Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: TGF-β1-induced ROS accumulation leads to G2 arrest through ATM signaling. (A). RT-qPCR analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (B). Western blot analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (C). Western blot analysis of ATM signaling phosphorylated activation in the condition of ATM or CHK2 knockdown; (D). Western blot analysis of ATM signaling phosphorylated activation under N-acetyl-l-cysteine (NAC) treatment; (E). Western blot analysis of ATM signaling phosphorylated activation under decitabine treatment. Reagent doses and duration: TGF-β1 (10 ng/ml, 48 h), NAC (4 mM, 48 h), decitabine (10uM, 48 h). Predicted molecular weight: ATM 350 kDa, pATM 370 kDa, CDC25C&pCDC25C 60 kDa, CHK1&pCHK1 54 kDa, CHK2&pCHK2 62 kDa, β-Tubulin 55 kDa; GAPDH 37 kDa. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns denotes not significant by one-way analysis of variance followed by the Tukey’s test. Error bars represent means ± SEM of at least three independent experiments.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, Knockdown, Molecular Weight

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/elife.104718

Figure Lengend Snippet: Figure 4. Isobavachalcone (IBC) inhibits the CHK2 kinase. (A) Selected protein kinases were incubated with the indicated range of IBC concentrations, and kinase activity in vitro was determined by using a radiometric assay. The IC50 of IBC for each kinase is indicated in the panel on the right. (B) MCF- 7 cells were pretreated with DMSO, 15 µM IBC, or 20 µM BML-277 for 2 hr, then camptothecin (CPT, 1 μM) was added for 2 hr. Phosphorylation of CHK2 on S516 (pCHK2) was detected by western blotting. The ratio of pCHK2-S516 induction, relative to the DMSO + CPT control, is indicated (n = 3). (C) MCF-7 cells were treated as indicated in (B). Phosphorylation of chromatin-bound BRCA1 at residue S988 was detected by western blotting. The relative ratio of pBRCA1-S988 signal, after normalization to Ponceau signal, is indicated. TBP was used as a marker of chromatin fraction. (D) MCF-7 cells

Article Snippet: DOI: https://doi.org/10.7554/eLife.104718 18 of 25 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- TBP 8515 Cell Signaling 1/1000 Antibody Mouse monoclonal anti- CHK1 2360 Cell Signaling 1/1000 Antibody Rabbit polyclonal anti- pRPA32 (S4/S8) A300- 245A Bethyl 1/1000 Antibody Rabbit monoclonal anti- CHK2 ab109413 Abcam 1/5000 Antibody Goat polyclonal Anti- RPA2 A303- 874 Bethyl 1/500 Antibody Rabbit polyclonal anti- pCHK2 (S516) 2669 Cell Signaling 1/1000 Antibody Rabbit polyclonal anti- pChk1 (S296) 2349 Cell Signaling 1/1000 Antibody Mouse monoclonal antipBRCA1 (S988) sc- 166793 Santa Cruz 1/200 Antibody Rabbit polyclonal anti- RAD51 PC130 Millipore 1/500 Antibody Rabbit polyclonal anti- pAKT (S473) 9271 Cell Signaling 1/1000 Continued

Techniques: Incubation, Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Control, Residue, Marker

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/eLife.104718

Figure Lengend Snippet: ( A ) MCF-7 cells treated with DMSO, 15 μM IBC, 20 μM bakuchiol (BKC), the combination IBC + BKC or AKT inhibitor, MK-2206, (AKTi, 10 μM) for 24 hr. Autophosphorylation of AKT was detected by immunoblotting analysis. Densitometric quantification of phosphor-AKT signal is shown. (n = 2). ( B ) MCF-7 cells treated overnight with DMSO, IBC, or AKT inhibitor (AKTi). Total RNA was isolated. Reverse transcription and quantitative PCR was performed with specific primers targeting the gene bodies of PCNA, E2F1, and E2F2 (n = 2). ( C ) MCF-7 cells were treated with IBC or AKT inhibitor (AKTi) for 24 hr. They were then sequentially labeled with IdU and CldU for 20 min. Replication fork progression was measured using DNA fiber spreading. The median length of CldU tracks of two biological replicates is indicated in red. ( D ) In vitro kinase assay of 43 cell cycle-related kinases following treatment with 30 µM IBC. The CHK2(I157T) mutation is linked to an increased risk of breast and colorectal cancers. CHK2(R145W) is associated with Li–Fraumeni syndrome. Both mutations do not affect the basal kinase activity of CHK2. ( E ) Percentage inhibition of kinase activity by IBC treatment is shown. ( F ) AKT kinase peptides were incubated with a half-log range dilution series of Isobavachalcone and in vitro kinase activity was measured using a radiometric assay. Data are presented as mean ± SD with a technical triplicate. ( G ) Densitometric quantification of the pCHK2-S516 induction by CPT for . The relative induction of pCHK2-S516 by CPT after IBC or BML-277 treatment compared to DMSO control is indicated. Mean ± SD of three independent experiments for DMSO + CPT and IBC + CPT are shown. The p-value was determined using two-tailed unpaired t -test. ( H ) Densitometric quantification of the pCHK2-S516 induction by CPT for . The relative induction of pBRCA1-S988 by CPT after IBC or BML-277 treatment compared to DMSO control is indicated. Mean ± SD of three independent experiments for DMSO + CPT and IBC + CPT are shown. The p-value was determined using two-tailed unpaired t -test. ( I ) Densitometric quantification of the pCHK1-S296 induction by HU for . The relative induction of pCHK1-S296 by HU after IBC or BML-277 treatment compared to DMSO control is indicated (n = 2). Figure 4—figure supplement 1—source data 1. Original membranes corresponding to . 4 and 7 are our codes for BKC and IBC, respectively. d: DMSO control; 7+4: means BKC + IBC. Figure 4—figure supplement 1—source data 2. Original membranes corresponding to with labels.

Article Snippet: Antibody , Rabbit polyclonal anti-pCHK2 (S516) , 2669 , Cell Signaling , 1/1000.

Techniques: Western Blot, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Labeling, In Vitro, Kinase Assay, Mutagenesis, Activity Assay, Inhibition, Incubation, Control, Two Tailed Test

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/eLife.104718

Figure Lengend Snippet: ( A ) Selected protein kinases were incubated with the indicated range of IBC concentrations, and kinase activity in vitro was determined by using a radiometric assay. The IC 50 of IBC for each kinase is indicated in the panel on the right. ( B ) MCF-7 cells were pretreated with DMSO, 15 µM IBC, or 20 µM BML-277 for 2 hr, then camptothecin (CPT, 1 μM) was added for 2 hr. Phosphorylation of CHK2 on S516 (pCHK2) was detected by western blotting. The ratio of pCHK2-S516 induction, relative to the DMSO + CPT control, is indicated (n = 3). ( C ) MCF-7 cells were treated as indicated in (B). Phosphorylation of chromatin-bound BRCA1 at residue S988 was detected by western blotting. The relative ratio of pBRCA1-S988 signal, after normalization to Ponceau signal, is indicated. TBP was used as a marker of chromatin fraction. ( D ) MCF-7 cells were treated with 15 μM IBC for 2 hr, then 4 mM HU was added for 2 hr. CHK1 autophosphorylation on S296 (pCHK1) was detected by western blotting. ( E, F ) In silico molecular docking of IBC in the active sites of CHK2 and CHK1, respectively. ( G, H ) Cellular thermal shift assay (CETSA) of IBC on the thermal stability of CHK2 and CHK1. MCF-7 cells were treated with 15 µM IBC or 20 μΜBML-277 for 2 hr. Cells were proceeded to CETSA as described in the ‘Materials and methods’. The amount of CHK2 and CHK1 present in the supernatant was detected by western blotting. The relative CHK2 and CHK1 signal was quantified. The p-values were determined using two-tailed paired t -test (n = 3). Figure 4—source data 1. Original membranes corresponding to with labels. Figure 4—source data 2. Original membranes corresponding to .

Article Snippet: Antibody , Rabbit polyclonal anti-pCHK2 (S516) , 2669 , Cell Signaling , 1/1000.

Techniques: Incubation, Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Control, Residue, Marker, In Silico, Thermal Shift Assay, Two Tailed Test

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/eLife.104718

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-pCHK2 (S516) , 2669 , Cell Signaling , 1/1000.

Techniques:

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/eLife.104718

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-pCHK2 (T68) , 2661 , Cell Signaling , 1/1000.

Techniques:

Journal: eLife

Article Title: Synergistic effect of inhibiting CHK2 and DNA replication on cancer cell growth

doi: 10.7554/elife.104718

Figure Lengend Snippet: Figure 4. Isobavachalcone (IBC) inhibits the CHK2 kinase. (A) Selected protein kinases were incubated with the indicated range of IBC concentrations, and kinase activity in vitro was determined by using a radiometric assay. The IC50 of IBC for each kinase is indicated in the panel on the right. (B) MCF- 7 cells were pretreated with DMSO, 15 µM IBC, or 20 µM BML-277 for 2 hr, then camptothecin (CPT, 1 μM) was added for 2 hr. Phosphorylation of CHK2 on S516 (pCHK2) was detected by western blotting. The ratio of pCHK2-S516 induction, relative to the DMSO + CPT control, is indicated (n = 3). (C) MCF-7 cells were treated as indicated in (B). Phosphorylation of chromatin-bound BRCA1 at residue S988 was detected by western blotting. The relative ratio of pBRCA1-S988 signal, after normalization to Ponceau signal, is indicated. TBP was used as a marker of chromatin fraction. (D) MCF-7 cells

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) Immortalized BJ fibroblasts Dr. D. Peeper The Netherlands Cancer Institute, Amsterdam Foreskin (normal neonatal mal) Cell line (H. sapiens) MCF- 7 HTB- 22 ATCC Mammary gland adenocarcinoma Cell line (H. sapiens) A549 CRM- CCL- 185 ATCC Lung carcinoma Cell line (H. sapiens) HCC827 CRL- 2868 ATCC Lung adenocarcinoma Cell line (H. sapiens) PC3 CRL- 1435 ATCC Prostate adenocarcinoma Cell line (H. sapiens) DU145 HTB- 81 ATCC Prostate carcinoma Cell line (H. sapiens) U937 CRL- 1593.2 ATCC Histiocytic lymphoma Cell line (H. sapiens) HCT116 CCL- 247 ATCC Colorectal carcinoma Cell line (H. sapiens) OVCAR8 NIH:OVCAR8 Ovarian carcinoma Cell line (H. sapiens) DLBCL cell lines Dr. J. Moreaux Institute of Human Genetics, Montpellier Cell line (H. sapiens) SUM159 SUM159PT Asterand Bioscience Triple- negative breast cancer cell line Antibody Mouse monoclonal anti- BrdU clone B44 347580 BD Biosciences 1/100 Antibody Rat monoclonal anti- BrdU clone BU1/75 ABC117- 7513 Eurobio Abcys 1/100 Antibody Mouse monoclonal antissDNA MAB3868 Millipore 1/250 Antibody Rabbit monoclonal antipCHK1 (S345) 2348 Cell Signaling 1/1000 Antibody Rabbit polyclonal anti- pCHK2 (T68) 2661 Cell Signaling 1/1000 Antibody Mouse monoclonal anti-γH2AX (S139) 05- 636 Millipore 1/500 Antibody Mouse monoclonal anti- actin A4700 Sigma 1/500 Antibody Rabbit monoclonal anti- RPA1 Ab79398 Abcam 1/300 Coquel et al. eLife 2024;13:RP104718.

Techniques: Incubation, Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Control, Residue, Marker