2 depletion. (** P < 0.01, *** P < 0.001). ( E ) Schematic diagram to show striking increase of reads mapping to the first exon and intron of POLR2A gene after RNA-seq. Cells were harvested after 48 hours of siRNA treatment. ( F ) Number of the categorized splicing defects after XAB2 depletion. The number in the brackets indicated the overlap between two siRNAs. ( G ) In vitro splicing assay showing splicing efficiency of T7-Ftz transcripts when the reactions were incubated with nuclear extract from HeLa cells over-expressing or depleting XAB2. ( H ) Quantification of splicing efficiency in G ( n = 2, n.s.: no significance, * P < 0.05, ** P < 0.01, *** P < 0.001). ( I ) Western blot to show the levels of XAB2 after over-expression or depletion.

Article Snippet: HeLa and FRT HeLa cells were gifts from Reed Lab in Harvard Medical School, 293T, MDA-MB-231 and HFF1 cells were purchased from the American Type Culture Collection (USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, RNA Sequencing, In Vitro, Splicing Assay, Incubation, Expressing, Western Blot, Over Expression

Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Experimental workflow. ( a ) ORFs of METTL proteins were cloned as GFP fusions under the control of a DOX-inducible promoter for targeted single copy integration into HeLa FRT cells. Whole cell extracts and/or nuclear extracts were prepared from GFP-METTL or GFP (control) expressing cells. GFP pull-downs were performed in triplicate followed by mass spectrometry analysis. Analysis of raw data was performed in MaxQuant (version 1.5.1.0) and Andromeda. Data filtering and generation of volcano plots was done essentially as described using a one-way ANOVA test with log2 fold change (FC) > 8 and false discovery rate (FDR) < 0.05 as thresholds. ( b ) Volcano plot of GFP-MBD3 interacting proteins as an example of successful complex identification using this protocol. Significant interactors are indicated. Volcano plots of ( c ) METTL3 and ( d ) METTL14 interactors. The log2 FC of GFP fusions to control in label-free quantification are plotted against the −log10 of the FDR calculated by a permutation-based FDR adapted t-test. The baits are indicated in red.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Clone Assay, Control, Expressing, Mass Spectrometry, Quantitative Proteomics

Journal: Scientific Reports

Article Title: The interactome of a family of potential methyltransferases in HeLa cells

doi: 10.1038/s41598-019-43010-2

Figure Lengend Snippet: Confirmation of METTL9 interactor and enzymatic activity of GFP-METTLs. ( a , b ) Validation of interaction between METTL9 and CANX by co-IP. ( a ) CANX is detected in GFP-METTL9 IP (a, lane 5) but not GFP IP (a, lane 2). 10 μl of GFP trap and 2 mg of whole cell extract were used. 20 μg of Input material were loaded for a comparison. ( b ) GFP-METTL9 (left panel, line 2) but not GFP (right panel, line 3) can be detected by immuno-blot with GFP antibody in the CANX IP. 10 μg of calnexin antibody and 4 mg of whole cell extract were used. 200 μg of Input material were loaded for comparison. No antibody (beads alone) used as control. ( c ) In vitro methyltransferase assays demonstrating that our GFP-METTL8 and GFP-METTL16 purifications have the expected RNA methyltransferase activity. GFP (as a control) and GFP-fusion proteins were purified from corresponding DOX-induced HeLa FRT cell lines and used in an in vitro methyltransferase assay on total RNA from HeLa cells as a substrate and 3H-SAM as a methyl-donor. After purification of the RNA, counts per minute (CPM) were quantified by liquid scintillation counting. Ratio of CPM measured for reactions with GFP-METTL fusion proteins relative to GFP control are plotted. Data are shown as mean ± SD from three replicates.

Article Snippet: GFP, GFP-METTL8 and GFP-METTL16 were purified from stable HeLa FRT cell lines using GFP nanobody sepharose beads (Chromotek).

Techniques: Activity Assay, Biomarker Discovery, Co-Immunoprecipitation Assay, Comparison, Control, In Vitro, Purification