cervical epithelial carcinoma cell line hela  (ATCC)

Journal: BMB Reports

Article Title: Intron retention decreases METTL3 expression by inhibiting mRNA export to the cytoplasm

doi: 10.5483/BMBRep.2023-0069

Figure Lengend Snippet: Protein expression of METTL3-IR differs from its RNA expression. (A) HeLa cells were transfected with Myc-METTL3 or Myc-METTL3-IR. The cell extracts were subjected to immunoblot analysis using anti-Myc and anti-METTL3 antibodies. GAPDH served as a loading control. (B, C) METTL3 and METTL3-IR exhibit different subcellular localizations. Immunofluorescence images showing Myc-tagged METTL3 isoforms (anti-Myc, green) transfected HeLa cells (B). Nuclei were stained with DAPI (blue). Arrows indicate nucleus and cytoplasm regions. Scale bar = 10 μm. The relative fluorescence intensities of the METTL3 (anti-Myc, green) and METTL3-IR (anti-Myc, green) proteins in the nucleus and cytoplasm (C). (D) Immunofluorescence images showing endogenous METTL3 localization. Nuclei were stained with DAPI (blue). Arrows indicate nucleus and cytoplasm regions. Scale bar = 10 μm. (E) Immunoblot analysis of endogenous METTL3 protein in human cell lines. The cell extracts were subjected to immunoblot analysis using an anti-METTL3 antibody. GAPDH served as a loading control. METTL3 (Ab1) indicates antibody targeting amino acid 1-250 of METTL3; METTL3 (Ab2) indicates antibody targeting amino acid 172-453 of METTL3. Asterisk indicates nonspecific bands.

Article Snippet: The human epithelial cervical carcinoma cell line (HeLa), human adenocarcinoma alveolar epithelial cell line (A549), human keratinocyte cell line (HaCaT), human erythroleukemic cell line (K562), and human colon cancer cell line (HCT116) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, RNA Expression, Transfection, Western Blot, Control, Immunofluorescence, Staining, Fluorescence

Journal: BMB Reports

Article Title: Intron retention decreases METTL3 expression by inhibiting mRNA export to the cytoplasm

doi: 10.5483/BMBRep.2023-0069

Figure Lengend Snippet: Intron retention results in the inhibition of METTL3 mRNA export to the cytoplasm. (A) Fractionation of METTL3 variants in the nucleus and cytoplasm. Cell lysates were obtained from 293A and HeLa cells. M, molecular size marker in base pairs (bp). RTase+ indicates the presence of reverse transcriptase, and RTase− is a control containing samples without reverse transcriptase to confirm the absence of genomic DNA contamination. (B, C) METTL3 overexpression was confirmed using immunoblot analysis with anti-Myc and anti-METTL3 antibodies. Diagram showing METTL3 constructs with IR and IR-UTR regions (B). UTR, untranslated region; S, stop codon. HeLa cells were transfected with Myc-METTL3-IR or Myc-METTL3-IR UTR (C). GAPDH served as a loading control. METTL3 (Ab1) indicates antibody targeting amino acid 1-250 of METTL3. (D, E) RNA Stability of METTL3 transcripts at different times point after 5 μg/ml actinomycin-D treatment. HCT116 cells were treated with 5 μg/ml actinomycin D and RNA was extracted for the indicated times. METTL3 transcripts analyzed via PCR and GAPDH was used as an internal control. Values are expressed as means ± SD. **P < 0.01 compared to GAPDH stability. (F) Schematic of the alternative splicing process involving METTL3 intron 8 and 9 retention.

Article Snippet: The human epithelial cervical carcinoma cell line (HeLa), human adenocarcinoma alveolar epithelial cell line (A549), human keratinocyte cell line (HaCaT), human erythroleukemic cell line (K562), and human colon cancer cell line (HCT116) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Fractionation, Marker, Reverse Transcription, Control, Over Expression, Western Blot, Construct, Transfection, Alternative Splicing

Journal: Cell reports

Article Title: Streptococcus pneumoniae Infection Promotes Histone H3 Dephosphorylation by Modulating Host PP1 Phosphatase.

doi: 10.1016/j.celrep.2020.02.116

Figure Lengend Snippet: Figure 4. PP1 Is the Host Phosphatase Mediating H3S10 Dephosphorylation (A) Representative immunoblot images are shown on the left and quantifications on the right. A549 cells pretreated with the PP1 inhibitor Tautomy- cetin, the PP2A inhibitor okadaic acid, or DMSO as the control and infected with R6 (MOI = 50) for 3 h. (B) A549 cells are transfected with PP1 siRNA or control siRNA before 3 h R6 (MOI = 50) infection. (C) A549 cells are transfected with PP1 siRNA or control siRNA before PLY treatment for 30 min. (D) A549 cells are transfected with PP1 siRNA or control siRNA before H2O2 treatment for 1 h. (E) HeLa cells are pretreated with PP1 inhibitor Tautomycetin before LLO treatment for 20 min. (F) HeLa cells are transfected with PP1 siRNA or control siRNA before LLO treatment for 20 min. All quantification graphs are of at least three in- dependent experiments. All quantifications in graphs show the means ± SD, and statistical sig- nificance was calculated by one-way ANOVA method (Turkey post hoc test). **p < 0.01, ***p < 0.001. See also Figure S4.

Article Snippet: The human cervical carcinoma epithelial cell line HeLa (ATCC CCL-2) cells and human colon carcinoma cell line CaCO2 (ATCC HTB-37) cells were cultured in MEM culture medium supplemented with 1% glutamine, 1 mM sodium pyruvate (GIBCO), 0.1 mM nonessential amino acid solution (GIBCO), and 10% (HeLa) or 20% (CaCO2) FCS.

Techniques: De-Phosphorylation Assay, Western Blot, Control, Infection, Transfection

Journal: Cell reports

Article Title: Streptococcus pneumoniae Infection Promotes Histone H3 Dephosphorylation by Modulating Host PP1 Phosphatase.

doi: 10.1016/j.celrep.2020.02.116

Figure Lengend Snippet: Figure 5. Bacterial Infection Induces Dephosphorylation of PP1 (A) A549 cells were infected for 3 h with WT and the indicated mutants of S. pneumonia strain TIGR4 (MOI = 25). A representative immunoblot (left) and a quantification (right) of four independent experi- ments are shown. Anti-phospho-PP1a (Thr320) antibody (Cell Signaling, 2581s) recognizes all PP1 isoforms (Shimada et al., 2010). The PP1 T320ph levels are normalized to PP1a and to the unin- fected control condition. S. pneumonia. Error bars represent SD, and statistical significance was calculated by one-way ANOVA method (Dunnett’s post hoc test, uninfected as control group). **p < 0.01. (B) The correlation of H3S10ph levels and PP1 T320ph levels of infected cells from four indepen- dent experiments is calculated by the nonpara- metric Spearman’s correlation coefficient method (C) Immunofluorescence of PP1 T320ph in A549 cells under uninfected, 3-h WT TIGR4 infection, 3-h DplyDspxB double mutant infection, and 3-h WT infection at MOI = 25 with 3-h Tautomycetin pretreatment conditions. Size bars represent 50 mm. Quantification of nuclear PP1 T320ph fluorescence intensity from at least two indepen- dent experiments, for more than 300 cells counted for each condition. (D) HeLa cells were infected for 3 h with the WT Listeria (EGD strain) and its hly mutant at MOI = 50. A representative immunoblot of three independent experiments is shown. See also Figure S5.

Article Snippet: The human cervical carcinoma epithelial cell line HeLa (ATCC CCL-2) cells and human colon carcinoma cell line CaCO2 (ATCC HTB-37) cells were cultured in MEM culture medium supplemented with 1% glutamine, 1 mM sodium pyruvate (GIBCO), 0.1 mM nonessential amino acid solution (GIBCO), and 10% (HeLa) or 20% (CaCO2) FCS.

Techniques: Infection, De-Phosphorylation Assay, Western Blot, Control, Mutagenesis

Journal: International Journal of Peptide Research and Therapeutics

Article Title: Statistical and Artificial Neural Network Approaches to Modeling and Optimization of Fermentation Conditions for Production of a Surface/Bioactive Glyco-lipo-peptide

doi: 10.1007/s10989-020-10094-8

Figure Lengend Snippet: Dose-dependent anti-cancer activities of Pseudomonas aeruginosa strain IKW1 glycolipopeptide on a human breast cancer cell line-MCF-7, b human cervical cancer cell line-HeLa and c human leukemic cell line-HL-60. GLP glycolipopeptide

Article Snippet: The glycolipopeptide was tested against human breast cancer (MCF-7), leukemia (HL-60) and cervical epithelial carcinoma (HeLa) cell lines (ATCC) at concentrations ranging from 5.20 to 665.6 μg/mL.

Techniques: