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. Author manuscript; available in PMC: 2023 Sep 26.
Published in final edited form as: Nat Cell Biol. 2023 Jan 12;25(1):159–169. doi: 10.1038/s41556-022-01049-w

Figure 3. Wild-type HRAS and NRAS constrain the growth of human KRAS-driven cancer cell lines.

Figure 3.

a. Wildtype (sgSAFE) or HRAS- or NRAS-knockout cells were seeded in 96 well plates and cultured under limited serum (1%). Cell numbers were measured via CCK8 assay. Points are Mean±SD of 12 wells normalized to Day 0 (one-way ANOVA).

b. Re-expression of wild-type HRAS suppresses proliferation of HRAS-null H23 and H727 cells. TRE-HRAS cells were seeded in 96-well plates and cultured under limited serum (1%) with or without 50 ng/ml Doxycycline (Dox) and cell numbers were measured via CCK8 assay. Points are Mean±SD of 8 wells normalized to Day 0 (one-tailed t-test).

c-d. Inactivation of HRAS or NRAS increases H23 colony formation. Wild-type (sgSAFE), HRAS-knockout (sgHRAS), or NRAS-knockout (sgNRAS) H23 cells were seeded at 1000 cells/well in 6-well plates and grown for two weeks. Cells were stained with crystal violet. C.

Representative images. Scale bar: 5mm. d. Mean±SD of colony number from 12 fields (one-way ANOVA).

e-f. Re-expression of wild type HRAS suppresses HRAS-null H23 cell colony formation. Cells were seeded at 1000 cells/well in 6-well plates and grown +/-50 ng/ml Dox for two weeks. Cells were stained with crystal violet. e. Representative images. Scale bar: 5mm. f. Mean±SD of colony number from 12 fields (one-way ANOVA).

g-k. Inactivation of wild-type HRAS or NRAS increases H23 cell growth after transplantation. g. Schematic of tumor initiation by transplantation of H23 cells with inactivation of HRAS or NRAS in NSG mice. h. Tumor weight from SubQ transplantation of indicated cells. Each dot represents a mouse. Mean value is shown. i. Ki67pos cell number in tumor sections from SubQ transplantation of indicated cells, shown as Mean±SD value of 20 view fields. j. Tumor area (percentage of human mitochondriapos area) from IV transplantation of indicated cells. Each dot represents a tumor. Mean value is shown. k. Ki67pos cell number in tumor sections from IV transplantation of indicated cells, shown as Mean±SD value of 15 view fields (200x). ns: not significant (one-way ANOVA).