Journal: Cancer Communications

Article Title: CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer

doi: 10.1002/cac2.12125

Figure Lengend Snippet: CHR‐6494 potentiates the killing efficacy of MLN8237 on breast cancer cells. A. Colony formation assay shows the effects of CHR‐6494 on MLN8237 sensitivity in MDA‐MB‐231 and SKBR3 cells. B and C. Relative apoptosis of MDA‐MB‐231 and SKBR3 cells treated with indicated drugs for 72 hours. The concentrations of MLN8237 were 200 nmol/L in MDA‐MB‐231 cells and 70 nmol/L in SKBR3 cells. The concentrations of CHR‐6494 were 200 nmol/L in both cells. One‐way ANOVA and LSD tests were used to evaluate the difference between groups. ns, not significant; n = 3 independent experiments; ** P < 0. 01, *** P < 0.001. D and E. MDA‐MB‐231 and SKBR3 cells were treated with the indicated doses of MLN8237 and CHR‐6494 for 96 hours, followed by MTT assay to determine the growth‐inhibitory effects. n = 3 independent experiments. The combination index was analyzed with the CompuSyn software. Abbreviations: PI, Propidium iodide

Article Snippet: CHR‐6494 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Colony Assay, MTT Assay, Software

Journal: Cancer Communications

Article Title: CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer

doi: 10.1002/cac2.12125

Figure Lengend Snippet: Synergistic inhibition of Aurora‐A and Haspin attenuates kinetochore‐microtubule attachment. A. Representative immunofluorescent images show the morphology of monopolar, bipolar, multipolar spindles, and small asters. B. Percentage of each kind of spindles with indicated treatments in MDA‐MB‐231 cells (≥ 60 cells per condition). C. The distance between centrioles and the size of pericentriolar material (PCM) measured in the MLN8237 treatment group or co‐treatment group (≥ 60 cells per condition). D. Immunofluorescence shows the tubulin intensity in cold‐stable microtubule assay after releasing MDA‐MB‐231 cells from G2/M synchronization to indicated drugs for 30 min. The concentrations of MLN8237 and CHR‐6494 were 200 nmol/L, respectively. E. The intensity of tubulin in cold‐stable microtubule assay was analyzed. Two‐tailed unpaired Student t ‐test were performed (≥ 28 cells per condition). *** P < 0.001. F. Cell cycle analysis of MDA‐MB‐231 cells treated with indicated drugs for 48 hours. G. Percentage of polyploids and ratio of G2/G1 in MDA‐MB‐231 cells treated with indicated drugs for 48 hours. n = 3 independent experiments; ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Abbreviations: MLN, MLN8237; CHR, CHR‐6494

Article Snippet: CHR‐6494 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Immunofluorescence, Two Tailed Test, Cell Cycle Assay

Journal: Cancer Communications

Article Title: CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer

doi: 10.1002/cac2.12125

Figure Lengend Snippet: Synergistic inhibition of Aurora‐A and Haspin disrupts the mitotic centromere aggregation of Aurora‐B and MCAK. A. The kinase activity of Aurora‐A and Aurora‐B was examined using Western blotting assays after releasing MDA‐MB‐231 cells from G2/M synchronization to indicated drugs for 30 min. The concentrations of MLN8237 and CHR‐6494 were 200 nmol/L. The repeated‐measures ANOVA, followed by the least significant difference test was used to evaluate the difference between groups. n = 3 independent experiments; ns, not significant; * P < 0.05; ** P < 0.01. B. Left panel, immunofluorescence shows the location of Aurora‐B and CENPA. Right panel, the fluorescence intensity of Aurora‐B and CENPA was measured. C. The localization of Aurora‐B at centromere was analyzed ( n = 3 independent experiments; ≥ 60 cells per condition). D. Immunofluorescence shows the location of MCAK. The arrows represent the direction of the line scan of fluorescence intensity for the entire cell. E. The localization of MCAK at the centromere was analyzed. “Strong” means that more than 50% of the centromeres show MCAK localization, while “weak” indicates that less than 50% of the centromeres show MCAK localization ( n = 3 independent experiments; ≥ 123 cells per condition) Abbreviations: MLN, MLN8237; CHR, CHR‐6494; DAPI, 4', 6‐diamidino‐2‐phenylindole

Article Snippet: CHR‐6494 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Activity Assay, Western Blot, Immunofluorescence, Fluorescence

Journal: Cancer Communications

Article Title: CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer

doi: 10.1002/cac2.12125

Figure Lengend Snippet: Combined inhibition of Aurora‐A and Haspin synergistically regresses breast tumors in vivo . A. Nude mice bearing MDA‐MB‐231 xenograft tumors were treated with MLN8237 and CHR‐6494 alone or in combination. The measured tumor volume from days 0 to 21 after treatment is plotted versus time. The repeated‐measures ANOVA, followed by the least significant difference test were used to evaluate the difference between groups, n = 12; * P < 0.05; ** P < 0.01; *** P < 0.001. B. Tumors removed from 6 mice in each group are shown. C. Statistical analysis of the weights of dissected tumors. The ANOVA test, followed by the least significant difference test, was used to evaluate the difference between groups, n = 12; ns, not significant; ** P < 0.01; *** P < 0.001. D. The body weights of mice were measured and plotted against time, n = 6. E. Expression of AURKA and GSG2 in 994 breast cancer patient samples from the TCGA Pan‐Cancer Atlas. Pearson's correlation and linear regression analyses were employed. F and G. Relapse‐free survival and overall survival for AURKA / GSG2 transcription levels in breast cancer patients using the Kaplan–Meier plotter online tool. Abbreviations: HR, Hazard Ratio; TCGA, The Cancer Genome Atlas; 95% CI, 95% Confidence Interval

Article Snippet: CHR‐6494 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, In Vivo, Expressing

Journal: bioRxiv

Article Title: Dual cholinergic mechanisms for sculpting striatal dopamine in vivo

doi: 10.64898/2025.12.19.695021

Figure Lengend Snippet: (a) Average DA across dorsal striatal FOV evoked by full-field CIN stimulation under baseline conditions and following systemic β 2 -nAChR antagonism (DHβE, 1 mg/kg, i.p.). These traces are averaged across all trials for each animal (N = 20 trials, 5 mice per condition). Data displayed as Mean ± SEM. (b) Quantification of peak evoked DA within time windows indexing the first (0 - 0.1 sec; blue) and second (0.1 - 0.3 sec; yellow) peaks illustrated as shaded regions in . Data displayed as Mean ± SEM. DHβE selectively abolished the first CIN-evoked DA peak (two-way rmANOVA, significant main effect of DHβE: F(1,4) = 24.35, P = 0.002; drug x peakTime interaction: F(1,4) = 3.1, P = 0.01; post-hoc paired t-test: Early peak magnitude is different between control vs DHβE: t(8) = 7.326, P = 0.0001), while producing only a modest reduction in the second peak (post-hoc paired t-test: Late peak magnitude was not different between control vs DHβE: t(8) = 1.852, P = 0.10). (c) Top: Population-averaged CIN-evoked DA timecourse across all animals (N = 5 mice) under various systemic pharmacological manipulations. All data displayed as Mean ± SEM, aligned to laser onset. Each panel examines the respective contributions of various cholinergic receptor subtypes. First panel : contribution of all cholinergic receptors by systemic injection of a cocktail containing muscarinic antagonist scopolamine (Sco) and mecamylamine (Mec), either at low (1mg/kg each) or high (5mg/kg) doses. Note a non-cholinergic DA peak that is observed with the drug cocktail on board at either dosage. Second panel : contribution of β 2 -nAChRs with muscarinic receptors intact displayed in panel a . Third panel : contributions of β 2 -nAChRs without muscarinic receptors available by comparing timecourse of evoked DA between administration of scopolamine (Sco; 1mg/kg) or a cocktail of scopolamine and DHβE (1mg/kg each). Fourth panel : contributions of all nicotinic receptors examined by contrasting scopolamine (1mg/kg) alone against a cocktail of scopolamine and mecamylamine (1mg/kg each). Fifth panel : contributions of muscarinic receptors by comparing DHβE (1mg/kg) against a cocktail of scopolamine and DHβE (1mg/kg each). Sixth panel : contributions of non-DHβE sensitive nicotinic receptors by contrasting DA timecourse of scopolamine and DHβE (1mg/kg each) and scopolamine (Sco) and mecamylamine (Mec) at two doses. Directly testing the contributions of α 7 - or α 6 *-nAChRs with their respective antagonists (MLA or bPiDl, 1 mg/kg each) administered in a cocktail with scopolamine and DHβE (1mg/kg each) reduces evoked DA to a level indistinguishable from scopolamine (Sco) and mecamylamine (Mec). Bottom panels emphasize these contributions by directly subtracting the pharmacological conditions to isolate the contributions of each cholinergic receptor class. Shaded regions indicate windows where first and second peaks were detected, same as . (d) Quantification of pharmacological effects on evoked DA release area under the curve (AUC) of the predefined time windows indexing the first (0 - 0.1 sec; blue) and second (0.1 - 0.3 sec; yellow) peaks illustrated as shaded regions in . Each dot is average for each mouse tested, and the box plots show mean across animals (red horizontal line) and 95% confidence interval (top and bottom box boundaries). Pharmacological manipulations differentially affect the early versus late components of CIN-evoked DA release (two-way rmANOVA, significant main effect of Drug (F(7,14) = 19.65, P = 3.15 × 10 −6 ), a significant main effect of PeakTime (F(1,2) = 69.53, P = 0.014), and a significant Drug × PeakTime interaction (F(7,14) = 12.04, P = 5.77 × 10 5 ). Post hoc paired t-test comparisons for each drug condition are indicated with a star for significance, shown below. Early peak AUC difference: Control vs Sco+Mec: t(3) = 8.083, P = 0.004; Control vs DHβE: t(4) = 10.332, P = 0.0004951; Sco vs Sco+DHβE: t(3) = 6.333, P = 0.00796; Sco vs Sco+Mec: t(3) = 3.948, P = 0.02899; DHβE vs Sco+DHβE: t(4) = 1.000, P = 0.3739; Sco+DHβE vs Sco+Mec: t(3) = 0.816, P = 0.4741; Sco+DHβE+MLA vs Sco+Mec: t(2) = 0.525, P = 0.6518; Sco+DHβE+bPiDl vs Sco+Mec: t(2) = 1.728, P = 0.2261. Late peak AUC difference: Control vs Sco + Mec: t(3) = 6.976, P = 0.006044; Control vs DHβE: t(4) = 3.671, P = 0.02138; Sco vs Sco+DHβE: t(3) = −1.071, P = 0.3627; Sco vs Sco+Mec: t(3) = 7.904, P = 0.004221; DHβE vs Sco+DHβE: t(4) = 1.000, P = 0.3739; Sco+DHβE vs Sco+Mec: t(3) = 3.283, P = 0.04633; Sco+DHβE+MLA vs Sco+Mec: t(2) = −0.056, P = 0.9607; Sco+DHβE+bPiDl vs Sco+Mec: t(2) = −3.043, P = 0.09316. (e) Schematic summarizing pharmacological experiments to isolate the contributions of various receptors to the biphasic cholinergic evoked DA release.

Article Snippet: The respective receptor contributions to cholinergic evoked DA release used the following antagonists: Dihydro-β-erythroidine hydrobromide (DHβE) for β 2 -subunit-containing nicotinic receptor blockade (1 mg/kg; Tocris cat#: 234910); scopolamine for broad muscarinic receptor blockade (1 or 5 mg/kg; Sigma-Aldrich cat#: S0929); mecamylamine for broad nicotinic receptor blockade blockade (1 or 5 mg/kg; Sigma-Aldrich cat#: M9020); Methyllycaconitine citrate (MLA) for α 7 -nicotinic receptor blockade (1mg/kg; MedChem Express cat#: HY-N2332A); 1,1’-(decane-1,10-diyl) bis (3-methylpyridin-1-ium) iodide (bPiDI) for α 6 *-nicotinic receptor blockade (1mg/kg; MedChem Express cat#: HY-152170).

Techniques: Control, Injection