Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2024 Hsu et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 9. — IMPA1/inositol/IMPDH2 confers enzalutamide resistance. (A and G) Relative cell viability of 22RV1 cells stably expressing shLuc, shIMPA1 (#1 and #2) (A), or shIMPDH2 (#1, #2 and #3) (G) upon vehicle (Veh), 5, or 10 μM of enzalutamide (Enza) treatment for 48 h were determined by CCK-8. The data are shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; non-significant by two-tailed unpaired t test. (B) Colony formation assay of 22RV1 cells stably expressing shLuc or shIMPA1 upon vehicle (Veh), 5 or 10 μM of enzalutamide (Enza) treatment every 2 days for 14 days. (C) Quantification of colony number from B by ImageJ was shown. The data were shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01; non-significant by two-tailed unpaired t test. (D) Cancer sphere formation in 22RV1 cells stably expressing shLuc and shIMPA1 #1 upon vehicle (Veh) and 10 μM of enzalutamide (Enza) treatment. Scale bar, 100 μm. (E and K) Quantification of the number of cancer sphere formations with over 50 μm of diameter from D and J to E and K. 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. (F) Immunoblotting of 22RV1 cells stably expressing IMPDH2 (#1, #2, and #3) with indicated antibodies. (H) Colony formation assay of 22RV1 cells stably expressing shLuc or shIMPDH2 (#1 and #2) upon vehicle (Veh), 5, 10, or 20 μM of enzalutamide (Enza) treatment every 2 days for 14 days. (I) Quantification of colony number from H by ImageJ was shown. (J) Cancer sphere formation in 22RV1 cells stably expressing shLuc and shIMPDH2 #1 upon vehicle (Veh) and 10 μM of enzalutamide (Enza) treatment. Scale bar, 100 μm. (L) Colony formation assay of 22RV1 scramble (Scram.) or IMPA1 knockout (KO) cells by CRISPR/Cas9 upon Flag vector, Flag-IMPA1 (WT) and Flag-IMPA1 D220A (D220A) overexpression in response to vehicle (Veh), 5 or 10 μM of enzalutamide (Enza) treatment every 2 days for 14 days. (M) Quantification of colony number from L by ImageJ was shown. The data were shown as the mean ± SEM of three independent experiments for each group. **, P < 0.01; ***, P < 0.001; non-significant by two-tailed unpaired t test. (N) Cancer sphere formation in 22RV1 cells upon Scramble (Scram.) with Flag, IMPA1 knockout (KO) with Flag, Flag-IMPA1^WT (WT), and Flag-IMPA1^D220A (D220A) in response to the treatment of vehicle (Veh) and 10 μM of enzalutamide (Enza) treatment. Scale bar, 100 μm. (O) Quantification of the number of cancer sphere formations with over 50 μm of diameter from N–O. 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. (P) Male nude mice subcutaneously injected with 22RV1 cells stably expressing shLuc or shIMPA1 were intraperitoneally injected with vehicle (Veh) or 5 mg/kg of enzalutamide (Enza) every 2 days for 40 days, and tumor volume was measured with indicated days. Five mice were included in each experimental group. (Q) Surgically castrated NOD/SCID mice subcutaneously injected with 22RV1 cells upon shLuc and shIMPA1 were intraperitoneally injected with vehicle or 5 mg/kg of enzalutamide (Enza) three times a week for 6 wk and tumor volume was measured with indicated days. Five mice were included in each experimental group. (R) Relative cell viability of 22RV1 cells stably expressing shLuc or shIMPA1 upon vehicle (Veh), 5 μM of enzalutamide (Enza), 80 μM of inositol (Ins), or 80 μM of guanosine (Gua) for 72 h treatment was determined by CCK-8. The data are shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; NS, non-significant by two-tailed unpaired t test. (S) Relative cell viability of 22RV1 cells stably expressing shLuc or shIMPDH2 upon vehicle (Veh), 5 μM of enzalutamide (Enza), 80 μM of inositol (Ins), or 80 μM of guanosine (Gua) for 72 h treatment was determined by Cell Counting Kit-8 (CCK-8). The data are 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. Source data are available for this figure: SourceData F9.