clonogenic survival assays  (KUB Technologies Inc)

Journal: Hepatology (Baltimore, Md.)

Article Title: HMGB1 Controls Liver Cancer Initiation through YAP-dependent Aerobic Glycolysis

doi: 10.1002/hep.29663

Figure Lengend Snippet: (A) Q-PCR analysis of mRNA expressions of indicated genes in Hepa1-6 and HuH7 cells after knockdown of HMGB1 (n=3, *p<0.05 versus control shRNA group). (B, C) Western blot analysis of indicated protein expression in Hepa1-6 and HuH7 cells after knockdown of HMGB1 or YAP. (D) Forced expression of HMGB1 cDNA restored HMGB1 and YAP expression in HMGB1−/− MEFs. (E) Forced expression of YAP cDNA restored YAP expression, but not HMGB1 expression in indicated HMGB1-knockdown HCC cells. (F) Forced expression of YAP reversed cell proliferation inhibition in HMGB1 knockdown HCC cells (n=3, *p < 0.05). (G–H) Clonogenic cell survival assay determines the reproductive ability of indicated HCC cells. A representative image is shown in panel G. Relative reproductive ability is semi-quantified in panel H (n=3, *p < 0.05).

Article Snippet: Cell viability and clonogenic cell survival assay Cell viability was assayed using Cell Counting Kit-8 kits (Dojindo Laboratories).

Techniques: Knockdown, Control, shRNA, Western Blot, Expressing, Inhibition, Clonogenic Cell Survival Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: HMGB1 Controls Liver Cancer Initiation through YAP-dependent Aerobic Glycolysis

doi: 10.1002/hep.29663

Figure Lengend Snippet: (A) Indicated mouse and human HCC cells were treated with glycyrrhizin (0.625, 1.25, 2.5, 5, 10 mM) or verteporfin (2.5, 5, 10, 20, and 40 µM) for 24 hours. Cell viability was assayed (n=3, *p<0.05 versus control untreated group). (B) Clonogenic cell survival assay determined the reproductive ability of Hepa1-6 and HuH7 cells following treatment with glycyrrhizin (2.5 mM) or verteporfin (10 µM). A representative image is shown in the left panel. Relative reproductive ability is semi-quantified in the right panel (n=3, *p < 0.05 versus control untreated group). (C) Q-PCR analysis of mRNA expressions of indicated genes in HCC cells after glycyrrhizin (2.5 mM) or verteporfin (10 µM) treatment for 24 hours (n=3, *p<0.05). (D–F) ECAR levels in indicated HCC cells (n=3, *p < 0.05). (G) HIF1α DNA binding activity in indicated HCC cells with or without glycyrrhizin (2.5 mM) or verteporfin (10 µM) treatment for 24 hours (n=3, *p<0.05 versus control in the treated group). (H) Immunoprecipitation analysis of HIF1α-YAP complex in hepatocytes from DEN-induced Hmgb1f/f mice or primary human HCC (pHCC) cells or HuH7 cells. (I–K) YAP-knockdown HuH7 cells were transfected with YAP-cDNA or YAP-5SA-S94A mutant for 48 hours, and then treated with hypoxia (1% O2) for 24 hours. HIF1α DNA binding activity (I), gene expression (J), and YAP-HIF1α complex (K) were assayed.

Article Snippet: Cell viability and clonogenic cell survival assay Cell viability was assayed using Cell Counting Kit-8 kits (Dojindo Laboratories).

Techniques: Control, Clonogenic Cell Survival Assay, Binding Assay, Activity Assay, Immunoprecipitation, Knockdown, Transfection, Mutagenesis, Gene Expression

Journal: ACS chemical biology

Article Title: Identification of small molecule translesion synthesis inhibitors that target the Rev1-CT/RIR protein-protein interaction

doi: 10.1021/acschembio.6b01144

Figure Lengend Snippet: Compounds 4 and 5 sensitize HT1080 cells to killing by cisplatin and reduce mutagenesis. (A) Compounds 4 and 5 enhance sensitivity to cisplatin in a colony survival assay. Cells were incubated with cisplatin (0.6 μM) for 24 hrs, followed by the addition of either 4 or 5 (1.5 μM) and colonies were allowed to form for seven days. Colonies were stained and counted with coomassie blue. *P<0.05 and ***P<0.001 were calculated with the Student t-test using comparisons as indicated. (B) Compounds 4 and 5 reduce the cisplatin-induced HPRT mutations in HT1080 cells. Data are the Ave ± SD of two separate experiments with 3 replicates each. All experiments were performed at least two separate times and results were comparable across all replicates. **P<0.01 and ***P<0.001 versus cisplatin treated cells, Student t-test.

Article Snippet: Clonogenic Survival Assay HT1080 cells were purchased from ATCC and grown in RPMI 1640 (Gibco), supplemented with 10% FBS (HyClone) and 1% Penicillin/Streptomycin.

Techniques: Mutagenesis, Clonogenic Cell Survival Assay, Incubation, Staining

Journal: ACS chemical biology

Article Title: Identification of small molecule translesion synthesis inhibitors that target the Rev1-CT/RIR protein-protein interaction

doi: 10.1021/acschembio.6b01144

Figure Lengend Snippet: Enhanced sensitivity to UV light in HT1080 cells treated with Rev1-CT/RIR PPI inhibitors. In (A) and (B) cells were plated and exposed to UV light (5 J/m2) or left untreated. The next day, 4 or 5 was added and cell viability was measured 24 hrs later. **P<0.01 and ***P<0.001 versus cells treated with compound alone, Student t-test. (C) Cells were treated with UV light followed by dosing with 4 or 5 (1.5 μM). Cells were incubated to allow for the formation of colonies that were counted with commassie blue. *P<0.05 were calculated with the Student t-test using comparisons as indicated. Data are the Ave ± SD of two separate experiments performed in triplicate. Experiments were performed at least two separate times and results were comparable across all replicates.

Article Snippet: Clonogenic Survival Assay HT1080 cells were purchased from ATCC and grown in RPMI 1640 (Gibco), supplemented with 10% FBS (HyClone) and 1% Penicillin/Streptomycin.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21

doi: 10.1016/j.jbc.2023.104900

Figure Lengend Snippet: Influence of Dyrk1A on NER and cell survival post-UV. A , left: knockout of Dyrk1A by CRISPR/Cas9 in LF-1 primary lung fibroblasts. sgRNA against the adeno-associated virus integration site (AAVS1) was used as a negative control. On this and all subsequent immunoblots, “ns” indicates a nonspecific band; Right: quantification of 6-4PP removal as in Figure 1 C . B , evaluation of unscheduled DNA synthesis (UDS) post-UV in HeLa cells treated with siRNA against Dyrk1A (siDyrk1A) or XPA (siXPA). Left : immunoblots showing knockdown of Dyrk1A and XPA. Right : quantification of EdU incorporation in G1/G2 after 20 J/m 2 UV or mock treatment. C , clonogenic survival post-UV in HeLa cells treated with siDyrk1A and/or siXPA. Left : immunoblot showing protein knockdown. Middle : clonogenic survival. Right : LD90 values were determined from clonogenic survival curves using GraphPad Prism v8. Data are reported as the mean ± SD for at least three independent experiments. p -values were obtained using the two-tailed unpaired Student t test, adjusted for multiple tests by the Holm-Sidak method; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant. 6-4PP, 6-4 pyrimidine-pyrimidone photoproduct; NER, nucleotide excision repair.

Article Snippet: Right : LD90 values were determined from clonogenic survival curves using GraphPad Prism v8.

Techniques: Knock-Out, CRISPR, Virus, Negative Control, Western Blot, DNA Synthesis, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21

doi: 10.1016/j.jbc.2023.104900

Figure Lengend Snippet: Dyrk1A promotes NER by regulating cyclin D1 stability. A , left : immunoblot from total HeLa cell extracts, following treatment with siRNA against Dyrk1A (siDyrk1A) and/or cyclin D1 (siCyclin D1). Right : cell cycle distribution assessed by DNA content analysis (DAPI) and EdU as in Figure 3 F . B , quantification of 6-4PP removal in HeLa cells ± siDyrk1A and/or siCyclin D1. VE-821 (10 μM) was employed as ATR inhibitor (ATRi). C , clonogenic survival post-UV (5 J/m 2 ) in HeLa cells ± siDyrk1A and/or siCyclin D1. D , expression of Flag-HA-cyclin D1 (WT or T286A) in WM1366 using a retroviral construct. Flag-HA-GFP is used as a control. E , left : immunoblot of retinoblastoma protein (Rb) and phospho-Rb (p-Rb) in WM1366 ± cyclin D1(T286A), pretreated or not for 4 h with 10 μM palbociclib. Right : overexpression of cyclin D1 (T286A) in WM1366 decreases the % of cells in G1 in a CDK-dependent manner. Cell cycle was assessed by flow cytometry of cells labeled with EdU and DAPI, with or without pretreatment with the CDK4/6 inhibitor palbociclib for 24 h. F , effect of cyclin D1(T286A) overexpression on 6-4PP removal. Cells were pretreated (or not) with palbociclib for 4 h, and the drug was maintained in the medium during post-UV incubation. G , effect of cyclin D1 overexpression on CPD removal. Cultures were pulsed with BrdU to label cells that were in S phase at the time of irradiation. Post-UV incubations were carried out in the presence of nocodazole to prevent cell division. Left : BrdU(−) and BrdU(+) cells were gated to select cells remaining in G1 and S, respectively, as indicated. Right : % of CPD remaining in G1 and S at 10 h and 20 h post UV. The XPA-null human fibroblast line GM04429 was used as a control. Data are reported as the mean ± SD for at least three independent experiments. p values were obtained using the two-tailed unpaired Student t test, adjusted for multiple tests by the Holm-Sidak method where applicable; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant. 6-4PP, 6-4 pyrimidine-pyrimidone photoproduct; CDK, cyclin-dependent kinase; CPD, cyclobutane pyrimidine dimer; NER, nucleotide excision repair; Rb, retinoblastoma protein.

Article Snippet: Right : LD90 values were determined from clonogenic survival curves using GraphPad Prism v8.

Techniques: Western Blot, Expressing, Retroviral, Construct, Control, Over Expression, Flow Cytometry, Labeling, Incubation, Irradiation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21

doi: 10.1016/j.jbc.2023.104900

Figure Lengend Snippet: Overexpression of cyclin D1 (T286A) sensitizes WM1366 to UV killing. A , UV sensitivity measured by clonogenic survival. B , detection of phospho-53BP1(S1778) and γ-H2AX in WM1366 cells at 16 h after irradiation with 20 J/m 2 UV. C , apoptosis assessed by cleavage of caspase 3 and of poly(ADP-ribose) polymerase 1 (PARP1) at 16 h post-UV. Cells treated with 1 μM staurosporine for 3 h was used as a positive control. Data are reported as the mean ± SD for at least three independent experiments. p values were obtained using the two-tailed unpaired Student t test, adjusted for multiple tests by the Holm-Sidak method where applicable; ∗ p < 0.05, ∗∗∗ p < 0.001. ns, not significant.

Article Snippet: Right : LD90 values were determined from clonogenic survival curves using GraphPad Prism v8.

Techniques: Over Expression, Irradiation, Positive Control, Two Tailed Test