Prism 8.0 software

Data are presented as the mean with error bars showing the standard deviation (SD). Statistical analyses were performed using Prism 8.0 GraphPad software (GraphPad). Data were analyzed using a un/paired two‐sided Student's t‐test, One‐Way analysis of variance (ANOVA) with Holm–Sidak's post‐test, Two‐Way ANOVA with Sidak's multiple comparisons test, or as indicated in figure legends. p‐values <0.05 were considered statistically significant and are reported in figures using the following notation: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (with * referring to the comparisons specified in each figure legend).

Data were reported as mean values ± SEM of at least three independent experiments (n=3) routinely performed in triplicate (three wells/condition). Results were analyzed using Prism 8.0 GraphPad Software (GraphPad, San Diego, CA, United States). Statistical significance was determined using ANOVA followed by Bonferroni post-hoc test. Differences were considered as statistically significant when p<0.05.

Assays were performed in base reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). YKL-05–099 was dissolved in 100% DMSO in a 10 mM stock. Serial dilution was conducted by Integra Viaflo Assist in DMSO. Recombinant CSF1R (Invitrogen, Carlsbad, CA) was used at a concentration of 2.5 nM. The substrate used was pEY (Sigma-Aldrich, St. Louis, MO) at a concentration of 0.2 mg/ml. Kinase assays were supplemented with 2 mM Mn²⁺, and 1 µM ATP was added. Assays were performed for 20 min at room temperature, after which time ³³P-ATP (10 µCi/µl) was added followed by incubation for another 120 min at room temperature. Thereafter, radioactivity incorporated into the pEY peptide substrate was detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to DMSO reactions. IC₅₀ values and curve fits were obtained using Prism 8.0 GraphPad Software (GraphPad Software, San Diego, CA).

Real-time semi-quantitative PCR (qPCR) values are presented as arbitrary units, mean ± SEM, normalized to housekeeping genes and expressed as 2^−ΔΔCT in arbitrary units. GraphPad 8.0 Prism software, all data are presented as mean ± SEM. Analysis of the groups were determined using an unpaired or paired t-test as appropriate; where data were not normally distributed, a non-parametric Mann–Whitney or Wilcoxon matched-pairs test was completed. Significance was accepted when p ≤ 0.05.