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