Fig 2: H-Ras transformed fibroblasts (Ras) have elongated mitochondria and increased OXPHOS that is supported by mitochondrial biogenesis.(A) Immunoblotting of whole-cell lysates shows H-Ras expression in transformed fibroblasts, but not in empty vector (EV) controls. (B) Growth curve shows the loss of contact inhibition in H-Ras transformed fibroblasts. The proliferation of EV controls remains cell-density dependent (n = 4). (C) Ras cells exhibit a morphological change and gain the ability to grow on top of each other to form multiple cell layers. (D) Mitochondrial stress test of EV controls and Ras cells (n = 3). The respiration of Ras cells is statistically increased compared to EV controls. (E) Measured and calculated parameters of mitochondrial respiration (using results from Figure 4D) (n = 3). (F) Statistical analysis of mitochondrial length in EV controls and Ras cells. In each condition, 100 random mitochondria were measured from EM images. Data are presented as mean ±SD. (G) RT-PCR shows that Ras cells have over a 20-fold increase in mRNA levels of PGC1α (n = 3). (H) Ras cells have increased mitochondrial mass, as indicated by an increased mtDNA/gDNA compared to EV controls (n = 3). (I) Immunoblotting of whole-cell lysates shows that Ras cells have higher expression levels of ETC subunits compared to EV controls. (J) Mfn2 knockdown decreases cellular proliferation in both EV controls and Ras cells. Proliferation was assessed by manual counting after cells were treated with scrambled siRNA control (SSC) or Mfn2 siRNA (Mfn2KD) for 72 hr. Relative proliferation was normalized to either an EV SSC or a RAS SSC for each condition (n = 4). (K) Mfn2 knockdown decreases cellular proliferation in H460 (lung cancer cells), HeLa (cervical cancer cells), BT549 (breast cancer cells), and MCF7 (breast cancer cells). Proliferation was assessed by using a CyQUANT proliferation assay after cells were treated with scrambled siRNA control (SSC) or Mfn2 siRNA (Mfn2KD) for 72 hr (n = 5). Unless specified, data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001, n.s. not statistically significant. OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone; AA, Antimycin A.

Fig 3: PRC2 transfers from chromatin to nascent pre-mRNA during gene activation.(a) Hypothesis: Upon activation of polycomb target genes, PRC2 switches from binding chromatin to binding nascent RNA.(b) SUZ12, H3K27me3 and total H3 chromatin occupancy (with IgG control) at Adcy7, Sorcs2 and Actb before and after HRasV12 expression (mean and S.D., n=3 independent ChIPs. Adcy7 SUZ12 P=0.0051, H3K27me3 P=2.1x10-4. Sorcs2 SUZ12 P=0.0041, H3K27me3 P=4.9x10-4, Welch’s one-tailed t-test).(c) SUZ12 binding to Adcy7, Sorcs2 and Actb nascent pre-mRNA before and after HRasV12 expression, measured by RIP-qPCR with and without UV-crosslinking of cells (mean and S.D., n=3 independent RIPs. Adcy7 SUZ12 +/-Ras P=0.011, Sorcs2 SUZ12 +/-Ras P=0.0019, Welch’s one-tailed t-test).(d) Change in HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy at Adcy7 and Sorcs2 after dox-mediated induction of HA-dCas9 expression in cells containing sgRNA specific for Adcy7 (mean and S.D., n=3 independent dox inductions. Adcy7 G-tract RNA: SUZ12 P=0.014, H3K27me3 P=0.024, Welch’s one-tailed t-test).(e) As (d), except in cells containing sgRNA specific for Sorcs2. Sorcs2 G-tract RNA: SUZ12 P=0.017, H3K27me3 P=2.9x10-4, Welch’s one-tailed t-test).

Fig 4: KRAS-G12V over-expression alongside hPRLrL + I co-expression in vitro.a MCF10A hPRLr over-expression transfectants received an additional stable transfection of KRAS-G12V, and expression was confirmed via IB. Cells were then grown in soft agar, and both b colony number and c colony size were determined. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. n = 3. Data is shown as mean ± SEM.

Fig 5: Effects of mutant KRAS or EGFR expression on phenotype and neuroendocrine markers in small cell lung cancer cells.(a) Overview of representative somatic alterations, protein expression profiles, MAPK pathway activity, and neuroendocrine differentiation in small cell lung cancer (SCLC), lung adenocarcinoma (LUAD), and transformed SCLC from EGFR-mutated LUAD. (b) Induction of EGFRL858R or KRASG12V as assessed by western blot and of GFP assessed by fluorescence phase contrast images in SCLC cell lines, H2107, H82, and H524 cells, upon treatment with 100 ng/mL doxycycline (dox) for 3 and 7 days. GAPDH was used as a loading control. (c) Photomicrographs showing the growing morphology in suspending aggregates of GFP-overexpressing H82 cells (left) and in mixed adherent and suspended states of EGFRL858R- (middle) or KRASG12V- (right) overexpressing H82 cells upon treatment with 100 ng/mL dox for 7 days. Yellow and white arrowheads indicate suspending aggregates and adherent cells, respectively. Scale bars, 400 µm. (d) Crystal violet assay of adherent cells with or without induction of GFP, EGFRL858R, or KRASG12V in H2107 (on day 7), H82 (on day 7), and H524 (on day 5) cells. Medium containing suspended cells was removed, and adherent cells were washed with PBS and then fixed and stained with crystal violet. (e) Quantification of cell attachment after GFP, EGFRL858R, or KRASG12V induction in H2107 (on day 7), H82 (on day 7), and H524 (on day 5) cells. After incubation, medium containing suspended cells was removed and adherent cells were washed with PBS. Adherent cells were then cultured in fresh media and viability was assessed using an alamarBlue cell viability agent. Values relative to a no dox control for each cell line are graphed as mean (three biological replicates) ± SEM. The Student’s t test, ****p<0.0001; **p<0.01; and *p<0.05. (f) Representative immunoblot showing the effects of induction of GFP, EGFRL858R, or KRASG12V on neuroendocrine markers as well as phosphorylation status of ERK and AKT upon treatment with 100 ng/mL dox for 3 and 7 days in H2107, H82, and H524 cells. GAPDH was used as a loading control. Numbers below blots show the amounts of respective band relative to the corresponding non-dox-treated control values after normalization to ERK1/2 (for pERK1/2) or GAPDH (for INSM1, BRN2, ASCL1, NEUROD1, CD56, SYP, and MYC) in each condition for each cell line. (g) Gene set enrichment analysis (GSEA) neuroendocrine differentiation scores of H2107-KRASG12V and H82-KRASG12V cells pre- and post-dox treatment (day 7). (h) Enrichment plots of the 50-gene lung-cancer-specific neuroendocrine expression signature gene sets in H2107-KRASG12V and H82-KRASG12V cells post-dox treatment for 7 days compared with GFP-overexpressing controls. Immunoblots are representative of at least two biological replicates.