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: MEK1 is required for Ventx2 clearance and asymmetric distribution during cell division.(A,B) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs, and 25 ng Mk-MO, as indicated. Embryos were fixed at blastula stage 9, or gastrula stage 11, cryosectioned and processed for anti-Myc (red), and anti-GFP (green) immunostaining, and DNA was stained with DAPI (blue). Graphs show the percentage of Myc positive nuclei (DAPI positive) over the total number of injected cells (GFP positive) from four independent experiments. (B) 3D reconstruction of confocal slices of mitotic Myc positive nuclei labeled by DAPI from stage nine sectioned embryos. Sister mitotic chromosomes are referred to as a (more intense Myc staining), and ß (less intense Myc staining). The A-V arrows indicate the animal-vegetal axis. Note the asymmetric cortical Ventx2-Myc signal in the MEK1 morphant cell. (C). The graph shows the ratios of Myc signal intensity betweena and ß sister nuclei.DOI: http://dx.doi.org/10.7554/eLife.21526.00910.7554/eLife.21526.010Figure 3—source data 1.Myc signal intensity ratios between daughter nuclei.Each value corresponds to the ratio calculated from one individual confocal slice between a and ß daughter nuclei (see legend to Figure 3 and Materials and methods for further details).DOI: http://dx.doi.org/10.7554/eLife.21526.010

Fig 4: Depletion of USP1 promotes apoptosis, DNA damage, and tumor growth arrest. Mock control, USP1-KO1, and USP1-KO1 cells reconstituted with either USP1 or MAST1 were used to perform the following experiments. (A) Western blot analyses to validate the expression of USP1 and MAST1 using USP1- and MAST1-specific antibodies. GAPDH was used as the loading control. (B) The cells were treated with either vehicle or cisplatin (2 µg/mL) for 24 h and subjected to immunofluorescence analysis to estimate ?H2AX foci formation. Green, ?H2AX; blue, nucleus stained by DAPI. Scale bar = 100 µm. The right panel depicts the percentage of ?H2AX-positive cells. (C) The cells were treated with cisplatin (2 µg/mL) for 24 h, and MEK1 activation and apoptosis-related factors were determined using Western blotting. GAPDH was used as the internal loading control. (D) The cells were treated with either vehicle or cisplatin (2 µg/mL) for 48 h and subjected to flow cytometry to measure the DNA content using PI staining and (E) annexin-V and 7-AAD staining. (F) The cells were treated with a sub-lethal dose of cisplatin (2 µg/mL) for 48 h, and cell viability was assayed using CCK-8 reagent. Data are presented as the mean and standard deviation of three independent experiments (n = 3). (G-I) Vehicle- or cisplatin-treated cells were subjected to a (G) colony formation assay, (H) wound-healing assay, and (I) Transwell cell-invasion assay. Data are presented as the mean and standard deviation of four independent experiments (n = 4). (J) Xenografts were generated by subcutaneously injecting the mentioned cell groups into the right flanks of NSG mice (n = 4/group). Mice were i.p. injected with either saline (vehicle) or cisplatin (2 mg/kg) twice a week beginning 7 days after xenograft implantation, and tumor size was monitored. Tumor volumes were recorded, and tissues were stored for IHC experiments. The right panel shows the tumors excised from the mice after the experiment. (K) Tumor volume was measured every other day and is 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. (L) Xenograft tumors were embedded in paraffin and sectioned. IHC analyses were performed with the indicated antibodies. Scale bar = 30 µm.

Fig 5: Effect of tissue culture format on EGFR pathway signaling in drug-resistant melanoma models. (A) A375 WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells growing in a 2D monolayer (top) and as 3D spheroids (bottom). For spheroid formation, 500 cells from each line were seeded in each well of a 96-well ultra-low attachment spheroid microplate and grown for five days before imaging; (B) Immunoblots tracking EGFR pathway signaling in engineered A375 melanoma model cells in 2D and 3D tissue culture. Protein was harvested from each cell line growing in 2D culture, and cellular protein from 3D spheroids was collected from spheroids seeded at 500 cells per 96-well and grown for seven days. Total protein (20 µg) was loaded in each lane and samples were blotted for total EGFR, total MEK1/2, phospho-MEK1/2 (Ser217/221), total ERK1/2 (p44/42 MAPK), phospho-ERK1/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), and GAPDH.