Figure 4. WRN dependency in MSI-H cancer cell lines is linked to its helicase function.

(A) Schematic representation of WRN domain structure. Location of nuclease- and ATPase-inactivating mutations in siRNA-resistant WRN (WRNr) expression constructs is indicated. (B) MSI-H CRC HCT 116 cells were stably transduced with FLAG-tagged wild-type or mutant forms of WRNr and monoclonal lines with similar WRNr expression levels were isolated. For WRNr wild-type, two clones with high and low transgene expression were selected to cover the expression range of WRNr variants. Anti-FLAG immunofluorescence analysis was performed to monitor homogenous expression of WRNr. Expression of WRNr wild-type and mutant forms and endogenous protein levels were determined using immunoblotting with anti-FLAG and anti-WRN antibodies. GAPDH expression was used to monitor equal loading. Scale bar, 20 µM. (C) WRNr-expressing HCT 116 cells were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to NTC siRNA. Data information: In (C), data are presented as mean ± SD of three independent experiments.

Figure 4—figure supplement 1. WRN dependency in MSI-H cancer cell lines is linked to its helicase function.

(A) MSI-H CRC RKO cells were stably transduced with FLAG-tagged wild-type or mutant forms of WRNr. Anti-FLAG immunofluorescence analysis was performed to monitor homogenous expression of WRNr. Overexpression of WRNr wild-type and mutant forms compared to endogenous protein levels was determined using immunoblotting with anti-FLAG and anti-WRN antibodies. GAPDH expression was used to monitor equal loading. Scale bar, 20 µM. (B) WRNr-expressing RKO cells were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to NTC siRNA. (C) MSI-H endometrial carcinoma HEC-265 cells were stably transduced with FLAG-tagged wild-type or mutant forms of WRNr. WRNr-expressing HEC-265 cells were transfected with the indicated siRNAs. Cell viability was determined 7 days after transfection and is shown relative to NTC siRNA. Overexpression of WRNr wild-type and mutant forms compared to endogenous protein levels was determined using immunoblotting with anti-FLAG and anti-WRN antibodies. Actin expression was used to monitor equal loading. Data information: In (B) and C), data are presented as mean ± SD of three independent experiments.