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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 S2. — IMPA1/inositol/IMPDH2 pathway maintains the proliferation of ALDH^high cells, but not ALDH^low/− cells. (A and B) Dot plot of sorted ALDH^high and ALDH^low/− LNCaP (A) and TRAMP-C2 (B) cells population. (C and D) Inositol levels were determined by K-INOSL assay kit according to the manufacturer’s instructions. The inositol levels were normalized by protein concentration in each experimental group. Three independent experiments were performed. *, P < 0.05; **, P < 0.01 by two-tailed unpaired t test. Immunoblotting of ALDH^high and ALDH^low/− LNCaP cells and ALDH^high and ALDH^low/− TRAMP-C2 cells with indicated antibodies. (E–H) Colony formation of ALDH^high (E and G) and ALDH^low/− (F and H) LNCaP cells and TRAMPC-C2 cells upon shLuc and shIMPA1 (#1 and #2). Immunoblotting of ALDH^high (E and G) and ALDH^low/− (F and H) LNCaP cells and TRAMP-C2 cells stably expressing shLuc and shIMPA1 (#1 and #2). (I–L) Quantification of number of colonies of ALDH^high (I and J) and ALDH^low/− (K and L) LNCaP cells (I and K) and TRAMPC-C2 cells (J and L). The data are shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; ***, P < 0.001; NS, non-significant by two-tailed unpaired t test. (M) Cancer sphere formation in ALDH^high LNCaP and ALDH^high TRAMP-C2 cells upon shLuc, shIMPA1 #1 and shIMPDH2 #1. Scale bar, 100 μm. (N and O) Quantification of cancer sphere formation of ALDH^high LNCaP cells (N) and ALDH^high TRAMP-C2 cells (O) from M. The data are shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. (P and Q) Immunoblotting of ALDH^high and ALDH^low/− LNCaP (P) and TRAMP-C2 (Q) cells stably expressing shLuc, shIMPA1 #1, or shIMPDH2 #1 with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (R and S) Colony formation of ALDH^high or ALDH^low/− LNCaP (R) and TRAMP-C2 (S) cells stably expressing shLuc, shIMPA1 #1, or shIMPDH2 #1 upon vehicle, 80 μM of inositol (Ins), and 80 μM of guanosine (Gua) treatment. (T–W) Quantification of number of colonies of ALDH^high (T and U) or ALDH^low/− (V and W) LNCaP (T and V) and TRAMP-C2 (U and W) cells upon shLuc, shIMPA1 #1, shIMPDH2 #1 with vehicle, 80 μM of inositol (Ins), and 80 μM of guanosine (Gua) treatment from R and S. The data were shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. Source data are available for this figure: SourceData FS2.