(A) Dose response curve of control BaF3 cells (Control), which was established after transfection of PIK3R1^WT but expressed low exogenous WT p85α level, or BaF3 cells stably expressing PIK3R1^E160*, PIK3R1^R348* or KRAS^G12D for PD0325901, SP600125 and SB203580. Means (±SD) of duplicates from 2 independent experiments are shown. (B) Heatmap of unsupervised cluster analysis of stable BaF3 cells and selected proteins by RPPA. Red indicates higher level relative to other samples. (C) The same set of lysates used in (B) was used for Western blotting. See also and and .

(A) Top, schematic showing the locations of p85α siRNA-targeted regions; Bottom, ovarian endometrioid cancer cells OVK18 transfected with p85α siRNAs or non-specific (NS) control for 72 hr were cultured in the absence of FBS (0% FBS) for additional 24 hr. The cells were then simulated with epidermal growth factor (EGF; 10 ng/ml) or insulin-like growth factor 1 (IGF-1; 10 ng/ml) for another 1 hr before being harvested for Western blotting. Lysates were loaded into two gels due to sample well limits but the membranes were probed with same antibodies for equal duration and the proteins were detected under same exposure time. (B) Western blotting of total lysates or subcellular fractions from SKUT2 endometrial cancer cells transfected with PIK3R1^WT, PIK3R1^E160*, PIK3R1^R348* or KRAS^G12D. α-tubulin (cytosolic), pan-cadherin (membrane) and histone H3 (nuclear) served as markers for purity of fractions. (C, D) Immunohistochemical staining for p-ERK (C) and p-JNK (D) in 4 endometrial carcinomas carrying PIK3R1^R348* and 12 endometrial carcinomas without PIK3R1^R348* (control). Nuclei were counterstained in hematoxylin. Bar, 50 μm. See also .

(A) Total lysates from transfected SKUT2 were analyzed by Western blotting. (B) Cells co-transfected with PIK3R1^WT, PIK3R1^R348* or KRAS^G12D and 20 nM siRNAs targeting the MKKs or non-specific (NS) control for 72 hr were harvested for subcellcular fractionation. Western blots shown were from nuclear lysates. (C) Transfected SKUT2 were harvested for subcellcular fractionation and Western blotting. (D) Cells were co-transfected with PIK3R1^WT, PIK3R1^R348* or KRAS^G12D and 10 nM siRNAs targeting B-Raf, c-Raf or NS control for 72 hr. Data shown are Western blots of cytosolic lysates. (E) Total lysates from transfected SKUT2 were harvested for immunoprecipitation (IP) with anti-B-Raf antibody and Western blotting (WB). IP with anti-IgG was used as control. (F) Cells co-transfected with PIK3R1^WT, PIK3R1^R348* or KRAS^G12D and 20 nM siRNAs targeting MLK2, MLK3, DLK (40 nM) or NS control for 72 hr were harvested for subcellular fractionation. Western blots shown were from nuclear lysates. (G) Western blots for MLK3 in total lysates, cytosolic and nuclear fractions from transfected SKUT2. (H) Western blots of IP with anti-MLK3 antibody using cytosolic and nuclear extracts from transfected SKUT2. See also

(A, B) SKUT2 cells transfected with HA-tagged PIK3R1^WT, PIK3R1^E160* or PIK3R1^R348* were harvested for subcellular fractionation and Western blotting (C, cytosolic; M, membrane; N, nuclear) (A) or immunostaining using anti-HA (green) antibody (B). DAPI was used for nuclear staining. ‘Merge’ indicates combined images. Bar, 10 μm. (C) Schematic illustration of PIK3R1 truncation mutations upstream of R348*. (D, E) Cell lysates from SKUT2 cells transfected with indicated mutants were subjected for subcellular fractionation as in panel A (D) or Western blotting (E). (F) BaF3 cells transfected with PIK3R1^WT or PIK3R1 mutant were cultured without IL3 for 4 weeks prior to viability assay. Means (±SD) of triplicates from 3 independent experiments are shown. (G, H) SKUT2 transfected with PIK3R1^WT, PIK3R1^N324*, PIK3R1^R348* or PIK3R1^N324* and PIK3R1^R348* fused with nucleus export signal (NES) were harvested for subcellular fractionation and Western blotting (WB) (G) or immunoprecipitation (IP) with anti-MLK3 antibody in nuclear extract and analyzed by WB (H). *p < 0.05, compared with PIK3R1^WT. control, parental SKUT2. See also .

(A) Total lysates from SKUT2 transfected with HA-tagged PIK3R1^WT or mutants were harvested for immunoprecipitation (IP) with anti-HA antibody and Western blotting (WB). IP with anti-IgG was used as control. (B, C) Cells co-transfected with PIK3R1^R348* and 20 nM each of the indicated siRNAs for 72 hr were harvested for subcellular fractionation (C, cytosolic; M, membrane; N, nuclear) and WB. (D-F) Cells co-transfected with PIK3R1^R348* and dominant negative (DN)-Cdc42, -Rac1 or -RhoA for 72 hr were harvested for subcellular fractionation (D) or total lysates used for IP with anti-B-Raf antibody (E) or nuclear lysates used for IP with anti-MLK3 antibody (F). See also .

(A) Nuclear lysates from SKUT2 cells transfected with HA-tagged PIK3R1^R348* were subjected to immunoprecipitation (IP) with anti-HA antibody and Western blotting (WB). (B) Nuclear lysates from SKUT2 transfected with HA-tagged PIK3R1^WT, PIK3R1^R348*, nucleus export signal (NES)-fused PIK3R1^R348* were subjected to IP with anti-JNK1 antibody. (C, D) Nuclear extract from SKUT2 transfected with HA-tagged PIK3R1^R348* was fractionated using gel filtration column. The indicated fractions were analyzed by WB (C) or pooled for IP with anti-HA antibody (D). (E, F) Nuclear extract from SKUT2 transfected with HA-tagged NES-PIK3R1^R348* was fractionated using gel filtration. The indicated fractions were analyzed (E) by WB or pooled for IP with anti-JNK1 antibody (F). F, fraction. MW, molecular weight. See also .

(A) SKUT2 stably expressing LacZ, PIK3R1^WT, PIK3R1^E160* or PIK3R1^R348* were cultured in medium containing 1% FBS with or without MEK inhibitor PD0325901 (PD) at 20 nM. DMSO served as negative control. Cell proliferation was determined at 24, 48, and 72 hr after seeding. (B) Stable SKUT2 were cultured in medium containing 5% FBS (control) or anti-Fas antibody (+FAS) with or without JNK inhibitor SP600125 (SP) at 20 μM for 72 hr before apoptosis assay. (C) Representative images (left) and mean number of invaded cells of five fields at magnification of 40x (right) of indicated SKUT2 cells in the presence of indicated inhibitors. (D) Representative images (left) and cell viability (right) of indicated SKUT2 cells grown in 3D culture for 6 days before being treated with SP or PD or combination for another 3 days. (E) Cells were subcutaneously injected into mouse flank regions (n=12/group). Tumor volume was measured weekly. (F) Tumor-bearing mice were treated with vehicles, MEK inhibitor GSK1120212b or GDC0973, JNK inhibitor BI78D3 or p38 MAPK inhibitor SB203580. Tumor growth inhibition percentages were calculated and presented in the graph. Scale bars represent 50 μm (C and D). All in vitro assays were performed with two independent stable clones yielding similar results. Data (Means (±SD)) of clone #1 of triplicates from 3 independent experiments are shown. All in vivo data are represented as mean ± SEM. *p < 0.05; **p < 0.05 compared with PIK3R1^R348* DMSO control; ***p < 0.05 compared with PIK3R1^WT DMSO control. ^#p < 0.05 compared with PIK3R1^WT at same time point; ^##p < 0.01 compared with PIK3R1^WT at same time point. ^###p < 0.05 compared with PIK3R1^WT treated with corresponding inhibitor. See also .