Fig 2: GLuc monitoring of SCLC induced by adenoviral sgRNA delivery to Cas9 mice. a Experimental scheme for SCLC induction and monitoring with adenoviral vectors (AV) expressing Cre and Trp53/Rb1/Rbl2-targeting sgRNAs. b Validation of sgRNA function by T7 endonuclease I Assay using genomic DNA from uninfected and AV-PRL.Cre infected NIH3T3-Cas9 cells. NTC, no template control. c Western Blot of LSL-Cas9 fibroblasts infected with AV-PR.Cre and AV-PRL.Cre showing downregulation of p53, Rb1 and Rbl2/p130 protein levels. ß-actin is shown as loading control. d Immunohistochemistry for GLuc and Cas9 expression in the lung of mice 2 weeks post infection. Scale bar, 50 µm. e Mutation spectrum of Trp53, Rb1 and Rbl2 sgRNA target loci (flanked by dashed lines). f Kaplan–Meier survival plot of LSL-GLuc mice infected with indicated CRISPR-AVs (AV-C.Cre: n = 14; AV-PR.Cre: n = 12; AV-PRL.Cre: n = 8). Shown are median survival and P-values from Log-rank (Mantel-Cox) test. g Temporal development of blood GLuc activity in mice from f following infection with indicated AVs. Shaded area represents the GLuc background activity. h Total fold change in blood-GLuc activity over the course of tumor development. P-values from Tukey’s multiple comparisons test. i Time point when blood-GLuc activity reached its maximum. P-value from an unpaired, two-sided t-test. j Time difference between the time of sacrifice (survival) and the time point when blood-GLuc activity was first elevated, i.e. exceeded the background range. k Correlation between time of sacrifice (survival) and the time point when blood-GLuc activity was first elevated. Shown is the linear regression with 95% confidence interval All error bars indicate SD, all data points represent biological replicates/individual mice

Fig 3: SCLC induction by adenoviral delivery of CRISPR nucleases. a Experimental scheme for SCLC induction and monitoring with adenoviral vectors (AV) expressing Cre and Trp53/Rb1-targeting Cas9 nucleases. b Validation of sgRNA function by T7 endonuclease I assay using genomic DNA from uninfected and AV-PR.CC9 infected NIH3T3 cells. NTC, no template control. c Western Blot of mouse embryonic fibroblasts (MEF) infected with AV-PR.CC9 showing Cas9 expression and downregulation of p53 and Rb1 protein levels. ß-actin is shown as loading control. d Immunohistochemistry for GLuc and Cas9 expression in the lung of mice 1 week after intratracheal AV-PR.CC9 infection. e,f Mutation spectrum of e Trp53 exon 6 and f Rb1 exon 17 sgRNA target loci (flanked by dashed lines). Shown are sequencing reads of the most abundant indel mutations at the sgRNA target site. g Sequential MRI of a representative mouse showing tumor progression. Shown are frontal and transversal sections with tumors marked in blue. h Kaplan–Meier survival plot of WT and LSL-GLuc mice infected with indicated CRISPR-AVs (AV-C.CC9 + GLuc: n = 12; AV-PR.CC9 + GLuc: n = 8; AV-PR.CC9 no GLuc: n = 3). Shown are median survival and P-values from Log-rank (Mantel-Cox) test

Fig 4: Expression of oncogenic H-RAS induces senescence in Rh28 aRMS cells. (A) To assess senescence, Rh28 cells expressing H-RAS mutants were stained for ß-gal. Representative images are shown (magnification ×100). Expression of oncogenic H-RAS increased ß-gal staining whereas the other H-RAS mutants did not. (B) Expression of RB and p53 pathway members was examined by immunoblot in both Rh28 and RD cells expressing H-RAS mutants. Representative blots are shown. While the expression of oncogenic H-RAS led to inconsistent changes in the levels of total RB, total p53, and phospho-p53 over multiple replicates, p16 and p21 were consistently elevated and pRB levels were decreased with the expression of oncogenic H-RAS in aRMS Rh28 cells; this effect was not seen to the same degree in R-eRMS RD cells. Actin used as a loading control. Densitometry quantitation shown below blots and is normalized to Rh28 Vector control.

Fig 5: Death of DIS cells after p21 knockdown ensued by TNF-a secretion Survival of DIS BJ cells transduced with siRNAs targeting p21, p53 or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots of p21, p53, cleaved PARP, and cleaved caspase-3 after siRNA treatment.Survival of DIS BJ cells transduced with siRNAs targeting p21, pRb or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots depict p21 and pRb protein levels following siRNA treatment.Western blot analysis of p21, p65, p-p65, JNK, p-JNK, cleaved PARP, and cleaved caspase-3 proteins 4 days after sip21 or siCtrl treatment of G and DIS cells; representative blots of at least two independent experiments are depicted.mRNA expression levels of TNF-a and IL-8 in DIS cells at the indicated times after sip21 or siCtrl treatment. Data are presented as means ± SEM of three repeats.Survival of DIS BJ cells transduced with siRNAs targeting p21, p65 or their combinations, as indicated, 4 days after siRNA washout. Western blots depict levels of p21, p65, JNK, p-JNK, cleaved PARP, and cleaved caspase-3 following siRNA treatment. Data are presented as means ± SEM of three repeats, each performed in triplicate.mRNA expression levels of TNF-a relative to control following treatment with siRNAs targeting p21, p65, or their combinations. Data are presented as means ± SEM of three repeats.Data information: Data were analyzed using Student's t-test. *P < 0.05, **P < 0.005; ***P < 0.0005. n.s, not significant. Source data are available online for this figure.