Figure 9. Knockdown of Elmo1 diminishes RAC1 expression and markers of invasion in Tgfbr2-deficient SCC.

(A) Immunofluorescence staining with antibodies against YFP and RAC1 revealed a reduction in RAC1 staining at the tumor-stroma border in Tgfbr2 cKO tumors with knockdown of Elmo1 (A’), compared to SH02 control tumors (A). Tumors expressing the hairpin-resistant ELMO1* construct show restoration of RAC1 compared to tumors with Elmo1 knockdown (A’’). All pictures have been taken at the same exposure time in the RAC1 channel. Three tumors for each condition have been analyzed. DAPI counterstains all nuclei in blue. Scale bars = 10 μm. (B) qPCR analysis of genes implicated in EMT, invasion and metastasis revealed a significant reduction in their mRNA expression in CD34+ cells isolated from Tgfbr2 cKO tumors with knockdown of Elmo1 compared to SH02 control tumors (see Figure 9—figure supplement 1 and 2). Data represent the mean ± standard deviation. Asterisks denote statistical significance using two way-ANOVA and Bonferroni post tests to compare each Elmo1 shRNA to SHO2 control. All p-values are <0.001.

DOI: http://dx.doi.org/10.7554/eLife.22914.027

Figure 9—figure supplement 1. Knockdown of Elmo1 in vivo.

(A) FACS analysis of tumors generated from orthotopic transplantation of 100,000 Tgfbr2 cKO CD34+ SCC cells infected with SH02 control or Elmo1 shRNA selected with puromycin and infected again with pLVX-IRES-mCherry or with the hairpin-resistant Elmo1 construct (ELMO1*) and selected based on mCherry expression. Three different tumors for each construct were analyzed. Approximately 32% of the cKO SCC SH02 or Elmo1 shRNA + empty vector total tumor bulk expressed mCherry, whereas less than 1.5% of the cKO SCC Elmo1 shRNA + ELMO1* expressed mCherry at the time of analysis. (B) Tumors were dissociated and YFP+mCherry-CD34+ and YFP+mCherry-CD34− cells were isolated by FACS and subjected to RNA extraction and qPCR analysis. We observed a 60% reduction in Elmo1 mRNA compared to CD34+ cells isolated from SH02 tumors. Infection of CD34+ Elmo1 shRNA SCC cells with the hairpin-resistant ELMO1* construct efficiently restored Elmo1 mRNA. Asterisks denote statistical significance using two-way ANOVA and Bonferroni post tests; p***<0.001. (C) 100,000 CD34+ cKO SCC cells which were infected with SH02 control, Elmo1 shRNA construct#1 or Elmo1 shRNA construct #2 were orthotopically transplanted into recipient mice and YFP+CD34+ cells were isolated from the resulting tumors using the same FACS strategy as employed in Figure 1, and subjected to qPCR. Elmo1 mRNA was reduced by 53–61% in CD34+ cells isolated from tumors with knockdown of Elmo1 compared to SH02 tumors. *p=0.045 for construct #1 and **p=0.004 for construct #2.

DOI: http://dx.doi.org/10.7554/eLife.22914.029

Figure 9—figure supplement 2. Knockdown of Elmo1 diminishes markers of invasion in Tgfbr2- deficient SCC.

(A) Infection of CD34+ Elmo1 shRNA SCC cells with the hairpin-resistant ELMO1* construct efficiently restored ELMO1 protein expression in the generated tumors as shown by immunofluorescence with an antibody against ELMO1. Scale bars: 10 μm. (B) 100,000 CD34+ cKO SCC cells which were infected with SH02 control, Elmo1 shRNA construct #2 or Elmo1 shRNA construct #2 with the hairpin-resistant ELMO1* were orthotopically transplanted into recipient mice and CD34+ YFP+mCherry+ cells were isolated from the resulting tumors using the same FACS strategy as employed in Figure 1, and subjected to qPCR. qPCR analysis of genes implicated in EMT, invasion and metastasis revealed a significant reduction in their mRNA expression in CD34+ cells isolated from Tgfbr2 cKO tumors with knockdown of Elmo1 compared to SH02 control tumors. See also Figure 9—figure supplement 1. The level of mRNA expression of these genes is restored or increased in the cells expressing the hairpin-resistant ELMO*1 construct. Data represent the mean ± standard deviation. Asterisks denote statistical significance using two-way ANOVA and Bonferroni post tests compared to SHO2; p*<0.05, p**<0.01, p***<0.001..

DOI: http://dx.doi.org/10.7554/eLife.22914.030