Journal: bioRxiv

Article Title: ALS driven by mutant NEK1 aggregation is accelerated by Pml loss, but clinically reversed through pharmacologic induction of Pml -mediated degradation

doi: 10.1101/2024.11.23.622051

Figure Lengend Snippet: Therapeutic benefit of Pml induction by IFNα or poly(I:C) through NEK1 t clearance in vivo . A) NEK1 t modification by SUMO1 and SUMO2/3. HEK293 cells were transfected with the indicated expression vectors and treated or not with IFNα. IP: immuno-precipitation, WCL: whole cell lysate. B) IFNα treatment induces proteasome-dependent degradation of NEK1 t in HEK293 cells. MG132: proteasome inhibitor, n=3. C) PML silencing impedes IFNα-induced NEK1 t degradation. Quantifications (right), n=3. D) Western blot of NEK1 t protein from the spinal cord of age-matched Nek1 t/t or Nek1 t/t Pml −/− mice following IFNα or poly(I:C) therapy, (*) non-specific band. E) IFNα or poly(I:C) therapy eliminates NEK1 t aggregates in αMNs of Nek1 t/t mice. Quantifications: ≥30 αMNs from ≥3 mice F) IFNα or poly(I:C) therapies selectively extend lifespan in Pml -proficient Nek1 t/t mice. Arrowhead: therapy initiation, continued until death. G) IFNα or poly(I:C) therapies improve muscle strength and neuromuscular function selectively in Pml -proficient Nek1 t/t ALS mice. Trendlines in Supplementary Fig.S2E.

Article Snippet: The following antibodies were used for Western blots in HEK293 or Neuro2A cells: anti-GST (Cell signaling, Cat # 2624S), human anti-GAPDH (Santa Cruz, Cat # sc-32233;), human anti-actin (BioLegend, Cat # 622102), human anti-YY1 (Santa Cruz, H-10, Cat # sc-7341), human anti-PML (Santa Cruz, H-238, Cat # sc-5621 and Santa Cruz, E-11, Cat # sc-377390), human anti-SUMO1 (CST, Cat # 4930), human anti-SUMO2/3 (Abcam, Cat # ab3742).

Techniques: In Vivo, Modification, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: NPJ Precision Oncology

Article Title: RNF4 mediated degradation of PDHA1 promotes colorectal cancer metabolism and metastasis

doi: 10.1038/s41698-024-00724-5

Figure Lengend Snippet: A Co-transfection of FLAG-PDHA1 and EGFP-SUMO (SUMO1, SUMO2, or SUMO3) in SW620 cells, with or without treatment of 20 μM MG132 for 6 h. Ni-NTA analyzed SUMOylation of PDHA1; B Co-transfection of FLAG-PDHA1-His, HA-Ub, and T7-SUMO3 in SW620 cells, followed by treatment with CHX (20 μg/ml), MG132 (20 μM), or CHX + MG132 for 6 h. SUMOylation and ubiquitination of PDHA1 were examined by Ni2+ pull-down assay; C Co-transfection of SW620 cells with FLAG-PDHA1-His (WT, 2 KR (UMO-deleted double mutation), K77 (SUMO-deleted single mutation), K186 (another SUMO-deleted single mutation)), HA-Ub, and T7-SUMO3, followed by treatment with 20 μM MG132 for 6 h and Ni2+ pull-down experiments to detect the SUMOylation and ubiquitination of PDHA1; D Performance of Western blot analysis to evaluate the protein expression of PDHA1, SAE1, SAE2, Ubc9, SUMO-1, and SUMO-2 in different SW620 cell groups; E Transfection of SW620 cells with Flag-PDHA1 WT, Flag-PDHA1 2 KR, Flag-PDHA1 K77, and Flag-PDHA1 K186 for 48 h. The protein expression of PDHA1 was measured by Western blot after different incubation durations with 20 μg/ml CHX. * indicates difference compared to the Flag-PDHA1 WT group, P < 0.05. Cell experiments were repeated three times.

Article Snippet: The PVDF membrane was immersed in 5% skim milk powder and sealed at room temperature for 1 h. Afterward, it was incubated overnight with primary antibodies, which included an anti-PDHA1 antibody (rabbit; 1:1000, ab110330, Abcam, UK), anti-RNF4 antibody (rabbit; 1:1000, 17810-1-AP, Proteintech, Wuhan Sanying, China), anti-Flag antibody (rabbit; 1:1000, F7425, Sigma-Aldrich, USA), anti-HA antibody (rabbit; 1:1000, #3724, Cell Signaling, USA), anti-Myc antibody (rabbit; 1:1000, SAB4501941, Sigma-Aldrich, USA), anti-T7 antibody (mouse; 1:1000, T8823, Sigma-Aldrich, USA), anti-EGFP antibody (mouse; 1:1000, MAB3580, Sigma-Aldrich, USA), anti-SAE1 antibody (rabbit; 1:1000, ab97523, Abcam, USA), anti-SAE2 antibody (rabbit; 1:1000, ab95835, Abcam, USA), anti-UBC9 antibody (goat; 1:1000, ab21193, Abcam, USA), anti-SUMO1 antibody (rabbit; 1:1000, S8070, Sigma-Aldrich, USA), anti-SUMO2/3 antibody (rabbit; 1:1000, 07-2167, Sigma-Aldrich, USA) and anti-α-Tubulin antibody (mouse; 1:5000, ab7291, Abcam, UK).

Techniques: Cotransfection, Pull Down Assay, Mutagenesis, Western Blot, Expressing, Transfection, Incubation

Journal: NPJ Precision Oncology

Article Title: RNF4 mediated degradation of PDHA1 promotes colorectal cancer metabolism and metastasis

doi: 10.1038/s41698-024-00724-5

Figure Lengend Snippet: A Co-transfection of FLAG-PDHA1 and EGFP-SUMO (SUMO1, SUMO2, or SUMO3) in SW620 cells, with or without treatment of 20 μM MG132 for 6 h. Ni-NTA analyzed SUMOylation of PDHA1; B Co-transfection of FLAG-PDHA1-His, HA-Ub, and T7-SUMO3 in SW620 cells, followed by treatment with CHX (20 μg/ml), MG132 (20 μM), or CHX + MG132 for 6 h. SUMOylation and ubiquitination of PDHA1 were examined by Ni2+ pull-down assay; C Co-transfection of SW620 cells with FLAG-PDHA1-His (WT, 2 KR (UMO-deleted double mutation), K77 (SUMO-deleted single mutation), K186 (another SUMO-deleted single mutation)), HA-Ub, and T7-SUMO3, followed by treatment with 20 μM MG132 for 6 h and Ni2+ pull-down experiments to detect the SUMOylation and ubiquitination of PDHA1; D Performance of Western blot analysis to evaluate the protein expression of PDHA1, SAE1, SAE2, Ubc9, SUMO-1, and SUMO-2 in different SW620 cell groups; E Transfection of SW620 cells with Flag-PDHA1 WT, Flag-PDHA1 2 KR, Flag-PDHA1 K77, and Flag-PDHA1 K186 for 48 h. The protein expression of PDHA1 was measured by Western blot after different incubation durations with 20 μg/ml CHX. * indicates difference compared to the Flag-PDHA1 WT group, P < 0.05. Cell experiments were repeated three times.

Article Snippet: EGFP-tagged SUMO1, SUMO2, and SUMO3 were created by inserting processed forms of human cDNAs encoding SUMO1 (1-97), SUMO2 (1-92), and SUMO3 (1-93) into the pEGFP vector (165830, addgene, USA).

Techniques: Cotransfection, Pull Down Assay, Mutagenesis, Western Blot, Expressing, Transfection, Incubation