Journal: Frontiers in Oncology

Article Title: Chk1 Inhibition Hinders the Restoration of H3.1K56 and H3.3K56 Acetylation and Reprograms Gene Transcription After DNA Damage Repair

doi: 10.3389/fonc.2022.862592

Figure Lengend Snippet: Modulation of acetylated K9, K14, K27, and K56 of histone H3.1 or H3.3 by the Chk1 inhibitor MK8776 and UV radiation. (A) H3.1-SNAP U2OS cells were pretreated with DMSO (-) or MK8776 (+) for 1 h and then irradiated with 20 J/m 2 UV radiation. The insoluble nuclear protein samples were collected at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h after radiation. The acetylation levels of H3K9, H3K14, H3K27, and H3K56 were examined. SNAP-tagged histone H3 was used as an internal reference. (B) H3.3-SNAP U2OS cells were pretreated with DMSO or MK8776 and then irradiated with 20 J/m 2 UV radiation. The insoluble nuclear protein samples were collected, and the acetylation levels of H3K9, H3K14, H3K27, and H3K56 were examined. SNAP-tagged histone H3 was used as an internal reference. (C) Time-course alteration of acetylated K9, K14, K27, and K56 of histone H3.1 after 20 J/m 2 UV radiation and MK8776 treatment. The data are normalized immunoblotting results by ImageJ analysis from three separate experiments. (D) Time-course alteration of acetylated K9, K14, K27, and K56 of histone H3.3 after 20 J/m 2 UV radiation and MK8776 treatment. The data are normalized immunoblotting results by ImageJ analysis from three separate experiments.

Article Snippet: For the serum starvation experiment, cells were seeded and grown to 70% confluence and then the medium was changed to serum-free DMEM medium for 24 h. The ATM inhibitor KU55933, the Chk1 inhibitor UCN-01, and the Chk1&Chk2 inhibitor AZD7762 were obtained from Sigma-Aldrich (St. Louis, MO 63103, USA), while the ATR inhibitor VE-821, the Chk1 inhibitor MK8776, and the ATM & ATR inhibitor AZD6738 were obtained from Cayman Chemical company (Ann Arbor, Michigan 48108, USA).

Techniques: Irradiation, Western Blot

Journal: Frontiers in Oncology

Article Title: Chk1 Inhibition Hinders the Restoration of H3.1K56 and H3.3K56 Acetylation and Reprograms Gene Transcription After DNA Damage Repair

doi: 10.3389/fonc.2022.862592

Figure Lengend Snippet: mRNA sequencing of Chk1-inhibited samples after UV radiation and verification of Chk1 inhibition. (A) Heatmap visualization of 105 genes that were expressed in all libraries. Data are expressed as the mean FPKM, standardized, and visualized by the DESeq R package (version 1.18.0). Columns: samples; rows: genes; red: relatively high expression; blue: relatively low expression. DMSO: DMSO mock group; LY: Ly2606368 treated group; 2 hours: 2 h after UV radiation; 8 hours: 8 h after UV radiation; 24 hours: 24 h after UV radiation. (B) Calculations of differentially expressed genes in the 105 genes. (C) qRT-PCR results of HOXB6, DTX3L, SSTR2, MYC, and HOXC1 mRNA expression levels after DMSO or Ly2606368 treatment and UV radiation. (D) Numbers of changed genes after chemical treatment and UV radiation. At 2 h, 8 h, and 24 h after UV radiation and chemical treatment, the normalized gene expression of Ly2606368-treated U2OS cells and DMSO-mock U2OS cells was compared, and the numbers of differentially expressed genes classified by functional annotation are shown. (E) Numbers of genes that changed more than 2-fold after chemical treatment and UV radiation. The numbers of genes that increased more than 2-fold or decreased more than 0.5-fold were calculated. (F) Western blot detection of phosphorylated Chk1 after DMSO, MK8776, or Ly2606368 treatment after UV radiation. The data are normalized immunoblotting results by ImageJ analysis from three separate experiments. (G) Western blot detection of phosphorylated Chk1, Tlk-1, and Asf1a after DMSO or MK8776 treatment under serum starvation conditions and UV radiation. For the serum starvation experiment, U2OS cells were seeded and grown to 70% confluence, and then the medium was changed to serum-free DMEM for 24 h.

Article Snippet: For the serum starvation experiment, cells were seeded and grown to 70% confluence and then the medium was changed to serum-free DMEM medium for 24 h. The ATM inhibitor KU55933, the Chk1 inhibitor UCN-01, and the Chk1&Chk2 inhibitor AZD7762 were obtained from Sigma-Aldrich (St. Louis, MO 63103, USA), while the ATR inhibitor VE-821, the Chk1 inhibitor MK8776, and the ATM & ATR inhibitor AZD6738 were obtained from Cayman Chemical company (Ann Arbor, Michigan 48108, USA).

Techniques: Sequencing, Inhibition, Expressing, Quantitative RT-PCR, Gene Expression, Functional Assay, Western Blot

Journal: Cancers

Article Title: Promises of Protein Kinase Inhibitors in Recalcitrant Small-Cell Lung Cancer: Recent Scenario and Future Possibilities

doi: 10.3390/cancers16050963

Figure Lengend Snippet: CHK1 kinase signaling, and inhibition involved in SCLC immunotherapy. ( A , B ) CHK1, a DNA-repair protein, is overexpressed in SCLC. It participates in the DNA repair process. The combination of the CHK1 inhibitor SRA737 and a low dose of gemcitabine (chemotherapeutic drug) augments the impact of PD-L1 IC blockade (via the use of anti-PD-L1 antibody). This augmentation is achieved by significantly elevating the presence of CD8 + T cells, DCs, and M1 macrophages within the TME. [Created with BioRender.com (accessed on 18 February 2024)].

Article Snippet: To achieve the intended objective, the use of the CHK1 inhibitor Prexasertib and PARP inhibitor Olaparib (manufactured by KuDOS Pharmaceuticals, Cambridge, UK) was employed.

Techniques: Inhibition

Journal: Cancers

Article Title: Promises of Protein Kinase Inhibitors in Recalcitrant Small-Cell Lung Cancer: Recent Scenario and Future Possibilities

doi: 10.3390/cancers16050963

Figure Lengend Snippet: A list of ICI clinical trials with their protein kinase (PK) inhibitors for SCLC.

Article Snippet: To achieve the intended objective, the use of the CHK1 inhibitor Prexasertib and PARP inhibitor Olaparib (manufactured by KuDOS Pharmaceuticals, Cambridge, UK) was employed.

Techniques: Clinical Proteomics, Activation Assay, Expressing, In Vivo, Inhibition

Journal: Scientific Reports

Article Title: Synthesis and new DNA targeting activity of 6- and 7- tert -butylfascaplysins

doi: 10.1038/s41598-024-62358-8

Figure Lengend Snippet: Effect of DNA damage response inhibitors on the anticancer activity of 6-TB and 7-TB. Effect was examined using a colony formation assay. Cells were seeded into 6-well plates and treated with tested compounds with or without specific inhibitor at the indicated concentrations for 10–14 days. The colonies were fixed, stained and counted. The inhibitors used are indicated as ATRi (VE-822), Chk1i (LY2603618), ATMi (KU-60019), Chk2i (PV1019). Statistically significant difference between group treated with 6-/7-TB alone and group co-treated with either inhibitor is indicated as: * p < 0.05 (ANOVA); otherwise, the difference is not statistically significant. Experiments were performed in triplicates (number of biological replicates n = 3).

Article Snippet: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma (Taufkirchen, Germany), annexin-V-FITC from BD Bioscience (San Jose, CA, USA); tariquidar (p-glycoprotein inhibitor) from MedChemExpress (Monmouth Junction, NJ, USA); calcein-AM from BIOZOL (Eching, Germany); VE-822 (ATR inhibitor), LY2603618 (Chk1 inhibitor), KU-60019 (ATM inhibitor), and PV1019 (Chk2 inhibitor) from LC Laboratories (Woburn, MA, USA); Anisomycin—from NeoCorp (Weilheim, Germany).

Techniques: Activity Assay, Colony Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Identification of kinase inhibitor influencing ERα levels and cell proliferation in luminal primary and metastatic breast cancer cells. Robust Z scores ( Z* ) graphs for kinase inhibitor-treated samples (100 nM final concentration) for 48 h in Tam Res and Y537S cells. The ERα levels were detected by in-cell WB (A and B) while cell number was detected by in-cell propidium iodide (PI) staining ( A’ and B’ ); red arrows indicate the Z* for inhibitors considered as positive hits. Red lines indicate the threshold used for analysis. Venn diagrams showing the positive hits for ERα and PI lists in Tam Res ( C ) and Y537S ( C’ ) cells. ( D ) Venn diagram showing the combined positive hits of the inhibitors (explicated in D’ – compound names are given in brackets) in the indicated cell lines. Kaplan–Meier plots show the relapse-free survival (RFS) probability in women carrying ERα-negative ( E and G ) or ERα-positive ( F and H ) as a function of CHK1 ( E and F ) or CHK2 ( G and H ) mRNA levels. All possible cutoff values between the lower and upper quartiles are automatically computed ( i.e., auto select best cutoff on the website), and the best performing threshold is used as a cutoff . Details of the parameters of the curves are given in supplementary table . Significant differences between the RFS are given as p- value in each panel

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Concentration Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Correlation between ERα and CHK1 and CHK2 activation status. A Volcano plot showing the protein expression as a function of the p -value of the indicated proteins ( i.e., ERα—red, CHK1—blue and CHK2 – purple) in ERα-negative or ERα-positive tumors as indicated in the breast cancer landscape and downloaded by https://www.breastcancerlandscape.org/ . B Level of ERα S118 phosphorylation (yellow), CHK1 S345 phosphorylation (blue), and CHK2 T68 phosphorylation (purple) in ERα-negative or ERα-positive breast cancer cell lines. Significant differences were obtained by unpaired two-tailed Student’s t-test. °°°° ( p < 0.0001) indicates significant differences in ERα S118 phosphorylation between ERα-negative or ERα-positive breast cancer cell lines. °°° ( p < 0.001) indicates significant differences in CHK2 T68 phosphorylation between ERα-negative or ERα-positive breast cancer cell lines. * ( p < 0.05) indicates significant differences in CHK1 S345 phosphorylation and CHK2 T68 phosphorylation in ERα-positive breast cancer cell lines. Linear regression and Spearman Correlation values (r) between ERα S118 phosphorylation and CHK2 T68 phosphorylation ( C ) or ERα S118 phosphorylation and CHK1 S345 phosphorylation ( D ) in ERα-positive breast cancer cell lines classified as luminal A (LumA – green) or luminal B (LumB – orange). r and p values are given in the main panel. Z scores for the antiproliferative effects of either ERα (red), CHK1 (blue), or CHK2 (purple) depletion by CRISPR/CAS9 ( E ) or small interference RNA (siRNA) ( F ) in ERα-positive breast cancer cell lines. The dotted lines represent the threshold for cell line sensitivity in each kind of procedure. Plots in ( B - F ) have been generated by downloading the experimental data by the Broad Institute through the DepMap portal https://depmap.org/portal . Each dot of the plots in ( B - F ) represents the value of the indicated parameter in a single breast cancer cell line. Crude data are given in supplementary table

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Activation Assay, Expressing, Phospho-proteomics, Two Tailed Test, CRISPR, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Dissection of the pathway inducing ERα degradation and preventing proliferation in MCF-7 and Y537S cells.Western blot and relative densitometric ( E and L ) analyses of ERα expression levels in ( A - D ) MCF-7 and ( F – H ) Y537S cells treated for 24 h with the indicated doses of the specific inhibitors of either CHK1 ( i.e., MK8776—MK), CHK2 ( i.e., CCT241533—CCT), ATR ( i.e., VE822—VE) or ATM ( i.e., KU60019—KU). The loading control was done by evaluating vinculin expression in the same filter. Significant differences with respect to control (0) were obtained by unpaired two-tailed Student’s t-test. Data show the mean ± the standard deviations. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. The number of replicates is given as solid dots in the bar graph. ( M – N ) The inhibitor concentration 50 (IC 50 ) was calculated for the indicated cell lines at 5 days after initial treatment with the indicated inhibitors. Significant differences were obtained by unpaired two-tailed Student’s t-test. Data show the mean ± the standard deviations. * ( p < 0.05) and ** ( p < 0.01) indicate significant differences between VE and KU IC 50 while °° p < 0.01 and °°° p < 0.001 indicate significant differences between MK and CCT IC 50 . Growth curve analyses in MCF-7 ( O ) and Y537S ( P ) cells were performed as indicated in the material and method section for 2 days after the cell transfection with the small interference RNA (siRNA) oligonucleotides against either CHK1 or CHK2. The graphs show the normalized cell index ( i.e., cell number), which is detected with the xCelligence DP device and calculated at each time point with respect to the control sample. Each sample was measured in a quadruplicate. For details, please see the material and methods section. Controls for CHK1 and CHK2 siRNA experiments are available in supplementary Fig. C and D

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Dissection, Western Blot, Expressing, Control, Two Tailed Test, Concentration Assay, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Inhibitor-dependent effect on DNA damage and replication stress in MCF-7 and Y537S cells.Western blot and relative densitometric ( G and H ) analyses of ERα, RPA2 and phosphorylated γH2AX expression levels in MCF-7 ( A , C , and E ) and Y537S ( B , D , and F ) cells treated for 24 h with the indicated doses of the specific inhibitors of either CHK1 ( i.e., MK8776—MK), CHK2 ( i.e., CCT241533—CCT), ATR ( i.e., VE822—VE) or ATM ( i.e., KU60019—KU) as well as with AZD7762 (AZD). The loading control was done by evaluating vinculin expression in the same filter. Significant differences are given in the heatmaps ( G and H ) with red being a significant increase and green being a significant decrease with respect to control (0). Analyses were performed by using the unpaired two-tailed Student’s t-test. Data are the mean ± the standard deviations, and blots show representative images of three different experiments. Histograms relative to the heatmaps are available in supplementary Fig.

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Western Blot, Expressing, Control, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Impact of clinically relevant CHK1 inhibitors in inducing ERα degradation and preventing proliferation in MCF-7 and Y537S cells. A Sensitivity scores of ERα-negative (black) and ERα-positive (red) breast cancer cell lines to AZD7762 (AZD), prexasertib (Prexa), MK8776 (MK), PF-477736 (PF), Ly-2603618 (Ly), PD-407824 (PD), CHIR-124 (CHIR), and SB-218078 (SB). A’ Sensitivity scores of ERα-negative (black) and ERα-positive (red) breast cancer cell lines to 4OH-Tamoxifen (Tam), VE822 (VE), and KU60019 (KU). * ( p < 0.05), ** ( p < 0.01) and **** ( p < 0.001) indicate significant differences to drugs among ERα-negative or ERα-positive breast cancer cell lines. The graphs have been generated by downloading the experimental data from the Broad Institute through the DepMap portal https://depmap.org/portal . Each dot of the plot in ( A and A’ ) represents the value of the indicated parameter in a single breast cancer cell line. Crude data are given in supplementary table . Western blot ( B ) and relative densitometric analyses ( B’ ) of ERα expression levels in MCF-7 (yellow) and Y537S (red) cells treated for 24 h with the indicated doses of prexasertib (Prexa). The loading control was done by evaluating vinculin expression in the same filter. Significant differences with respect to control (0) were obtained by unpaired two-tailed Student’s t-test. Data show the mean ± the standard deviations, **** p < 0.0001. The number of replicates is given as solid dots in the bar graph. C Growth curve analyses in MCF-7 (yellow) and Y537S (red) cells were performed as indicated in the material and method section for 5 days with different doses of prexasertib (Prexa). The graph shows only one concentration for each cell line and the normalized cell index ( i.e., cell number), which is detected with the xCelligence DP device and calculated at each time point with respect to the control sample. Each sample was measured in a quadruplicate. For details, please see the material and methods section. D The inhibitor concentration 50 (IC 50 ) was calculated for each cell line at 5 days after initial treatment

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Generated, Western Blot, Expressing, Control, Two Tailed Test, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: The impact of ATR:CHK1 and ATM:CHK2 pathways on E2:ERα signaling to cell proliferation. A Western blot of ERα and ERα S118 phosphorylation expression levels in MCF-7 pre-treated with the specific inhibitors of either CHK1 ( i.e., MK8776 – MK 1 μM), CHK2 ( i.e., CCT241533 – CCT 1 μM), ATR ( i.e., VE822 – VE 1 μM) or ATM ( i.e., KU60019 – KU 1 μM) as well as with AZD7762 (AZD 1 μM) for 24 h and then treated for 30 min with 17β-estradiol (E2 -10 nM). ( A’ ) Densitometric analysis is relative to panel ( A ). The loading control was done by evaluating vinculin expression in the same filter. Panels show representative blots of three independent experiments. Significant differences with respect to - sample are calculated by Student t-test and indicated by * p -value < 0.05. B Estrogen response element promoter activity in MCF-7 ERE-NLuc cells pre-treated the specific inhibitors of either CHK1 ( i.e., MK8776 – MK 1 μM), CHK2 ( i.e., CCT241533 – CCT 0.5 μM), ATR ( i.e., VE822 – VE 1 μM) or ATM ( i.e., KU60019 – KU 1 μM) as well as with AZD7762 (AZD 1 μM) for 24 h and then treated with 17β-estradiol (E2 10 nM) for additional 24 h. The experiments were performed three times in quintuplicate duplicate. Significant differences with respect to untreated ( i.e., -) samples are calculated by Student t-test and indicated by **** p -value < 0.0001. Significant differences with respect to CTR E2 sample are calculated by Student t-test and indicated by °°°° p -value < 0.0001. C Western blot of presenilin 2 (pS2) expression levels in MCF-7 pre-treated with the specific inhibitors of either CHK1 ( i.e., MK8776 – MK 1 μM), CHK2 ( i.e., CCT241533 – CCT 0.5 μM), ATR ( i.e., VE822 – VE 1 μM) or ATM ( i.e., KU60019 – KU 1 μM) as well as with AZD7762 (AZD 1 μM) for 24 h and then treated for 24 h with 17β-estradiol (E2 -10 nM). The loading control was done by evaluating vinculin expression in the same filter. Panels show representative blots of three independent experiments. The present blot has been merged by different blots; dotted lines represents points where gel images have been merged. Histogram depicting the densitometric analysis is available in supplementary Fig. B. Original Western blots are provided in supplementary table . ( D , D’ and D’’ ) Real-time growth curves in MCF-7 cells treated with AZD7762 (AZD) at the indicated doses in the absence and the presence of 17β-estradiol (E2 10 nM). The graphs show the normalized cell index ( i.e., cell number), which is detected with the xCelligence DP device and calculated at each time point with respect to the control sample. Each sample was measured in a quadruplicate. For details, please see the material and methods section. ( E ) Bromodeoxyuridine (BrdU) incorporation assay in MCF-7 cells treated with 17β-estradiol (E2 10 nM – 24 h) after 24 h pre-treatment with AZD7762 (AZD) at the indicated doses. The experiments have been performed twice in duplicate

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Western Blot, Phospho-proteomics, Expressing, Control, Activity Assay, BrdU Incorporation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: CHK1 inhibitors synergic effects with 4OH-Tamoxifen in MCF-7 cells. Kaplan–Meier plots showing the relapse-free survival (RFS) probability in women carrying breast tumors expressing either ERα, progesterone receptor (PR) but not HER2 ( A ) or ERα, but not PR or both expressing or not HER2 ( B ) as a function of CHK1 mRNA levels. All possible cutoff values between the lower and upper quartiles are automatically computed ( i.e., auto select best cutoff on the website), and the best performing threshold is used as a cutoff . Details of the parameters of the curves are given in supplementary table . Significant differences between the RFS are given as p -value in each panel. Synergy map of 12 days-treated MCF-7 ( C ) and T47D-1 ( D ) cells with different doses of 4OH-Tamoxifen (Tam) and AZD7762 (AZD) or prexasertib (Prexa) ( C , and D left and right panels, respectively). Growth curves in MCF-7 ( C’ ) or T47D-1 ( D’ ) cells show the synergic effect of each combination of compounds with selected doses. Significant differences with respect to untreated ( i.e., -,-) samples are calculated by Student t-test and indicated by **** p -value < 0.0001. Significant differences between Tam + AZD with respect to Tam alone or AZD alone are calculated by Student t-test and indicated by ° p -value < 0.05. Significant differences between Tam + Prexa with respect to Tam alone or Prexa alone are calculated by Student t-test and indicated by ^ p -value < 0.05. For details, please see the material and methods section

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: CHK1 inhibitors synergic effects with palbociclib and abemaciclib in Y537S cells. A Kaplan–Meier plots showing the relapse-free survival (RFS) probability in women carrying breast tumors expressing ERα, PR, or both expressing or not HER2 as a function of CHK1 mRNA levels. All possible cutoff values between the lower and upper quartiles are automatically computed ( i.e., auto select best cutoff on the website), and the best performing threshold is used as a cutoff . Details of the parameters of the curves are given in supplementary table . Significant differences between the RFS are given as p-value in each panel. B mRNA expression levels of CHK1 and CHK2 in patient-derived xenograft (PDX) immortalized cell lines stratified as indicated in the graph. Data have been extracted by the supplementary materials given in . Volcano plot showing the mRNA expression as a function of the p -value of the indicated proteins ( i.e., presenilin2 -pS2, TFF—red, CHK1 – blue) in CRISPR/CAS9 engineered MCF-7 cells expressing the Y537S ( C ) or the D538G ( D ) ERα mutant as extracted by the supplementary materials given in . Synergy map of 5 days-treated Y537S cells with different doses of MK8776 (MK) ( E ) or prexasertib (Prexa) and palbociclib (Palbo) or abemaciclib (Abe) ( E and F left and right panels, respectively). Significant differences with respect to untreated ( i.e., -,-) samples are calculated by Student t-test and indicated by **** p -value < 0.0001. Significant differences between MK + Abe with respect to MK alone or Abe alone are calculated by Student t-test and indicated by °°°° p -value < 0.0001. Significant differences between MK + Palbo with respect to MK alone or Palbo alone are calculated by Student t-test and indicated by ^^^^ p -value < 0.0001. Significant differences between Prexa + Abe with respect to Prexa alone or Abe alone are calculated by Student t-test and indicated by °°°° p -value < 0.0001. Significant differences between Prexa + Palbo with respect to Prexa alone or Palbo alone are calculated by Student t-test and indicated by ^^^^ p -value < 0.0001. Growth curves in Y537S cells showing the synergic effect of each combination of compounds with selected doses are shown in ( E’ and F’ ). For details, please see the material and methods section

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Expressing, Derivative Assay, CRISPR, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells

doi: 10.1186/s13046-022-02360-y

Figure Lengend Snippet: Effect of CHK1 inhibitors in 3D-models of breast cancer. Images ( A , B ) and quantitation ( A’ , B’ ) of tumor spheroids surface area ( A , A’ ) and alginate-based cultures ( B , B’ ) generated in MCF-7 and Y537S cells, treated at time 0 with CHK1 inhibitors ( i.e., AZD7762, AZD—1 μM; prexasertib, Prexa—1 μM; MK8776—MK 5 μM) or left untreated (CTR), for 7 days. The number of replicates is given as solid dots in the graphs. Significant differences with respect to the CTR sample were determined by unpaired two-tailed Student’s t-test: **** p -value < 0.0001; *** p -value < 0.001. Scale bars equal to 200 µm for panel A and 50.0 mm for panel B

Article Snippet: Finally, we studied the anti-proliferative effects of CHK1 inhibitors ( i.e., AZD, Prexa, MK) in MCF-7 and Y537S tumor cell spheroids [ , ] as well as in alginate-based cultures [ , ] to understand if differences in the effect of these drugs exist in cells grown in 3D structures [ ].

Techniques: Quantitation Assay, Generated, Two Tailed Test

Journal: PLoS ONE

Article Title: Modulation of Early Mitotic Inhibitor 1 (EMI1) depletion on the sensitivity of PARP inhibitors in BRCA1 mutated triple-negative breast cancer cells

doi: 10.1371/journal.pone.0235025

Figure Lengend Snippet: (A) MDA-MB-436 cells, transfected as in , were subjected to the indicated concentrations of cisplatin for 72 h followed by a proliferation assay and an immunoblot of one of the replicates. The x-axis is in log 10 and the datapoints from samples containing no drug (vehicle only) were placed on the y-intercept. Results represent the mean and SEM of three replicate experiments. Datapoints at each concentration of cisplatin were analyzed by the two-sided students t test, and * indicates p<0.05, and ** indicates p<0.01. (B) MDA-MB-436 cells were transfected as in and subjected to the CHK1 inhibitor (SB218078) in the growth medium for 96 h and analyzed as described above. Results represent the mean and SEM of three replicate experiments. (C) MDA-MB-436 cells were transfected with control siRNA and EMI1-specific siRNA2, followed by inclusion of the MEKi (selumetinib) in culture medium at the indicated concentrations for 96 h, followed by analysis for proliferation and protein abundance as described above. Results represent the mean and SEM of three replicate experiments.

Article Snippet: The drugs olaparib (Cat. No. A10111-10), Talazoparib (BMN-673; Cat. No. A11243), CHK1 inhibitor (SB218078; Cat. No. A1548), and MEK inhibitor selumetinib (AZD6244; Cat. No. A10257) were purchased from AdooQ.

Techniques: Transfection, Proliferation Assay, Western Blot, Concentration Assay, Control, Quantitative Proteomics

Journal: PLoS ONE

Article Title: A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

doi: 10.1371/journal.pone.0111714

Figure Lengend Snippet: Percentage of cells in G1, S, or G2 phase.

Article Snippet: PRKDC was detected in Chk1 immunoprecipitations in CEM cells which had detectable levels of phosphorylation of Chk1 S345 in response to HU treatment ( ).

Techniques:

Journal: PLoS ONE

Article Title: A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

doi: 10.1371/journal.pone.0111714

Figure Lengend Snippet: % H2AX phosphorylation.

Article Snippet: PRKDC was detected in Chk1 immunoprecipitations in CEM cells which had detectable levels of phosphorylation of Chk1 S345 in response to HU treatment ( ).

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

doi: 10.1371/journal.pone.0111714

Figure Lengend Snippet: Cells were transfected with Chk1, RRM1, RRM2, RRM1/Chk1, RRM2/Chk1, and Luciferase control (untreated or treated with 1 mM HU for the last 8 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) extracts were prepared and immunoblotted with the indicated antibodies. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. (c) Cells were collected at indicated time points indicated and analyzed for activated caspases. Data performed in duplicates. Error bars represent SD between experiments. (d) Cell proliferation was assessed with clonogenicity assay.

Article Snippet: PRKDC was detected in Chk1 immunoprecipitations in CEM cells which had detectable levels of phosphorylation of Chk1 S345 in response to HU treatment ( ).

Techniques: Transfection, Luciferase, Control, Phospho-proteomics, Flow Cytometry

Journal: PLoS ONE

Article Title: A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

doi: 10.1371/journal.pone.0111714

Figure Lengend Snippet: (a) At 30 h after siRNA transfections of the luciferase, RRM1, and RRM2 siRNA duplexes, extracts were prepared for immunoprecipitations. Cells transfected with Luciferase were incubated with 1 mM HU for 30 min. Chk1 was immunoprecipitated from luciferase (positive control), Luciferase + HU, RRM1, and RRM2 depleted cells with Chk1 antibodies (MAb58D7) cross-linked to protein A and were immunoblotted as indicated. Chk1 immunoprecipitations were peptide blocked as a negative control. (b) Polα was immunoprecipitated from luciferase (positive control), luciferase +HU, RRM1, and RRM2 depleted cells depleted cells with Polα antibody (SJK132-20) cross-linked to protein G and Western blots were immunoblotted as indicated. (c) Whole cell extracts were immunoblotted as indicated.

Article Snippet: PRKDC was detected in Chk1 immunoprecipitations in CEM cells which had detectable levels of phosphorylation of Chk1 S345 in response to HU treatment ( ).

Techniques: Transfection, Luciferase, Incubation, Immunoprecipitation, Positive Control, Negative Control, Western Blot