Fig 2: HECTD1 and USP7 regulate DLC1 ubiquitination. (A) HEK293T cells were transiently transfected with vectors encoding the indicated constructs. The next day, cells were treated with the proteasome inhibitor MG-132 (10 µM) before lysis. Lysates were subjected to pulldown with Ni–NTA-agarose. Pulldowns and lysates were analyzed by immunoblotting with the indicated antibodies. (B) MCF7 cells were transiently transfected with vectors encoding the indicated constructs. The next day, cells were treated with MG-132 (10 µM), P5091 (20 µM) as indicated or DMSO as a control for 6 h before lysis. Lysates were subjected to pulldown with Ni–NTA-agarose. Pulldowns and lysates were analyzed by immunoblotting with the indicated antibodies. (A, B) Western blots from three independent experiments were analyzed with ImageJ, the fold change in ubiquitinated GFP-DLC1 species was determined by normalizing their signal intensity to that of control sample, and is presented as mean ± s.d. Full-length western blots are provided as Supplementary Information.

Fig 3: HECTD1 and USP7 are novel regulators of DLC1 stability. (A, B, C) HEK293T cells were transiently transfected with vectors encoding the indicated constructs or FLAG empty vector (-). (A) The next day, cells were lysed and immunoprecipitation with an anti-FLAG antibody was performed. Cell lysates (WCL) and precipitates were analyzed by immunoblotting using the indicated antibodies. Cropped blots showing co-immunoprecipitation signals are derived from blots with different exposure compared to WCL. (B, C) The next day, cells were lysed and immunoprecipitation with an anti-GFP-nanobody was performed. Cell lysates (WCL) and precipitates were analyzed by immunoblotting using the indicated antibodies. Cropped blots showing co-immunoprecipitation signals are derived from blots with different exposure compared to WCL. (D) MCF7 cells were treated with P5091 (20 µM) or HBX 41108 (10 µM) as indicated and lysates were analyzed by immunoblotting with the indicated antibodies. The control sample was treated with DMSO for 6 h. Western blots from three independent experiments were analyzed with ImageJ, the fold change in DLC1 expression was determined by normalizing the DLC1/GAPDH ratio to that of control sample and is presented as mean ± s.d. (E) Cells were transfected with control siRNA or two independent siRNAs targeting HECTD1. 72 h post transfection cells were lysed and lysates were analyzed by immunoblotting with the indicated antibodies. Cropped blots shown from different cell lines are derived from separate blots. Western blots from three independent experiments were analyzed with ImageJ, the fold change in DLC1 expression was determined by normalizing the DLC1/GAPDH ratio to that of control sample and is presented as mean ± s.d. (F) MCF7 cells were transfected with control siRNA or two siRNAs targeting HECTD1. 72 h post transfection cells were treated with cycloheximide (CHX, 60 µg/ml) as indicated and lysates were analyzed by immunoblotting with the indicated antibodies. (G) Quantification of protein levels from (F) by ImageJ. n = 4, error bars represent mean ± s.d. (A, B, C, D, E, F) Full-length western blots are provided as Supplementary Information.

Fig 4: MiR-497a-5p targeted HECTD1. (A) The binding sites between miR-497a-5p and HECTD1 3'UTR were shown. The interaction between miR-497a-5p and HECTD1 was confirmed by dual-luciferase reporter assay (B), RIP assay (C), and RNA pull-down assay (D). (E, F) The mRNA and protein expression of HECTD1 was detected by qRT-PCR and western blot (WB) analysis in BV2 cells transfected with miR-497a-5p mimic or inhibitor. (G, H) The mRNA and protein expression of HECTD1 was measured by qRT-PCR and WB analysis in BV2 cells treated with or without LPS. **p < .01; ***p < .001.

Fig 5: Circ-Bnc2 targeted miR-497a-5p to upregulate HECTD1. (A, B) The mRNA and protein levels of HECTD1 were determined by qRT-PCR and western blot (WB) analysis in BV2 cells transfected with NC + miR-NC, circ-Bnc2 + miR-NC, or circ-Bnc2 + miR-497a-5p. (C) The main idea diagram of this study. **p < .01; ***p < .001.