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. 2019 Mar 25;8:e43333. doi: 10.7554/eLife.43333

Figure 2. Loss of WRN selectively impairs viability of MSI-H CRC and endometrial cancer cell models.

(A) MSS and MSI-H CRC cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. WRN siRNA knock-down efficacy was analyzed by immunoblotting. Protein lysates were prepared 72 hr after transfection. GAPDH expression was used to monitor equal loading. (B) Crystal violet staining of MSS and MSI-H CRC lines treated as in panel A. (C) Cell viability analysis of MSS and MSI-H endometrial cell lines treated as in panel A. Data information: In (A and C), data are presented as mean ± SD of three independent experiments.

Figure 2.

Figure 2—figure supplement 1. Non-transformed cells do not display WRN dependency.

Figure 2—figure supplement 1.

(A) WRN siRNA knock-down efficacy in endometrial carcinoma cell models was analyzed by qRT-PCR. RNA lysates were prepared 72 hr after transfection. WRN mRNA expression is normalized to 18S rRNA levels (n = 1 experimental replicate). (B) Non-transformed hTERT RPE-1 and HCT 116 cells were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to NTC siRNA. WRN siRNA knock-down efficacy was analyzed by immunoblotting. Protein lysates were prepared 72 hr after transfection. GAPDH expression was used to monitor equal loading. (C) TP53-wild-type CRC cell lines SK-CO-1 and HCT 116 cells were transfected with the indicated siRNAs. Cell viability and WRN siRNA knock-down efficacy was analyzed as in panel B. Data information: In (B and C) data are presented as mean ± SD of two independent experiments.
Figure 2—figure supplement 2. MLH1/MSH3 reconstitution in HCT 116 CRC cells does not alleviate WRN dependency.

Figure 2—figure supplement 2.

(A) HCT 116 cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. (B) WRN siRNA knock-down efficacy was analyzed by qRT-PCR. RNA lysates were prepared 48 hr after transfection. WRN mRNA expression is normalized to 18S rRNA levels. (C) Reconstitution of MLH1 and MSH3 was determined by immunoblot, Actin expression was used to monitor equal loading. Data information: In (A), data are presented as mean ± SD of three to six independent experiments. In (B), data are presented as mean ± SD of two independent experiments.
Figure 2—figure supplement 3. WRN inactivation does not elicit dependency on MLH1 or MSH3 in MSS SW480 CRC cells.

Figure 2—figure supplement 3.

(A) SW480 monoclonal cell lines were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to non-targeting control (NTC) siRNA. Knock-out of WRN was confirmed by immunoblot, Actin expression was used to monitor equal loading. (B) MLH1 and MSH3 siRNA knock-down efficacy was analyzed by qRT-PCR. RNA lysates were prepared 48 hr after transfection. MLH1 and MSH3 mRNA expression is normalized to 18S rRNA levels. Data information: In (A), data are presented as mean ± SD of three independent experiments. In (B), data are presented as mean ± SD of two independent experiments.