Fig 2: The combination of VCR and RCM1 is more efficient than single agents in reducing growth of human RD rhabdomyosarcoma cells in vitro. (A), Left: VCR reduces RD tumor cell viability in a dose-dependent manner after 24 hours of treatment compared to vehicle (saline) control. Right: RCM1 reduces RD tumor cell viability in a dose-dependent manner after 24 hours of treatment compared to vehicle (DMSO) control. Graphs show the percentage of live cells per group compared to vehicle control. (B), Combination therapy using IC50 concentrations of VCR (3.5nM) and RCM1 (3.0µM) reduced tumor cell viability compared to single drugs or vehicle (saline + DMSO) control after 24 hours of treatment. The graph shows the percentage of live cells per group compared to vehicle control. (C), Combination therapy reduces proliferation (Ki-67, red) of RD tumor cells compared to single drugs or vehicle control after 24 hours of treatment. (D), Combination therapy reduces mitosis (PH3, green) of RD tumor cells compared to vehicle control after 24 hours of treatment. (E), Combination therapy induces apoptosis (Cleaved-Caspase 3, red) of RD tumor cells compared to single drugs alone or vehicle control after 24 hours of treatment. 5 random fields per sample were used to quantify the number of Ki-67+, PH3+ and Cleaved-Caspase 3+ cells per group. Values are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Scale bar=50µm.

Fig 3: Genetic loss of EYA3-PTP activity attenuates hypoxia-induced PH. a Right ventricular systolic pressure (RVSP) measured after the Sugen-hypoxia (Su-Hx) protocol is reduced in Eya3D262N mice relative to Eya3+/+ control animals. RVSP was measured upon return to room air. Mice maintained in room air served as controls (CTL). Two independent Su-Hx experiments were conducted with a total of 21 C57BL/6J Eya3+/+ and 18 Eya3D262N mice. RVSP measurements reported here represent successful catheterization and are plotted as the mean ± SD. Statistical significance was assessed using pairwise t-tests. b Right ventricular hypertrophy measured after the Su-Hx protocol is reduced in Eya3D262N mice relative to Eya3+/+ control animals. Right ventricular hypertrophy (Fulton’s index = right ventricular (RV) weight/left ventricle (LV) + interventricular septum weight) measured upon return to room air after the Su-Hx protocol, and in mice maintained in room air for an equivalent period. Data plotted as the mean ± SD. Statistical significance was assessed using pairwise t-tests. c Quantitation of the percentage of Ki-67-positive cells. Each data point represents at least six sections per lung. For all groups n = 4. Data plotted as the mean ± SD, statistical significance determined by pairwise t-tests. d Cell proliferation and muscularization is reduced in Eya3D262N mice subject to the Su-Hx protocol. Lung sections were stained with antibodies to a-SMA and Ki-67, and with the nuclear marker DAPI. Scale bar = 100 µm. e Genetic loss of EYA3-PTP activity reduces muscularization of DPAs but does not affect the endothelial cell layer. Representative sections from lungs of Eya3+/+ and Eya3D262N mice either maintained in room air or subject to the Su-Hx protocol were stained for the smooth muscle cell marker a-SMA (green), the endothelial cell marker von Willebrand factor (vWF; red), and the nuclear marker DAPI (blue). Scale bar = 100 µm. Source Data are provided as a Source Data file

Fig 4: Immunohistochemical staining for Ki-67, Napsin A, and TTF1 expression in early LUAD samples from the NJDT cohort. Positive results (×400) of Ki-67(A), Napsin A (C), TTF1 (E) expression FFPE samples of early LUAD by IHC analysis. Negative results (×400) of Ki-67 (B), Napsin A (D), and TTF1 (F) expression FFPE samples of early LUAD by IHC analysis.

Fig 5: p57 is required for the maintenance of quiescence in Bmi1high crypt cells.a Experimental setup for deletion of the maternal allele of the p57 gene in the small intestine. Bmi1-GFP/Villin-CreERT2/p57+/+ (control) and Bmi1-GFP/Villin-CreERT2/p57+/F (p57 CKO) mice were injected intraperitoneally with tamoxifen (50 mg/kg) on 5 consecutive days and were analyzed 3 days after the final injection. b RT-qPCR analysis of relative p57 mRNA abundance in Bmi1high crypt cells isolated as in Fig. 1h from the small intestine of control or p57 CKO mice (n = 3 mice). c Representative immunofluorescence staining of EdU and Bmi1-GFP in the intestine of control and p57 CKO mice 6 h after intraperitoneal injection of EdU (20 mg/kg). Yellow and white arrowheads indicate Bmi1-GFPhigh cells positive or negative for EdU, respectively. Scale bar, 10 µm. d Quantitation of the frequency of EdU+ cells among Bmi1-GFPhigh crypt cells as in c (n = 3 mice). e Representative flow cytometric analysis of the frequency of quiescent (Ki67–PI–) cells in the Bmi1-GFPhigh fraction of intestinal epithelial cells from control and p57 CKO mice treated as in a. f Quantitative analysis for determinations as in e (n = 4 mice). Quantitative data in b are means + SEM, and those in d and f are means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001 (two-tailed Student’s t test). Source data are provided as a Source Data file.