Fig 2: Oncogenic RAS-driven mouse and human cancer cell lines are sensitive to dual MEKi and ERKi treatment. (A,B). Relative cell number (A) and cellular viability (B) assessed on day three of treatment of the indicated UPS cell lines with the indicated inhibitors (1 µM). Results are normalized to the untreated condition, mean ± std. dev. of 3 biological replicates is shown and statistical differences compared to treatment with DMSO are shown. Two-Way ANOVA, Turkey’s method: * 0.05 < p < 0.0332, ** 0.0332 < p< 0.0021, *** 0.0021 < p < 0002, **** p < 0.0001. (C–F). Short-term (3 days) response of UPS (C), KPC (D) and human CRC, NSCLC, RMS (E) and PDAC (F) cell lines to the indicated inhibitors (1 µM). Mean of three independent experiments is shown, each of which involved three technical replicates. Differences of treatments compared to DMSO were all statistically significant, Dunnett’s multiple comparison test (2way ANOVA): * 0.05 < p < 0.0332, ** 0.0332 < p < 0.0021, *** 0.0021 < p < 0002, **** p < 0.0001. An exception in panel E to the high significance is cell line NCI-H1299 where cell growth after treatment with GDC was reduced to a less significant extent (**). Statistical significance of the double treatment is shown only when significant over both the respective single treatments. Triple treatment was more effective than double treatment only when compared to RO + GDC, which was ineffective on its own (C). Squares without significance stars were all non-significant. No statistically relevant differences were measured in the efficiency of treatments between cell lines from primary tumors and corresponding metastatic lesions (C). (G,H). Curves of interaction between SCH and GDC with respective synergistic score (CI) in the indicated mouse (G) and human cell lines (H). X and Y axes represent both the fixed concentration of the individual drug as well as the 72 h anti-proliferative IC50 value of that drug at the fixed concentration of the other drug.

Fig 3: NGBR knockdown decreases the migration of HemSCs in vitro.(A) NGBR knockdown did not affect the cell viability of HemSCs. AO/EB staining was used for determining apoptotic HemSCs after treatment with siControl and siNGBR. The quantitative results show the average percentage of apoptotic cells (n = 3), 3 repeats. (B) The representative wound-healing images of HemSCs transfected with siControl or ​siNGBR at indicated times. Each condition was photographed in 4 separate fields. The quantitative results are presented as a bar graph (right). *P < 0.05 vs. control (siControl) cells (n = 3), 3 repeats. (C) Transwell migration assay of HemSCs transfected with either siControl or ​siNGBR with/without EGF, FGF2, and VEGF stimulation. The representative images of migrated cells are shown in the left panel. The bar graph in the right panel shows the quantitative numbers of migratory cells evaluated by ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. control (siControl) cells treated with EGF, FGF2, and VEGF (n = 3), 3 repeats. (D) Scratch wound healing assay shows reduced migration of either HRAS- or KRAS-deficient HemSCs. HemSCs transfected with siControl, siHRAS, or siKRAS were subjected to scratch wound healing assay. The representative images of wound healing are shown at indicated times. The bar graph in the right panel represents the percentage of the cell-covered area determined from each time point. *P < 0.05 vs. control (siControl) cells (n = 3), 3 repeats. (E) Transwell migration assay of HemSCs transfected with siControl, siHRAS, or siKRAS. Cell migration was determined using Corning Transwell chambers. The results presented are an average of migrated cell numbers in 4 random microscopic fields from 3 independent experiments. *P < 0.05 vs. control (siControl) cells (n = 3). Statistical analyses: 2-tailed unpaired Student’s t test (A, B, D, and E) and 1-way ANOVA with Bonferroni’s post hoc test (C); data are expressed as mean ± SEM. NGBR, NOGOB receptor; HemSCs, hemangioma stem cells; AO/EB, acridine orange/ethidium bromide; siControl, control siRNA; siNGBR, NGBR siRNA.

Fig 4: Nogo-B receptor (NgBR) knockdown attenuates epidermal growth factor receptor (EGF)-stimulated signaling and estrogen receptor alpha (ERa) phosphorylation in MCF-7-TamR cells. a MCF-7-TamR cells were transfected with siNgBR and treated with EGF (100 ng/mL) for 5 min. Downstream signaling of the EGF pathway was determined using western blot assay. b Quantitative analysis of phosphorylated proteins presented in Fig. 4b were carried out using ImageJ and were normalized to total proteins. c MCF-7-TamR cells were transfected with siNgBR and treated with 100 ng/mL EGF for 12 h. Survivin protein levels were determined using western blot analysis. d Quantitative analysis of survivin protein levels presented in Fig. 4c were carried out using ImageJ and were normalized to ß-actin. e NgBR is required for the EGF-stimulated activation of H-Ras and K-Ras in MCF-7-TamR cells. MCF-7-TamR cells were transfected with siNgBR and stimulated with 100 ng/mL EGF for 5 min. The complex of activated Ras (GTP-loaded Ras) was precipitated from total cell lysates using GST-RBD beads. Protein levels were detected by western blotting. Both Ras and NgBR were detected in the complexes precipitated by the Raf-pull-down method. The data are from three separate repeat experiments and are presented as the mean ± SD (*p < 0.05, n = 3)

Fig 5: Overexpression of constitutively activated HRAS/KRAS diminishes NGBR deficiency caused G1 phase arrest.(A and B) siControl and siNGBR cells were transiently transfected with empty vector or HRAS-G12V/KRAS-G12V expression vector for 24 hours. Protein levels of NGBR, p21, p53, cyclin D1, RB1, and phosphorylated RB1 were determined by Western blot. Quantitative analysis of proteins was carried out using ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. siNGBR cells (n = 3). (C) siControl and siNGBR cells expressing the FUCCI fluorescent CDT1-RFP (red) and geminin-GFP (green) were transiently transfected with HRAS-G12V or KRAS-G12V expression vector for 24 hours. The images were taken with a confocal microscope. Quantitative analysis of G1 phase cells was determined by ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. siNGBR cells (n = 4), 3 repeats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test; data are expressed as mean ± SEM. NGBR, NOGOB receptor; siControl, control siRNA; siNGBR, NGBR siRNA; RB1, retinoblastoma; FUCCI, fluorescence ubiquitination cell cycle indicator.