vcp inhibitor nms 873  (TargetMol)

Journal: Cell Death & Disease

Article Title: Targeting EGFR-binding protein SLC7A11 enhancing antitumor immunity of T cells via inducing MHC-I antigen presentation in nasopharyngeal carcinoma

doi: 10.1038/s41419-024-07327-9

Figure Lengend Snippet: A Bubble chart of GO enrichment analysis for downregulated genes in SUNE1 cells post-SLC7A11 knockdown. B Upper: qPCR analysis of FAF2 mRNA post-SLC7A11 knockdown. Lower: the effect of knockdown SLC7A11 on the protein expression of FAF2 and MHC-I were verified by Western blot. β-actin were used as loading controls. C Upper: qPCR analysis of FAF2 mRNA following SLC7A11 overexpression. Lower: the effect of SLC7A11 overexpression on the protein expression of FAF2 and MHC-I were verified by Western blot. β-actin were used as loading controls. D Left: qPCR confirmation of FAF2 mRNA knockdown using siFAF2-2/3. Right: the effect of FAF2 knockdown on the protein expression of SLC7A11 and MHC-I were verified by Western blot. Tubulin were used as loading controls. E Immunofluorescence assessment of MHC-I in SUNE1 and 6-10B cells post-FAF2 knockdown. Scale bars, 50 μm. * P < 0.05, *** P < 0.001. The Welch one-way ANOVA test and Games-Howell test in SUNE1 cells, Kruskal–Wallis Test and Dunn’s test in 6-10B cells. Error bars, mean ± SEM . F Immunoblot assessment of SLC7A11, FAF2, and MHC-I in SUNE1 cells post-transfection: control, siSLC7A11, siFAF2, or combined siSLC7A11 and siFAF2. Tubulin were used as loading controls. G Left: Immunoblot of ERAD pathway proteins (VCP, OS9, SYVN1, HERP) in SUNE1 cells post-SLC7A11 knockdown (left), and in 5-8F cells post-SLC7A11 overexpression (right). H Left: Immunoblot of MHC-I in SUNE1 cells transfected with control or FAF2 siRNA for 36 h, then treated with cycloheximide (CHX, 100 μg/ml) for specified durations. Tubulin were used as loading controls. Right: The half-life of the MHC-I protein was calculated. I Immunoblot assessment of MHC-I in SUNE1 and 6-10B cells under control, MG132, or Chloroquine treatments. Tubulin were used as loading controls. J In 5-8F cells overexpressing SLC7A11, IP complexes of MHC-I were immunoblotted for ubiquitination (Ub) under control, VCP inhibitors (NMS-873, 10 μM, MERCK, SML1128), or ERAD inhibitors (Eeyarestatin I, 1.25 μM, MERCK, E1286) treatments. β-actin was used as loading controls.

Article Snippet: J In 5-8F cells overexpressing SLC7A11, IP complexes of MHC-I were immunoblotted for ubiquitination (Ub) under control, VCP inhibitors (NMS-873, 10 μM, MERCK, SML1128), or ERAD inhibitors (Eeyarestatin I, 1.25 μM, MERCK, E1286) treatments. β-actin was used as loading controls.

Techniques: Knockdown, Expressing, Western Blot, Over Expression, Immunofluorescence, Transfection, Control

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in cells arrested following treatment with 1 µM palbociclib for 24 h. Cells were mock treated or treated with 50 µM proteasome inhibitor (MG132), 10 µM VCP inhibitor (VCPi; NMS873) or 20 µM neddylation inhibitor (NAEi; MLN4924) before treatment with 1 mM FA for 30 min. Values represent the mean ± s.e.m. n (left to right) = 25, 24, 34, 26, 24, 30, 40, 31, 27, 30, 39, 34, 27, 30, 39, 34, 27, 30, 40 and 33 cells from 3 (NAEi) or 4 (VCPi) independent experiments. Unpaired two-tailed t -test. b , Top: scheme of experiment. Bottom: IP of pSer2-modified elongating Pol II followed by immunoblotting with the indicated antibodies either directly after a 30 min FA pulse (1 mM) or after recovery for the indicated time. This experiment was performed twice with similar results. Untr., untreated. c , Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in siRNA-transfected cells, which were arrested in G1 with 1 μM palbociclib for 24 h before treatment with 1 mM FA for 30 min. Graphs represent the mean ± s.e.m. n (left to right) = 36, 28, 38, 24, 35, 28, 36, 28, 29 and 28 cells from 3 independent experiments. Unpaired two-tailed t -test. d , Quantification of recovery of transcription in RPE1 cells transfected with the indicated siRNA. Cells were treated with a 30 min FA (1 mM) pulse and left to recover for the indicated times, including a 30 min pulse labelling with EU. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 688, 652, 331, 633, 611, 466, 726, 391, 265, 721, 418 and 313 cells from 2 (siCSB) or 3 (siCtrl and siDDB1) independent experiments. Unpaired two-tailed t -test. e , Relative clonogenic survival assay in WT (MRC-5 sv40), CS-A (CS3BE sv40), CS-B (CS1AN sv40) and UVSS-A (TA-24 sv40) cells with the indicated doses of FA for a 1 h pulse. Graphs were normalized to the untreated colony number, which was set at 100%. Graphs represent the mean ± s.e.m. from five independent experiments. Unpaired two-tailed t -test. f , TC-DPC repair model. TC-DPC repair is initiated when DPC-stalled Pol II is recognized by CSB, which recruits CRL4 CSA E3 ligase. UVSSA stabilizes CSB through USP7. Ubiquitin (Ub)-DPC ubiquitylation by CRL4 CSA drives VCP-dependent and proteasome-dependent DPC degradation followed by transcription restart. Functional TC-DPC repair may explain the phenotypic differences in the TC-NER syndromes CS and UV-sensitive syndrome. Source numerical data ( a , c – e ) and unprocessed blots ( b ) are available in the source data. Scheme in f was created using BioRender ( https://biorender.com ).

Article Snippet: Cells were treated with the VCP inhibitor NMS873 (10 μM, Selleck Chemicals) directly together with FA treatment.

Techniques: Two Tailed Test, Modification, Western Blot, Transfection, Clonogenic Cell Survival Assay, Ubiquitin Proteomics, Functional Assay

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 50 µM proteasome inhibitor (MG132) and subsequently with 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 24, 30, 30, 30, 30 cells (up to down) from 3 independent experiments. b : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 10 µM VCP inhibitor (VCPi: NMS873) and subsequently with 1 mM FA for 30min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 34, 40, 39, 39, 40 cells (up to down) from 3 independent experiments. c : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 20 µM neddylation inhibitor (NAEi: MLN4924) and subsequently with 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 26, 31, 34, 34, 33 cells (up to down) from 3 independent experiments. d : Relative immobile fractions of mScarlet-I-CSB FRAP in HCT116 cells mock-treated, or treated with 50 µM MG132 and 10 µM VCP inhibitor (VCPi: NMS873) prior to treatment with 300 μM FA for 30 min. Values represent mean ± S.E.M., whereby 122, 50, 46, 82, 45, 48, 52, 48, 49, 56, 45, 52, 53, 51, 54 cells respectively (left to right) from 3 independent experiments were analyzed. Unpaired two-tailed t-test. e : CSB-mScarlet-I FRAP in HCT116 cells treated with 50 µM proteasome inhibitor MG132 prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( d ). Graph is an average of n = 46, 46, 48, 49, 52, 54 cells (up to down) from 3 independent experiments. f : CSB-mScarlet-I FRAP in HCT116 cells treated with 10 µM VCP inhibitor NMS873 (VCPi) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( d ). Graph is an average of n = 38, 50, 45, 48, 45, 51 cells (up to down) from 3 independent experiments. g : CSB-mScarlet-I FRAP in non-replicating RPE1 cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 2 μM SUMO inhibitor (SUMOi: ML792) and subsequently with 1 mM FA for 30 min. Graph is an average of n = 29, 28, 27, 28, 28, 27 cells (up to down) from 3 independent experiments. h : Relative immobile fractions of mScarlet-I-CSB FRAP in non-cycling RPE1 cells mock treated, or treated with 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 1 mM FA for 30 min quantified from ( h ). Values represent mean ± S.E.M. Unpaired two-tailed t-test. i : Immunostaining with the indicated antibodies of chromatin fraction after formaldehyde treatment. Cells were pre-treated for 2 hr pretreatment with 2 μM sumoylation inhibitor (SUMOi) before treatment with 1 mM FA for 30 min. Cells were harvested immediately after FA treatment for chromatin fractionation. This experiment was performed twice with similar results. j : Relative immobile fractions of mScarlet-I-CSB FRAP in WT and CSA KO HCT116 cells and WT cells treated with 20 µM neddylation inhibitor (NAEi: MLN4924) or 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 300 μM FA for 30 min. Values represent mean ± S.E.M., whereby 122, 129, 61, 25, 82, 96, 57, 37, 52, 67, 49, 26, 56, 68, 74, 30, 53, 57, 65, 27 cells respectively (left to right) from a 3 (NAEi and Sumoi) or 4 (CSA KO) independent experiments were analyzed. Unpaired two-tailed t-test. k : CSB-mScarlet-I FRAP in HCT116 cells treated with 20 µM neddylation inhibitor (NAEi: MLN4924) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( j ). Graph is an average of n = 66, 56, 61, 57, 49, 74, 65 cells (up to down) from 3 independent experiments. l : CSB-mScarlet-I FRAP in HCT116 cells treated with 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in (j) . Graph is an average of n = 25, 37, 26, 30, 27 cells (up to down) from 3 independent experiments. m : Immunoblot of cells transfected with siRNA against DDB1, stained with antibodies against DDB1 and Tubulin. This experiment was performed twice with similar results. n : CSB-mScarlet-I FRAP in cells transfected with siRNAs against DDB1. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 28, 24, 28, 28, 28 cells (up to down) from 3 independent experiments. o : Representative pictures of the recovery of transcription in siRNA transfected RPE1 cells. Quantification is shown in Fig. . Scale bar = 50 μm. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Cells were treated with the VCP inhibitor NMS873 (10 μM, Selleck Chemicals) directly together with FA treatment.

Techniques: Two Tailed Test, Immunostaining, Fractionation, Western Blot, Transfection, Staining

Journal: Molecular cell

Article Title: Tandem deubiquitination and acetylation of SPRTN promotes DNA-protein crosslinks repair and protects against aging

doi: 10.1016/j.molcel.2020.06.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ATPase VCP/p97 Inhibitor NMS-873 , TOCRIS , Cat# 6180.

Techniques: Recombinant, Staining, Mutagenesis, In Situ, Fractionation, Software