cdc7 in 1  (MedChemExpress)

Journal: Advanced Science

Article Title: CDC7 Inhibition Potentiates Antitumor Efficacy of PARP Inhibitor in Advanced Ovarian Cancer

doi: 10.1002/advs.202403782

Figure Lengend Snippet: Drug screening identifies CDC7 inhibitor as a potential sensitizer to Olaparib in OV. A,B) Representative images (A) and quantifications (B) of colony formation assay in OV cell lines treated with three PARPi (Olaparib, Niraparib, and Talazoparib) at indicated concentrations. The cut‐off values for sensitive and resistant were 50% based on the relative colony area (in comparison to the control group). C) Outline of the drug screening using 130 kinase inhibitors that target cell cycle‐related pathways in combination with Olaparib in OVCAR5 and OVCAR8 cells. D) Distribution of the S/C (single/combination) score for all 130 kinase inhibitors in OVCAR5 and OVCAR8 cells upon combinational drug screening. E) The top 5 overlapped drugs were identified as sensitizers of Olaparib in OVCAR5 and OVCAR8 cells. F) Heatmap showing the survival rate of OVCAR5 and OVCAR8 cells treated with the top 5 overlapped drugs (1.0 µ m ) from the drug screen in the presence or absence of Olaparib. G) Combination index analysis of XL413 and Olaparib in OVCAR5 and OVCAR8 cells (Combination index value < 1 was defined as synergy). H) Cell proliferation assay showing the growth effect of indicated treatments in OVCAR5 and OVCAR8 cells. I) Colony formation assay in OVCAR5 and OVCAR8 cells in response to combination treatment of Olaparib and XL413. J) Immunoblot analysis of CDC7 and GAPDH in lysates collected from OVCAR5 and OVCAR8 cells transfected with siNC or siCDC7. K) Sensitivity of OVCAR5 and OVCAR8 cells transfected with siNC or siCDC7 in response to indicated concentrations of Olaparib. L,M) Representative images (L) and quantifications (M) of the percentages of G2/M, S, and G1 phase in OVCAR5 and OVCAR8 cells. Cells were exposed to indicated treatments, stained with propidium iodide, and analyzed using flow cytometry. Data in (H,K) are shown as mean ± SD of triplicate (two‐way ANOVA). Data in (M) are shown as mean ± SD of triplicate (one‐way ANOVA). ns, no significance, * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: [ ] The following antibodies were used for immunoblot analysis: CDC7 (Santacruz, sc‐56275), GAPDH (Proteintech, 60004‐1‐1 g), ACTIN(CST, 8456S), γH2AX (CST, 2577S), H3 (CST, 3638S), P‐MCM2 (Abcam, ab133243), MCM2 (Abcam, ab108935), P‐TBK1 (CST, 5483S), TBK1 (CST, 3504S), P‐IRF3 (CST, 4947S), IRF3 (CST, 4302S), cGAS (CST, 31659), STING (CST, 13467), BRCA1 (CST, 9010) and a‐tublin (Proteintech, 66031‐I‐1 g).

Techniques: Drug discovery, Colony Assay, Comparison, Control, Proliferation Assay, Western Blot, Transfection, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: CDC7 Inhibition Potentiates Antitumor Efficacy of PARP Inhibitor in Advanced Ovarian Cancer

doi: 10.1002/advs.202403782

Figure Lengend Snippet: Enhanced genome instability and replication stress by CDC7 inhibition in combination with Olaparib. A) Heatmap of differentially expressed genes ( p < 0.05) in OVCAR5 cells following the indicated treatments (Duplicates). B) GSEA analysis using RNA‐seq data showed the significant enrichment of the Gene Ontology (GO) gene sets “the DNA REPLICATION” and the KEGG gene sets “DNA_DOUBLE_STRAND_BREAK_PROCESSING.” C–E) DNA fiber assay in OVCAR5 and OVCAR8 cells exposed to indicated treatments. C) Cells were treated with 20 µ m IdU for 30 min, followed by 200 µ m CIdU and indicated treatments. Representative images of DNA fibers (D) and quantifications of CIdU/ldU ratio (E) were shown. F,G) Representative images (F) and quantifications (G) of neutral comet assay in OVCAR5 and OVCAR8 cells following the indicated treatments. Scale bar, 20 µm. H,I) Representative images (H) and quantifications (I) of γH2AX foci in OVCAR5 and OVCAR8 cells following the indicated treatments. Scale bar, 2.5 µm. J) Immunoblot analysis of indicated proteins including γH2AX, Histone H3, total and phospho MCM2, and ACTIN in lysates collected from OVCAR5 and OVCAR8 cells following the indicated treatments. Data in (E,G,I) are shown as mean ± SD of triplicate (one‐way ANOVA). ** p < 0.01; *** p < 0.001.

Article Snippet: [ ] The following antibodies were used for immunoblot analysis: CDC7 (Santacruz, sc‐56275), GAPDH (Proteintech, 60004‐1‐1 g), ACTIN(CST, 8456S), γH2AX (CST, 2577S), H3 (CST, 3638S), P‐MCM2 (Abcam, ab133243), MCM2 (Abcam, ab108935), P‐TBK1 (CST, 5483S), TBK1 (CST, 3504S), P‐IRF3 (CST, 4947S), IRF3 (CST, 4302S), cGAS (CST, 31659), STING (CST, 13467), BRCA1 (CST, 9010) and a‐tublin (Proteintech, 66031‐I‐1 g).

Techniques: Inhibition, RNA Sequencing, Neutral Comet Assay, Western Blot

Journal: Advanced Science

Article Title: CDC7 Inhibition Potentiates Antitumor Efficacy of PARP Inhibitor in Advanced Ovarian Cancer

doi: 10.1002/advs.202403782

Figure Lengend Snippet: Induction of cell‐autonomous cGAS/STING response by Olaparib and CDC7 inhibitor. A,B) GSEA analysis showing the “CYTOSOLIC DNA SENSING PATHWAY” (A) and “INTERFERON ALPHA RESPONSE/INTERFERON GAMMA RESPONSE” (B) were enriched in combination treatment group using KEGG and Hallmarks gene sets. C) Heatmap of the significantly upregulated ISGs ( p < 0.05) upon combinational treatment of XL413 and Olaparib versus control in OVCAR5 cells (Duplicates). D,E) Representative images (D) and quantifications (E) of PicoGreen staining in OVCAR5 and OVCAR8 cells following the indicated treatments. DAPI (blue) was used to visualize the nuclei. Scale bar, 5 µm. Data are shown as mean ± SD (one‐way ANOVA). ** p < 0.01, *** p < 0.001. F) Immunoblot analysis of indicated proteins including total and phospho TBK1, total and phospho IRF3, total and phospho MCM2, and GAPDH in lysates collected from OVCAR5 and OVCAR8 cells following the indicated treatments. G) qRT‐PCR analysis of the indicated ISGs expression levels in OVCAR5 and OVCAR8 cells after treated with CTRL, XL413, Olaparib, and combination treatment. Data are shown as mean ± SD of triplicate (one‐way ANOVA). ns, no significance, * p < 0.05, ** p < 0.01; *** p < 0.001. H) qRT‐PCR analysis of the indicated ISGs expression levels in OVCAR5 cells treated with siNC, siSTING#1 or siSTING#2. Data are shown as mean ± SD of triplicate (two‐way ANOVA). ns, no significance, * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: [ ] The following antibodies were used for immunoblot analysis: CDC7 (Santacruz, sc‐56275), GAPDH (Proteintech, 60004‐1‐1 g), ACTIN(CST, 8456S), γH2AX (CST, 2577S), H3 (CST, 3638S), P‐MCM2 (Abcam, ab133243), MCM2 (Abcam, ab108935), P‐TBK1 (CST, 5483S), TBK1 (CST, 3504S), P‐IRF3 (CST, 4947S), IRF3 (CST, 4302S), cGAS (CST, 31659), STING (CST, 13467), BRCA1 (CST, 9010) and a‐tublin (Proteintech, 66031‐I‐1 g).

Techniques: Control, Staining, Western Blot, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: CDC7 Inhibition Potentiates Antitumor Efficacy of PARP Inhibitor in Advanced Ovarian Cancer

doi: 10.1002/advs.202403782

Figure Lengend Snippet: Targeting CDC7 with Olaparib triggers anti‐tumor immunity in vitro. A) Immunoblot analysis of indicated proteins including total and phospho TBK1, total and phospho IRF3, total and phospho MCM2, and GAPDH in lysates collected from ID8 and HGS1 cells following the indicated treatments. B) qRT‐PCR analysis of the indicated ISGs expression levels in ID8 and HGS1 cells after the indicated treatments. C) Expression levels of MHC‐I in ID8 and HGS1 cells after the indicated treatments. D) ID8‐OVA or HGS1‐OVA cells were treated with the indicated treatments, and then co‐cultured with B3Z cells, B3Z activation was determined by LacZ activity. E,F) Pretreatment of ID8‐OVA or HGS1‐OVA cells with the indicated treatments, and then co‐cultured with OT‐I cells, OT‐I activation was determined by expression of CD69 (E) and GZMB (F) by flow cytometry analysis. G) The cytotoxic effect of OT‐I of ID8‐OVA cells was detected after co‐culturing with OT‐I cells for 48 h. H,I) Immunoblot analysis of indicated proteins including total and phosphor‐TBK1, total and phosphor‐IRF3, STING, and GAPDH in lysates collected from HGS1 cells transfected with sgCtrl or sgSTING (‐1, ‐2) or sgcGAS (‐1, ‐2) following the indicated treatments. J) qRT‐PCR analysis of Ccl5 and Ifi27 in HGS1‐OVA WT or STING −/− cells after the indicated treatments. K) Surface levels of MHC‐I in HGS1‐OVA WT or STING −/− cells after treated with XL413 or Olaparib or their combination for 48 h. L) HGS1‐OVA WT or STING −/− cells were treated with XL413 or Olaparib or their combination for 48 h, then co‐cultured with B3Z for an additional 24 h, after which B3Z activation was determined by LacZ activity. M,N) HGS1‐OVA WT or STING −/− cells were treated with XL413 or Olaparib or their combination, then co‐cultured with OT‐I, after which OT‐I activation was determined by expression of CD69 (M) and GZMB (N) by flow cytometry analysis. Data in (B–G) are shown as mean ± SD of triplicate (one‐way ANOVA). Data in (J–N) are shown as mean ± SD of triplicate (two‐way ANOVA). ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: [ ] The following antibodies were used for immunoblot analysis: CDC7 (Santacruz, sc‐56275), GAPDH (Proteintech, 60004‐1‐1 g), ACTIN(CST, 8456S), γH2AX (CST, 2577S), H3 (CST, 3638S), P‐MCM2 (Abcam, ab133243), MCM2 (Abcam, ab108935), P‐TBK1 (CST, 5483S), TBK1 (CST, 3504S), P‐IRF3 (CST, 4947S), IRF3 (CST, 4302S), cGAS (CST, 31659), STING (CST, 13467), BRCA1 (CST, 9010) and a‐tublin (Proteintech, 66031‐I‐1 g).

Techniques: In Vitro, Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Activation Assay, Activity Assay, Flow Cytometry, Transfection