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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 4. — IMPA1/inositol promotes PCSCs, anchorage-independent growth, and tumorigenicity of CRPC in vivo. (A and E) Immunoblotting of DU145 (A) and 22RV1 (E) scramble (Scram.) or IMPA1 knockout (KO) cells by CRISPR/Cas9 upon Flag vector, Flag-IMPA1 (WT), and Flag-IMPA1 D220A (D220A) overexpression with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (B and F) The levels of inositol in DU145 (B) and 22RV1 (F) scramble (Scram.) or IMPA1 knockout by CRISPR/Cas9 cells upon Flag vector, Flag-IMPA1 (WT), and Flag-IMPA1 D220A (D220A) overexpression were determined by K-INOSL assay kit. The inositol levels were normalized by protein concentration in each experimental group. Three independent experiments were performed for the statistic of two-tailed unpaired t test. **, P < 0.01; NS, non-significant; ***, P < 0.001 by two-tailed unpaired t test. (C and G) Representative images of spheres from DU145 (C) and 22RV1 (G) scramble (Scram.) or IMPA1 knockout cells upon Flag vector, Flag-IMPA1 (WT), and Flag-IMPA1 D220A (D220A) overexpression. Scale bar, 100 μm. (D and H) Quantification of the number of spheres from DU145 (D) and 22RV1 (H) is 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. (I) Soft agar assay is shown in PC3 cells stably expressing shLuc or IMPA1-two specific shRNA lentivirus (#1 and #2). (J) Quantification of the number of colonies formation in soft agar by ImageJ in I is shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01 by two-tailed unpaired t test. (K and M) Tumorigenicity of PC3 (K) or DU145 (M) cells stably expressing shLuc or shIMPA1 was determined by tumor volume. At least four xenograft tumors in each group were quantified. **, P < 0.001 by two-tailed unpaired t test. (L and N) Tumor weight from PC3 (L) or DU145 (M) cells stably expressing shLuc or shIMPA1 was measured at day 48 (L) and day 38 (N). At least four xenograft tumors in each group were quantified. **, P < 0.01 by two-tailed unpaired t test. (O) Nude mice subcutaneously injected with 22RV1 cells stably expressing shLuc or shIMPA1 were intraperitoneally injected with vehicle or 30 mg/kg of inositol every 2 days until 41 days, and tumor volume was measured with indicated days. At least five xenograft tumors in each group were quantified. **, P < 0.001; ***, P < 0.001 by two-tailed unpaired t test. (P) Tumor weight from 22RV1 cells stably expressing shLuc or shIMPA1 upon intraperitoneal injection with vehicle or 30 mg/kg of inositol every 2 days at day 41 was measured. At least five xenograft tumors in each group were quantified. **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. (Q) Nude mice subcutaneously injected with DU145 cells stably expressing shLuc or shIMPA1 (#1 and #2) were intraperitoneally injected with vehicle or 50 mg/kg of inositol every 2 days until 34 days, and tumor volume was measured with indicated days. At least four xenograft tumors in each group were quantified. *, P < 0.05; **, P < 0.01 by two-tailed unpaired t test. Source data are available for this figure: SourceData F4.