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. 2024 Oct 29;221(11):e20231832. doi: 10.1084/jem.20231832

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© 2024 Hsu et al.

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Figure 7. — Inositol promotes PCSCs via directly binding to IMPDH2. (A) IMPDH activity of adherent and spheroid in 22RV1 cells. The activity was normalized by protein concentration in each experimental group. The data were shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01 by two-tailed unpaired t test. (B) The levels of guanosine and GMP in ALDH^high and ALDH^low/− of PC3 cells were determined by metabolomics analysis. *, P < 0.05 by two-tailed uppaired t test. (C) Spheres from DU145 stably expressing shLuc or shIMPDH2 (#1 and #2) upon the treatment of vehicle (Veh) or 40 μM of inositol (Ins). Scale bar, 100 μm. (D) Quantification of the number of spheres from DU145 is shown in C. The data are shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; NS, non-significant by two-tailed unpaired t test. (E) Spheres from PC3 cells upon 500 μM of LiCl, 10 μM of MPA, or 25 μM of Ins treatment for 4 days. Scale bar, 100 μm. (F) Quantification of sphere number from PC3 by ImageJ shown in E. The data are shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; NS, non-significant by two-tailed unpaired t test. (G) 22RV1 cells stably expressing shLuc or shIMPDH2 (#1 and #2) incubated with ALDEFLUOR for 45 min. FACS profiles represent DEAB control and ALDH staining in each group. The gate (P3) was set according to DEAB control. (H) Quantification of relative ALDH positive cells normalized by DEAB control according to the gate (P3) is shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01 by two-tailed unpaired t test. DEAB, ALDH inhibitor. (I) Cancer sphere formation in 22RV1 cells expressing shLuc and shIMPA1 #1 upon vehicle, 40 μM of inositol, 40 μM of guanosine, 100 μM of GTP, 100 μM of GDP, 50 μM of cGMP, 3 μM of xanthosine, 3 μM of xanthine and 40 μM of adenosine. The concentrations of treatment were close to the reported physiological ranges (Traut, 1994). Scale bar, 100 μm. (J) Quantification of the number of cancer sphere formations with over 50 μm of diameter from A. The data were shown as the mean ± SEM of three independent experiments for each group. ***, P < 0.001; NS, non-significant by two-tailed unpaired t test. (K and N) Immunoblotting of 22RV1 (N) stably expressing shLuc or shIMPA1 (#1 and #2) and DU145 (Q) stably expressing shLuc or shIMPDH2 (#1 and #2) with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (L and O) Spheres from 22RV1 (L) or DU145 (O) stably expressing shLuc, IMPA1-two specific shRNA lentivirus (#1 and #2), or IMPDH2-two specific shRNA lentivirus (#1 and #2) upon vehicle (Veh), 40 μM of inositol (Ins), 40 μM of guanosine (Gua) or 40 μM of adenosine (Ade) treatment for 4 days. Scale bar, 100 μm. (M and P) Quantification of the number of spheres from L and O for M and P, respectively, was shown. Data are shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; NS, non-significant by two-tailed unpaired t test. Source data are available for this figure: SourceData F7.