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. 2016 Oct 19;5:e17929. doi: 10.7554/eLife.17929

Figure 5. CypD activity is required for phenotypes associated with TP53 exon-6 truncating mutations.

(A) CypD is required for maintaining cells in a mesenchymal like state. The chart represents mRNA expression analysis of the indicated genes in A549 (p53-WT), Hop62 (p53-psi), DMS114 (p53-R213*) and Calu-6 (R196*) cell lines after CypD knockdown. Cells harboring p53-psi or TP53 exon-6 nonsense mutations are indicated in blue. mRNA expression was quantified by SYBR-green-based RT-qPCR. Each bar is the average of 3 replicates and represents mRNA expression of the indicated gene relative to GAPDH (p-value, *<0.05, **<0.005 and ***<0.0005, unpaired t-test). See Figure 5—figure supplement 1A for analysis in additional cell lines. (B) CypD is required for the survival of cells harboring p53-psi splice or TP53 exon-6 truncating mutations. The graph represents cell survival curve of indicated cell lines when treated with CypD inhibitor C-9 for 120 hr. (C) Crystal violet staining of the indicated cell lines upon CypD knockdown with two independent shRNAs. A scramble shRNA was used as negative control. The quantification of knockdown efficiency is provided in Figure 5—figure supplement 1B. (D) The chart depicts the percentage of viable cells 8 days after infection with the indicated CypD shRNA constructs relative to scramble shRNA control. Each bar represents the mean of 9 individual replicates (p-value *<0.0005 and **<0.00005, unpaired t-test). (E) Workflow of the transplantable model system used in this study. A549 (p53-WT) and Calu-6 (p53 R196*) cells were transduced with an inducible CRISPR-Cas9 (DD-Cas9) targeting CypD (CypD g.131) and Renila (Ren g.208). Cells were transplanted sub-cutaneously in immune-deficient mice. When the tumors reached an approximate size of 4–5 mm in diameter, mice were treated with Shield-1 (1 µg). Tumor volume was determined at the indicated time points. See Supplementary file 5 for sgRNA sequences. (F) The charts illustrate quantification of tumor volumes (mean ± SD) in the indicated cohorts at given time points (n = 4, p-value *<0.05, unpaired t-test). Validation of CypD inactivation is provided in Figure 5—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.17929.027

Figure 5.

Figure 5—figure supplement 1. Differential expression of indicated genes and knockdown efficiency of CypD shRNAs.

Figure 5—figure supplement 1.

(A) The chart represents mRNA expression analysis of the indicated genes in HCC1937, H2126 and H1299 cell lines. mRNA expression was quantified by SYBR-green based RT-qPCR. Each bar is the average of 3 replicates and represents mRNA expression of the indicated genes relative to GAPDH (p-value, *<0.05, **<0.005 and ***<0.0005 unpaired t-test). (B) The chart represents the knockdown efficiency of two CypD shRNAs in indicated cell lines at 96h post infection. mRNA expression was quantified by SYBR-green based RT-qPCR. Each bar is the average of 3 replicates and represents mRNA expression of the indicated genes relative to GAPDH (p‐value, *<0.005 and **<0.0005 unpaired t-test).
DOI: http://dx.doi.org/10.7554/eLife.17929.028
Figure 5—figure supplement 2. Inactivation of CypD by CRISPR-Cas9 in in vitro and in vivo systems.

Figure 5—figure supplement 2.

(A) Western blot analysis of the indicated cell lines using a CypD specific antibody and an antibody against RasGAP as loading control. (B) Mice were injected sub-cutaneously with A549 cells, after 9 days treated with Shield-1 and sacrificed at day 16. Sections of tumors transduced either with Renila sgRNA or CypD sgRNA were stained with a CypD specific antibody. Representative pictures are shown.
DOI: http://dx.doi.org/10.7554/eLife.17929.029