Fig 2: Decreased metabolic and growth kinases in crSTIM1-/- hearts. Immunoblots and densitometric analysis of AMPK (a), PDPK1 (b), ERK1/2 (c), and MEK1/2 (d), phosphorylation in hearts isolated from control and crSTIM1-/- mice treated subcutaneously with 15 min saline or phenylephrine. Protein expression is normalized to total protein and/or GAPDH. STIM1, stromal interaction molecule 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus age-matched control (ANOVA/ Tukey's post hoc), n = 3 per genotype

Fig 3: Chemerin induces intracellular calcium mobilization and stimulates MAPK and Akt signaling in neuroblastoma cellsIntracellular calcium mobilization was measured in SK-N-SH cells with confocal laser scanning microscopy following the stimulation with 10nM chemerin without (A) and with (B) the prior addition of the calcium chelator EDTA. The arrow indicates the time point when chemerin was added. (C) Chemerin stimulates the phosphorylation of Akt, ERK1/2 and MEK1/2 in SK-N-AS cells in a dose-dependent manner. The cells were serum-starved for 24h prior to stimulation and samples were taken 5, 10, 20 and 30min after stimulation. Densitometric analysis of the protein bands was performed and the ratios between p-ERK1/2 and total ERK1/2, p-Akt and total Akt as well as p-MEK1/2 and ß-actin were calculated. The values are displayed relative to the control=1. The experiments were performed three times with similar results.

Fig 4: Combination of pimozide and lestaurtinib inhibits MAST1 protein and cisplatin-resistant tumor growth more than either single treatment. (A) The effect of USP1 inhibition on MAST1 protein level was determined by treating HeLa-cisR cells with increasing concentrations of pimozide for 24 h. The protein expression of MAST1 was determined by Western blotting. GAPDH was used as an internal loading control. (B) The effect of combination treatment of pimozide and lestaurtinib on MAST1-mediated MEK phosphorylation. HeLa-cisR cells were treated with pimozide (50 µM) and lestaurtinib (200 nM) in the presence of sub-lethal doses of cisplatin (5 µg/mL) for 24 h. The activity of MAST1 was assessed by a Western blot analysis of the phospho-MEK1 and phospho-ERK levels. GAPDH was used as an internal loading control. (C, D) The effect of combined treatment with pimozide and lestaurtinib on (C) cisplatin sensitivity (n = 3) (D) and cell viability in A549-cisR and HeLa-cisR cells (n = 4). Data are presented as the mean and standard deviation of at least three independent experiments. Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. (E) Combination index (CI) plots for the synergistic effect of pimozide and lestaurtinib in A549-cisR and HeLa-cisR cells. (F-H) The effect of combination treatment with pimozide and lestaurtinib was validated using (F) colony formation assay, (G) wound-healing assay, and (H) Transwell cell-invasion assay. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. (I) Xenografts were generated by subcutaneously injecting A549-cisR cells into the right flanks of NSG mice (n = 4). Mice were treated with pimozide (10 mg/kg), lestaurtinib (20 mg/kg), and cisplatin (5 mg/kg) beginning 26 days after xenograft implantation, and tumor size was monitored. The right panel shows the tumors excised from the mice after the experiment. (J) Tumor volume and tumor weight were measured and are presented graphically. Data are presented as the mean and standard deviation of four independent experiments (n = 4). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. For brevity, statistical significance is shown only for comparisons between the groups of interest, except for the negative control group.

Fig 5: MEK1 Ablation Delays Return to Quiescence during Recovery from a Single 5-FU Injection and Promotes the Expression of Cell Cycle and OXPHOS Genes(A–C) BM cellularity and HSCs per femur (A), HSC cell cycle distribution (B), and blood parameters (C) during the recovery from 5-FU injection.(D and E) Heatmap (D) and scatterplots (E) of genes differentially expressed in F/F and cKO HSCs from untreated or 5-FU-treated mice (9 days prior isolation). Blue, increased expression in F/F HSCs; red, increased expression in cKO HSCs.(F) qRT-PCR of selected cell cycle and OXPHOS genes in FAC-sorted HSCs. Data are normalized to expression levels in F/F HSCs from untreated mice (dotted line).Error bars represent the SD of the mean. *p < 0.05, **p < 0.01, and ***p < 0.001 comparing CRE+ or F/F to cKO. White asterisks, comparison of 5-FU-treated versus untreated animals of the same genotype. See also Figure S2 and Table S1 for a complete gene list.