Journal: Oncogene

Article Title: BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

doi: 10.1038/s41388-023-02603-y

Figure Lengend Snippet: a Immunofluorescence detection of BMAL1 and γH2AX foci in U2OS cells transfected with NC or siBMAL1. Cells were treated with Zeocin or DMSO. Scale bar: 5 μm. b Quantification of panel a . The number of BMAL1 foci per cell ( n ≥ 100). c Quantification of panel a . The number of BMAL1 foci colocalized with γH2AX foci. d Location of I-PpoI cutting site at 28S rDNA. The positions of primers used for ChIP-qPCR are indicated by arrows. e , f ChIP-qPCR for 28S rDNA with or without cutting by I-PpoI and binding by BMAL1. g Flag-BMAL1-overexpressing HEK293T cells were treated with Zeocin or DMSO, followed by pulldown with anti-Flag beads and immunoblotting with antibodies against p-S/TQ, Flag and γH2AX. h Flag-BMAL1-overexpressing HEK293T cells with or without ATM inhibitor KU60019 or ATR inhibitor VE-821 pretreatment were treated with Zeocin, followed by pulldown with anti-Flag beads and immunoblotting with antibodies against p-S/TQ and Flag. i HEK293T cells with Zeocin pretreatment were overexpressed Flag-Wild type BMAL1 (BMAL1-WT) or Flag-S183A BMAL1 (BMAL1-S183A), followed by pulldown with anti-Flag beads and immunoblotting with antibodies against p-S/TQ and Flag. j Immunofluorescence detection of Flag and γH2AX foci in U2OS cells overexpressed with Flag-BMAL1-WT or Flag-BMAL1-S183A. Cells were treated with Zeocin. Scale bar: 5 μm. k Quantification of panel j . The number of Flag foci colocalized with γH2AX foci. l ChIP-qPCR for 28S rDNA with or without cutting by I-PpoI and binding by BMAL1-WT or BMAL1-S183A. All values are the average ± SEM of three independent experiments. Student’s unpaired two-tailed t-test was used to determine the statistical significance ( *** P < 0.001; **** P < 0.0001).

Article Snippet: Antibodies used: BMAL1 (1:100, NB100-2288, Novus Biologicals), CLOCK (1:100, NBP1-51610, Novus Biologicals), mouse-γH2AX (1:400, ab26350, Abcam), rabbit-γH2AX (1:400, 9718, CST), RPA1 (1:100, sc-28304, Santa Cruz), RAD51 (1:400, ab133534, Abcam), BrdU (1:300, 347580, BDIS), Flag (1:1000, M20008, Abmart).

Techniques: Immunofluorescence, Transfection, ChIP-qPCR, Binding Assay, Western Blot, Two Tailed Test

Journal: Oncogene

Article Title: BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

doi: 10.1038/s41388-023-02603-y

Figure Lengend Snippet: a Representative results of neutral comet assay. U2OS cells were transfected with siNC, siBMAL1-1 or siBMAL1-2. b Quantification of panel a . The percentages of DNA in the tail ( n ≥ 100). c Immunoblot analysis of phosphorylated ATM (p-ATM), total ATM and BMAL1 in U2OS cells transferred with siNC, siBMAL1-1 or siBMAL1-2. d Immunoblot analysis of γH2AX and BMAL1. HEK293T cells over-expressed with BMAL1-WT or BMAL1-S183A were treated with Zeocin and incubated in fresh culturing medium for 6 h. e FACS analysis the frequency of HR in BMAL1-WT or BMAL1-S183A overexpressed U2OS DR‐GFP cell line transfected with I-SceI. f FACS analysis the frequency of NHEJ in BMAL1-WT or BMAL1-S183A overexpressed U2OS EJ5-GFP cell line transfected with I-SceI. g Immunoblot analysis of γH2AX and BMAL1. B02 or NU7441 or DMSO was added to culturing medium and cells over-expressing vector or BMAL1-WT were collected 6 h after Zeocin treatment. All values are the average ± SEM of three independent experiments. Student’s unpaired two-tailed t-test was used to determine the statistical significance ( *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used: BMAL1 (1:100, NB100-2288, Novus Biologicals), CLOCK (1:100, NBP1-51610, Novus Biologicals), mouse-γH2AX (1:400, ab26350, Abcam), rabbit-γH2AX (1:400, 9718, CST), RPA1 (1:100, sc-28304, Santa Cruz), RAD51 (1:400, ab133534, Abcam), BrdU (1:300, 347580, BDIS), Flag (1:1000, M20008, Abmart).

Techniques: Neutral Comet Assay, Transfection, Western Blot, Incubation, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Oncogene

Article Title: BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

doi: 10.1038/s41388-023-02603-y

Figure Lengend Snippet: a Immunofluorescence detection of RAD51 and γH2AX foci in U2OS cells transfected with NC, siBMAL1-1 or siBMAL1-2. Cells were treated with Zeocin. Scale bar: 5 μm. b Quantification of panel a . The number of RAD51 foci per cell ( n ≥ 100). c Quantification of panel a . The number of RAD51 foci colocalized with γH2AX foci. d Immunofluorescence detection of RPA1 and γH2AX foci in U2OS cells transfected with NC, siBMAL1-1 or siBMAL1-2. Cells were treated with Zeocin. Scale bar: 5 μm. e Quantification of panel d . The number of RPA1 foci per cell ( n ≥ 100). f Quantification of panel d . The number of RPA1 foci colocalized with γH2AX foci. g Detection of the abundance of ssDNA in BMAL1 depleted HEK293T cells transfected with I-PpoI. qPCR was performed on the genomic DNA with the indicated primers amplifying regions including the positions at 0.1, 0.5 and 1 kb downstream from the DSB, as depicted. The values of %ssDNA was calculated as follows: %ssDNA=1/[(2ΔCt-1)+0.5] × 100. h Immunofluorescence detection of BrdU foci in U2OS cells transfected with NC, siBMAL1-1 or siBMAL1-2. Cells were treated with Zeocin. Scale bar: 5 μm. i Quantification of panel h . The percentage of cell with BrdU foci ( n ≥ 100). All values are the average ± SEM of three independent experiments. Student’s unpaired two-tailed t-test was used to determine the statistical significance ( * P < 0.05, *** P <0.001; **** P < 0.0001).

Article Snippet: Antibodies used: BMAL1 (1:100, NB100-2288, Novus Biologicals), CLOCK (1:100, NBP1-51610, Novus Biologicals), mouse-γH2AX (1:400, ab26350, Abcam), rabbit-γH2AX (1:400, 9718, CST), RPA1 (1:100, sc-28304, Santa Cruz), RAD51 (1:400, ab133534, Abcam), BrdU (1:300, 347580, BDIS), Flag (1:1000, M20008, Abmart).

Techniques: Immunofluorescence, Transfection, Two Tailed Test

Journal: Oncogene

Article Title: BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

doi: 10.1038/s41388-023-02603-y

Figure Lengend Snippet: a , b ChIP-qPCR showing the relative enrichment of Histone H4 acetylation (H4Ac) at 28S rDNA with or without cutting by I-PpoI. HEK293T cells were transfected with siNC, siBMAL1-1 or siBMAL1-2. c Immunofluorescence detection of CLOCK and γH2AX foci in U2OS cells transfected with NC or siBMAL1. Cells were treated with Zeocin or DMSO. Scale bar: 5 μm. d Quantification of panel c . The number of CLOCK foci per cell ( n ≥ 100). e Quantification of panel c . The number of CLOCK foci colocalized with γH2AX foci. f ChIP-qPCR for 28S rDNA with or without cutting by I-PpoI and binding by CLOCK. HEK293T cells were transfected with siNC or siBMAL1. g ChIP-qPCR for DAB1 with or without cutting by I-PpoI and binding by CLOCK. HEK293T cells were transfected with siNC or siBMAL1. h Co-IP assay to determine the interaction of Flag-BMAL1-WT or Flag-BMAL1-S183A with CLOCK in control and Zeocin treated HEK293T cells. HEK293T cells were immunoprecipitated with Flag-beads. All values are the average ± SEM of three independent experiments. Student’s unpaired two-tailed t -test was used to determine the statistical significance ( ** P < 0.01, *** P <0.001; **** P < 0.0001).

Article Snippet: Antibodies used: BMAL1 (1:100, NB100-2288, Novus Biologicals), CLOCK (1:100, NBP1-51610, Novus Biologicals), mouse-γH2AX (1:400, ab26350, Abcam), rabbit-γH2AX (1:400, 9718, CST), RPA1 (1:100, sc-28304, Santa Cruz), RAD51 (1:400, ab133534, Abcam), BrdU (1:300, 347580, BDIS), Flag (1:1000, M20008, Abmart).

Techniques: ChIP-qPCR, Transfection, Immunofluorescence, Binding Assay, Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Two Tailed Test

Journal: Oncogene

Article Title: BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

doi: 10.1038/s41388-023-02603-y

Figure Lengend Snippet: a Kaplan–Meier curve analysis of the overall survival probabilities of ACC patients. Patients were assigned to two groups based on low or high levels of BMAL1 expression. b The relative expression level of BMAL1 in specimens from patients with stage I to IV ACC. Data were obtained from the TCGA database. c Mice were injected subcutaneously with 1 × 10 7 normal (shNC) or BMAL1 stably depleted (shBMAL1) SW-13 cells and treated with 5 mg/kg of VP16 or vehicle control every two days starting on day 0 for 8 days. Representative images of xenograft tumors were showed. d Quantification of c . The volume of xenograft tumors ( n = 5 per group). e Representative histology staining images showing the signal of γH2AX in VP16 treated and untreated xenograft tumors driven by normal or BMAL1 depleted SW-13 cells. Scale bar: 20 μm. f Quantification of e to determine the percentage of γH2AX positive cells. g Representative histology staining images showing the apoptotic cells in VP16 treated and untreated xenograft tumors driven by normal or BMAL1 depleted SW-13 cells. Scale bar: 100 μm. h Quantification of g to determine the percentage of apoptotic cells. For each group, >1000 cells were examined. All values are the average±SEM of three independent experiments. The unpaired Student’s two-tailed t -test was used to determine the statistical significance ( * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used: BMAL1 (1:100, NB100-2288, Novus Biologicals), CLOCK (1:100, NBP1-51610, Novus Biologicals), mouse-γH2AX (1:400, ab26350, Abcam), rabbit-γH2AX (1:400, 9718, CST), RPA1 (1:100, sc-28304, Santa Cruz), RAD51 (1:400, ab133534, Abcam), BrdU (1:300, 347580, BDIS), Flag (1:1000, M20008, Abmart).

Techniques: Expressing, Injection, Stable Transfection, Control, Staining, Two Tailed Test

Journal: British journal of pharmacology

Article Title: Role of the CLOCK protein in liver detoxification.

doi: 10.1111/bph.14828

Figure Lengend Snippet: FIGURE 1 Clock deletion disrupts the rhythmicity of CYP expression in mouse liver. (a) Validation of Clock knockout (Clock−/−) mice by determining Clock transcript. (b) Heatmap showing relative mRNA expression of drug-metabolizing enzymes at ZT2 and ZT14 in Clock+/+ versus Clock−/−mice. (c) Diurnal mRNA expression of Cyp2a4/5 and Cyp2b10 in the livers from Clock+/+ and Clock−/−mice; Npas2+/+ and Npas2−/−mice; and Bmal1+/+ and Bmla1−/−mice. (d) Diurnal protein expression of CYP2A4/5 and CYP2B10 in the livers from Clock+/+ and Clock−/−mice. (e) CYP activities derived from liver microsomes of Clock+/+ and Clock−/−mice. The specific substrates used to determine the activities of CYPs were testosterone for CYP2A4, coumarin for CYP2A5, and pentoxyresorufin for CYP2B10. In Panels a, c, d, and e, data are mean ± SD (n = 5). *P < .05, significant differences between two genotypes; two-way ANOVA with Bonferroni post hoc test

Article Snippet: Clock−/−, Npas2−/−, Bmal1−/−, Rev-erbα−/−, and Rev-erbβ−/− (on a C57BL/6 background) mice were obtained from Cyagen Biosciences Inc (Guangzhou, China).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Derivative Assay