human carcinoma cell lines hela cervix uterine  (ATCC)

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 1. γ-tubulin associates with DSB-mimicking DNA in vitro, and is recruited to DNA damage sites in cells. (A) Pulldown with biotin-labeled dsDNA (500 bp with free ends, as described in Zhu et al., 2020) mimicking double-strand breaks (DSBs) was performed using HeLa cell lysates. A control (ctr) pulldown was performed using blank beads. The input, control pulldown and dsDNA pulldown samples were analyzed by immunoblotting (IB). n=3 independent experiments. (B) SCC38 cells were laser-microirradiated and analyzed by immunofluorescence for γ-tubulin and phospho-ATM S1981, as described in the Materials and Methods. The images were taken 3 min post laser treatment. The region of laser microirradiation is denoted by the white arrow. n=3 independent experiments. (C,D) HeLa cells with transient expression of mCherry–γ-tubulin and GFP–polymerase λ were laser microirradiated after pre-sensitization and imaged, as described in the Materials and Methods. The images were taken 5 min post laser treatment. Colocalization of mCherry–γ-tubulin and GFP– polymerase λ after laser microirradiation was quantified in panel C and shown in panel D. Arrows in the inset images in C indicate the points chosen for colocalization analysis. The regions of laser microirradiation are denoted by white arrows in D. n=3 independent experiments. (E) HeLa cells were treated with etoposide (1 µM, 10 min) and analyzed by immunofluorescence for γ-tubulin and 53BP1. The colocalization is shown. n=3 independent experiments. (F) Whole- cell extracts (WCE) and nuclear fractions were isolated from HeLa cells with or without etoposide treatment (1 µM, 3 h), as described in the Materials and Methods, and analyzed by IB for γ-tubulin and histone H3. (G) The IB results in panel F were quantified. Band intensities of γ-tubulin were normalized to those of histone H3. n=3 independent experiments. Bars show mean±s.d. Two-tailed unpaired Student’s t-test was performed; ns, not significant; *P<0.05.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: In Vitro, Labeling, Control, Western Blot, Immunofluorescence, Expressing, Isolation, Two Tailed Test

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 2. γ-tubulin associates with DNA repair proteins. (A) As described in the Materials and Methods, γ-tubulin immunoprecipitation (IP) was performed in lysates of HeLa cells that were untreated or treated with bleomycin (20 µM, 3 h) to induce DNA damage. The γ-tubulin IP products and control IP products with blank beads were subjected to proteomic analysis for identification of associated proteins. Poly-ADP-ribose polymerase 1 (PARP1) and the catalytic subunit (DNA-PKcs) of the DNA-dependent protein kinase complex were identified as γ-tubulin-associated proteins, as shown by the numbers of identified peptides, percentage of coverage and Mascot scores (via RAW MS data to MGF conversion and Mascot Server analysis). (B) γ-tubulin IP was performed in HeLa cells with or without etoposide. The input (10%), control IP using blank beads and γ-tubulin IP products were analyzed by IB for the indicated proteins. (C–E) Quantification of control or γ-tubulin IP products, as shown in panel B, was shown as the percentage of the input. Bars show mean±s.d. Two-tailed unpaired Student’s t-test was performed from >3 independent experiments; *P<0.05; **P<0.01; ***P<0.001. (F) Ku80 IP was performed in HeLa cells with bleomycin treatment. The input, control IP using blank beads, and Ku80 IP products were analyzed by IB. n=3 independent experiments.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: Immunoprecipitation, Control, Two Tailed Test

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 3. Inhibition of γ-tubulin leads to accumulation of endogenous DNA damage and delayed DNA repair. (A) HeLa cells were treated with control or γ-tubulin siRNA for 1 day. The cells were harvested and analyzed by IB. n=3 independent experiments. (B) HeLa cells were treated with control or γ- tubulin siRNA, and with or without transfection of a γ-tubulin-expressing vector, for 1 day. The cells were harvested and analyzed by IB. n=3 independent experiments. (C) For the repair kinetics analysis, HeLa cells with control or γ-tubulin siRNA were treated with 1 μM etoposide for 1 h, followed by repair/ recovery for 0, 3, 6 and 9 h. The cells were harvested and analyzed by IB. n=3 independent experiments. (D) HeLa cells were treated with or without 6 μM gatastatin G2 and irradiated (IR), followed by repair/recovery for 0, 3, 6 and 9 h. The cells were harvested and analyzed by IB. n=3 independent experiments.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: Inhibition, Control, Transfection, Expressing, Plasmid Preparation, Irradiation

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 4. γ-tubulin suppression reduces DSB repair efficacy. (A,B) Chromosome-integrated, I-SceI-induced homologous recombination (HR) (A) or non- homologous end joining (NHEJ) (B) assays were performed as described in the Materials and Methods. The reporter cells, HeLa-Dr or U2OS-EJ5, were transfected with control (ctr) or γ-tubulin siRNA. I-SceI endonuclease was expressed using a lentiviral vector. Following 48 h incubation, DNA repair was measured by IB to assess GFP expression relative to SMC1 expression. The GFP/SMC1 ratio in γ-tubulin-depleted cells was normalized to that in control cells for relative repair efficiency. The mean±s.d. values, calculated from three independent experiments, are shown. Statistical significance was analyzed using an unpaired two-tailed Student’s t-test; *P<0.05; **P<0.01. (C) HeLa cells treated with control or γ-tubulin siRNA were irradiated with 20 Gy of X-ray irradiation (IR), followed by repair/recovery for 30 min. Chromatin fractionation was performed as described in the Materials and Methods. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (D) HeLa cells were treated with control or γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with hydroxyurea (HU, 5 mM) for 2 or 4 h. Chromatin fractionation was performed. Whole-cell lysates and chromatin- enriched fractions were analyzed by IB. n=5 independent experiments. (E) HeLa cells were treated with control or γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with HU at the indicated concentrations for 3 h. Chromatin fractionation was performed. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (F) HeLa cells were treated with or without γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with 20 Gy IR. Cells were incubated post IR for the indicated durations (in minutes) and then subjected to chromatin fractionation. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (G) The chromatin recruitment of NHEJ and HR repair proteins, normalized to that of H3, was analyzed for control or γ-tubulin siRNA-treated groups. n=5 independent experiments. Bars show mean±s.d. Statistical analysis was performed using two-tailed unpaired Student’s t-test; ***P<0.001.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: Homologous Recombination, Non-Homologous End Joining, Transfection, Control, Plasmid Preparation, Incubation, Expressing, Two Tailed Test, Irradiation, Fractionation

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 5. γ-tubulin depletion reduces the mobilization and formation of DNA damage foci. (A–D) Wild-type (WT) or Kif2C knockout (KO) U2OS cells were treated with control or γ-tubulin siRNA (knockdown, KD). (A) Mean square displacement measurements of EGFP–53BP1 foci were carried out as described in the Materials and Methods, and are shown. (B) Examples of 10 min mobility traces of EGFP–53BP1 are shown. (C) The measured distances traveled by individual 53BP1 foci are shown. The central bar shows the mean. Statistical analysis was performed using two-tailed unpaired Student’s t-test; ns, not significant; **P<0.01; ***P<0.001. (D) The depletion of γ-tubulin was confirmed by IB. EGFP–53BP1 was transiently transfected, and expressed at a moderate level, as observed by immunofluorescence (Fig. S7C), relative to endogenous 53BP1. (E,F) HeLa cells expressing mApple–53BP1 were treated with control or γ-tubulin siRNA. Cells were irradiated with 20 Gy of X-ray irradiation (IR), followed by 30 min incubation. 53BP1 foci are shown in panel F and quantified in panel E (n>100). All data were collected from at least three independent experimental sets as mean±s.d. **P<0.01 by unpaired two-tailed Student’s t-test.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: Knock-Out, Control, Knockdown, Two Tailed Test, Transfection, Immunofluorescence, Expressing, Irradiation, Incubation

Journal: Journal of cell science

Article Title: γ-tubulin mediates DNA double-strand break repair.

doi: 10.1242/jcs.262255

Figure Lengend Snippet: Fig. 6. Inhibition of γ-tubulin enhances tumor cell responses to DNA damage. (A–D) HeLa cells were treated with control, etoposide (2 µM), gatastatin G2 (2 µM), doxorubicin (3 µM), MR-3 (15 µM) and combinations of drugs, as indicated. Cell numbers were quantified as described in the Materials and Methods. Relative cell viability was calculated by normalizing the final cell count relative to the count recorded on the initial day. The displayed results depict the mean±s.d. derived from at least three distinct experiments. *P<0.05; **P<0.01; two-way ANOVA with Tukey's multiple post hoc test. (E,F) SCC38 cells were treated with control, etoposide (3 µM), doxorubicin (4 µM), MR-3 (15 µM) and combinations of drugs, as indicated. Cell numbers were quantified, and relative cell viability was calculated by normalizing the final cell count relative to the count recorded on the initial day. The displayed results depict the mean ±s.d. derived from at least three distinct experiments. *P<0.05; **P<0.01; one-way ANOVA with Dunnett's multiple post hoc test. (G–I) SCC38 cells were maintained under anchorage-independent culture conditions. Cells were then incubated with gatastatin G2 (3 µM), MR-3 (15 µM), doxorubicin (4 µM) and combinations of drugs, as indicated. (G) Representative images of spheroid formation. (H,I) The relative diameters of spheroids in treated groups were normalized to that of the control. The data were analyzed from at least three independent experimental sets using one-way ANOVA with Dunnett’s multiple post hoc test and are depicted as mean±s.e.m. ***P<0.001.

Article Snippet: MATERIALS AND METHODS Cell culture, transfection and treatment The human cervix carcinoma HeLa cell line was obtained from and authenticated by American Type Culture Collection, and cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone).

Techniques: Inhibition, Control, Cell Counting, Derivative Assay, Incubation

Journal: Scientific Reports

Article Title: On the mechanism of miR-29b enhancement of etoposide toxicity in vitro

doi: 10.1038/s41598-024-70856-y

Figure Lengend Snippet: Effect of siRNA against Mcl-1 on etoposide cytotoxicity in different cell lines. (Panel a ): The cells were transfected (6 h) with siRNA against Mcl-1 or negative control. Cells were then treated with 60 μM etoposide 24 h post transfection and incubation continued until experiment was ended. The proliferation was calculated from doubling time value as explained in Materials and methods section. Each bar represents mean ± s.d. of at least three independent experiments (N HeLa = 4, N Hep G2 = 3, N Caco-2 = 3), in each experiment triplicate measurements were performed. **p < 0.01 versus corresponding negative control. (Panel b ): Evaluation of siRNA efficiency by western blot analysis in Hela cells. The cells were transfected (6 h) with anti-Mcl-1 siRNA or negative control. Shown are representative western blots of cells cultured for 24 h after transfection. (Panel c ): Evaluation of siRNA efficiency by western blot analysis in Hep G2 cells. Treatment was the same as for panel b. Panel d: Evaluation of siRNA efficiency by western blot analysis in Caco-2 cells. Treatment was the same as for (panel b ).

Article Snippet: HeLa cells were obtained from The European Collection of Authenticated Cell Cultures (ECACC, No. 93021013 Human Negroid cervix epitheloid carcinoma cell line) via Sigma-Aldrich as provider.

Techniques: Transfection, Negative Control, Incubation, Western Blot, Cell Culture