Fig. 4. FRAT1 expression sensitizes H1299 cells to SHIN1 treatment.

Cell lines were established with either EV or FRAT1 overexpression in NCI-H1299 cells. (A) Clonogenic assay was performed with cells expressing either EV or FRAT1 in the presence of the indicated concentrations of SHIN1, and (B) cell proliferation was estimated by quantification of clonogenic cell growth as described in Materials and Methods. (C and D) Cell lines were established with FRAT1 overexpression and overexpression of pTRIPZ-EV, pTRIPZ-HA-NES-GSK3β, pTRIPZ-HA-NLS-GSK3β, or pTRIPZ-HA-NLS-GSK3β-(KD) in NCI-H1299 cells. (C) Clonogenic assay was performed using the above cell lines in the presence of the indicated concentrations of SHIN1, and (D) cell proliferation was estimated by quantification of clonogenic cell growth as described in Materials and Methods. Values are expressed as means ± SEM. Student’s t test, **P < 0.001, n = 3. (E and F) 1 × 10⁶ of either EV- or FRAT1-expressing NCI-H1299 cells were injected into mice through subcutaneous inoculation as described in Materials and Methods. Mice were treated with either vehicle or SHIN1 (100 mg/kg) three times a week, and tumors were extracted after 3 weeks (n = 5 mice per group). Values are expressed as means ± SD. Two-tailed Student’s t test, **P < 0.01.