Figure 2. Asukamycin impairs breast cancer cell proliferation through targeting UBR7.

(a) Expression of UBR7 and loading control GAPDH levels in 231MFP shControl, shUBR7, or shUBR7 cells expressing FLAG-tagged wild-type (WT) or FLAG-tagged C374A mutant UBR7 protein, assessed by Western blotting. (b) Cell proliferation in 231MFP shControl or shUBR7 cells treated with DMSO vehicle or asukamycin for 24 h, assessed by Hoechst stain. (c) Cell proliferation in 231MFP shUBR7 cells expressing FLAG-WT or FLAG-C374A mutant UBR7 treated with DMSO vehicle or asukamycin (50 μM) for 24 h. (d) anti-FLAG, anti-UBR7, and loading control anti-GAPDH protein expression in 231MFP cells stably expressing FLAG-GFP, FLAG-UBR7 WT, or FLAG-UBR7 C374A mutant assessed by Western blotting. (e) Cell proliferation of 231MFP cells transiently expressing FLAG-GFP, FLAG-UBR7 WT or FLAG-UBR7 C374A mutant treated with DMSO or asukamycin (5 μM) for 48 h, assessed by Hoechst stain. Data shown in (b, c, and e) are shown as individual replicate values and average ± sem and are n=6 biologically independent samples/group. Gels shown in (a, d) are representative blots from n=3 biologically independent samples/group. Statistical significance was calculated with two-tailed unpaired Student’s t-tests and are shown as *p<0.05 compared to vehicle-treated controls within each group, #p<0.05 against respective concentrations of asukamycin-treatment in shControl groups in (b, c) or asukamycin-treatment in FLAG-GFP expressing groups in (e). Source data for cropped blots can be found in Source Data for Figure 2. Source data for bar graphs can be found in Source Data Tables for Figure 2.