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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 S3. — Inositol directly maintains cancer stemness in prostate cancer spheres and tumor organoids independently of its downstream PI cycle. (A) Spheres from DU145 stably expressing Flag vector or Flag-IMPA1 lentivirus. Scale bar, 100 μm. (B) The cell lysates of spheres from DU145 stably expressing Flag vector or Flag-IMPA1 lentivirus were subjected to immunoblotting with indicated antibodies. (C) Quantification of number of spheres from DU145 (A) was shown as the mean ± SEM of three independent experiments for each group. **, P < 0.001 by two-tailed unpaired t test. (D) Representative images of spheres from DU145 and 22RV1 upon 1, 2.5, 5, and 10 μM of inositol treatment for 4 days. Scale bar, 150 μm. (E–H) Quantification of the size and number of spheres from DU145 (E and G) and 22RV1 (F and H) upon the vehicle or 10 μM of inositol (Ins) treatment is shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01 by two-tailed unpaired t test. (I) Illustration of cDNA sequence of IMPA1 targeted by the gRNA pairs. (J) Photographs of spheres from 22RV1 cells stably expressing shLuc or shIMPA1 upon the treatment of vehicle (Veh), 25 μM of inositol (Ins), 4 μM of PI, phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 3,4,5-trisphosphate (PIP₃), 25 μM of inositol 1,3,4-trisphosphate (IP₃), inositol 1,4-bisphosphate (IP₂), or inositol 4-phosphate (IP). Immunoblotting of 22RV1 cells stably expressing shLuc or shIMPA1 with indicated antibodies. Scale bar, 100 μm. (K) Quantification of the number of spheres from 22RV1 (J) was shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; NS, non-significant by two-tailed unpaired t test. (L) Tumor organoids from TRAMP expressing shLuc or shIMPA1 upon the treatment of vehicle (Veh) or 25 μM of inositol (Ins), 4 μM of PI, phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 3,4,5-trisphosphate (PIP₃), 25 μM of inositol 1,3,4-trisphosphate (IP₃), inositol 1,4-bisphosphate (IP₂) or inositol 4-phosphate (IP) were shown. Scale bar, 100 μm. (M) Quantification of the number of tumor organoids from TRAMP was shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; NS, non-significant by two-tailed unpaired t test. (N) Immunoblotting of DU145 cells stably expressing shLuc, shIMPA1, shCDIPT, shPI4K2α, shPLCβ1, or shINPP1 with indicated antibodies. (O) Photographs of spheres from DU145 cells stably expressing shLuc, shIMPA1, shCDIPT, shPI4K2α, shPLCβ1, or shINPP1. Scale bar, 100 μm. (P) Quantification of the number of spheres from DU145 (O) was shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; ***, P < 0.001; NS, non-significant by two-tailed unpaired t test. (Q) The images of the EM represent the cell membrane from DU145 cells stably expressing shLuc and shIMPA1 (#1 and #2). The red arrow indicates cell membrane. (R) Insulin levels were determined by human insulin ELISA kit (Elabscience) in ALDH^high LNCaP cells upon shLuc and shIMPA1 (#1 and #2). NS, non-significant. (S and T) The levels of dopamine (S) and glutamine (T) were determined by LC-MC/MS in 22RV1 cells upon shLuc and shIMPA1 (#1 and #2). NS, non-significant. Triplicate samples in each group were performed for LC-MS/MS. Source data are available for this figure: SourceData FS3.