Fig. 6

Stem-like properties and drug resistance are dependent on MTHFD2. a ALDH activity in PC9 and PC9M2 cells was measured using the ALDEFLUOR^® assay. Cells positive for ALDH are depicted with green dots as strong intensities of fluorescence measured by fluorescein isothiocyanate isomer-I (FITC) and their proportion to total cells are described in red (right panels). DEAB, an inhibitor of ALDH activity, was used for quenching and DEAB-treated cells were used as negative controls (left panels). Horizontal and vertical axes indicate intensities of ALDH activity (FITC) and complexity of cells (side scatter), respectively. b mRNA expression levels of stem cell markers in PC9 and PC9M2 cells were measured by quantitative RT-PCR and data are normalized by the value of PC9 cells. The data are represented as mean ± SD, N = 3. c mRNA expression levels of stem cell markers in PC9M2 cells were measured by quantitative RT-PCR and data are normalized by the value of the negative control (NCT). shMTHFD2#50, shMTHFD2#53, MTHFD2 knockdown. The data are represented as mean ± SD, N = 3. d mRNA expression levels of β-catenin and its downstream targets AXIN2 and CD44 were measured by quantitative RT-PCR and data are normalized with the value of NCT. The data are represented as mean ± SD, N = 3. e Gefitinib sensitivity was measured by cell growth with increased doses of gefitinib in PC9M2 cells. Experiments were performed three times and the representative results were presented. The data are represented as mean ± SD, N = 4. f Western blot for MTHFD2 in empty vector-transduced control PC9 cells (CTL) and MTHFD2-overexpressing PC9 cells (OE). g Cell growth activity was measured with or without gefitinib treatment (1 μM). Experiments were performed three times and the representative results were presented. The data are represented as mean ± SD, N = 3. h Growth curves of tumors of CTL or OE PC9 cells. Mice were administered orally with vehicle or gefitinib (10 mg/kg/day) every day from 6 days after the inoculation (yellow arrow). Horizontal axis and vertical axis depict days after inoculation of cells and volume of tumors, respectively. The data are represented as mean ± SEM, N = 4. i mRNA expression levels of stem cell markers in PC9M2 cells were measured by quantitative RT-PCR with or without AICAR treatment (1 mM) and data are normalized by the value of control (without AICAR treatment) (left panel). Cell growth activity was measured with increased doses of AICAR treatment with or without gefitinib (1 μM) (right panel). Experiments were performed three times and the representative results were presented. The data are represented as mean ± SD, N = 4. j mRNA expression levels of stem cell markers in PC9M2 cells were measured by quantitative RT-PCR with or without metformin treatment (4 mM) and data are normalized by the value of control (without AICAR treatment). The data are represented as mean ± SD, N = 3. Statistical significance is calculated with a two-tailed unpaired t-test; N.S. not significant, *p < 0.05, **p < 0.01