Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet: Cadmium indirectly inhibits the proteasome (A) Relative proteasome activity of HepG2 cells treated with CdCl 2 (Cd; + 1 μM or ++ 5 μM) or carfilzomib (CFZ, 10 nM) during 6 h. Each bar represents the mean, and error bars denote SD of at least three independent experiments. (B) Relative proteasome activity of purified 26S proteasome complexes incubated with the substrate Suc-LLVY-AMC and treated with or without Cd (1 μM) or CFZ (10 nM) during the indicated time in minutes (n = 3).

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Activity Assay, Purification, Incubation

Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet:

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Recombinant, Purification, Bicinchoninic Acid Protein Assay, Activity Assay, MTS Assay, Expressing, Western Blot, Plasmid Preparation, Software

Journal: Cancers

Article Title: CUL4A , ERCC5 , and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

doi: 10.3390/cancers12051128

Figure Lengend Snippet: Gene expression results.

Article Snippet: Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems).

Techniques: Gene Expression, Expressing, Control

Journal: Cancers

Article Title: CUL4A , ERCC5 , and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

doi: 10.3390/cancers12051128

Figure Lengend Snippet: Survival analysis in accordance to gene expression.

Article Snippet: Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems).

Techniques: Expressing, Biomarker Discovery, Control

Journal: Cancers

Article Title: CUL4A , ERCC5 , and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

doi: 10.3390/cancers12051128

Figure Lengend Snippet: Prognostic and predictive value of DNA-damage repair genes. Samples were grouped taking into account the median of gene expression. ( A ) high expression of CUL4A significantly correlated with better progression-free survival (PFS) on trabectedin plus doxorubicin arm (6.53 months (95% CI: 0.00–13.39) vs 1.80 months (95% CI: 0.00–3.63); p = 0.038); ( B ) high expression of ERCC1 significantly correlated with better (PFS) on trabectedin plus doxorubicin arm (8.10 months (95% CI: 4.77–11.43) vs 2.63 months (95% CI: 0.41–4.86) p = 0.006) and ( C ) high expression of ERCC5 significantly correlated with better PFS on trabectedin plus doxorubicin arm (7.67 months (95% CI: 5.64–9.69) vs 1.70 months (95% CI: 1.05–2.35); p = 0.039).

Article Snippet: Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems).

Techniques: Gene Expression, Expressing

Journal: Cancers

Article Title: CUL4A , ERCC5 , and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

doi: 10.3390/cancers12051128

Figure Lengend Snippet: Prognostic and predictive value of CUL4A protein expression. Samples were grouped as CUL4A positive or negative, taking into account the nuclear expression levels evaluated by immunohistochemistry. Antibody: anti-CUL4A polyclonal antibody (1:50, 2699s, Cell Signaling Technology, Danvers, MA, USA). In the whole series CUL4A protein expression was associated with worse PFS ( A ): 2.60 months (95% CI: 0.58–4.62) vs 7.03 months (95% CI: 5.03–9.04), p = 0.009; and ( B ) and with worse OS ( B ): 10.57 months (95% CI: 5.95–15.18) vs 21.07 months (95% CI: 17.70–24.43), p = 0.001. In the doxorubicin arm, CUL4A expression was also associated with worse PFS ( C ): 2.53 months (95% CI: 1.12–4.00) vs 7.4 months (95% CI: 4.45–10.35), p = 0.025 and worse OS ( D ): 8.73 months (95% CI: 4.62–12.84) vs 27.03 months (95% CI: 16.99–37.08), p = 0.004. In the combination series, CUL4A protein expression did not correlate with PFS ( E ): 3.40 months (95% CI: 0.83–6.00) vs 5.77 months (95% CI: 4.25–7.28), p = 0.127, nor OS ( F ): 14.23 months (95% CI: 5.68–22.79) 19.70 months (95% CI: 8.82–30.58), p = 0.176.

Article Snippet: Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems).

Techniques: Expressing, Immunohistochemistry

Journal: Cancers

Article Title: CUL4A , ERCC5 , and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

doi: 10.3390/cancers12051128

Figure Lengend Snippet: Trabectedin IC50 and gene expression levels in soft-tissue sarcoma (STS) cell lines.

Article Snippet: Gene expression was measured by qRT-PCR using the following TaqMan assays on demand (Applied Biosystems, Foster City, CA, USA): BRCA1 (Hs01556190_m1), CUL4A (Hs00757716_m1), ERCC1 (Hs01012159_m1), ERCC5 (Hs01012159_m1) in a 7500 Fast thermocycler (Applied Biosystems).

Techniques: Gene Expression

Journal: Molecular Metabolism

Article Title: Hepatic FTO is dispensable for the regulation of metabolism but counteracts HCC development in vivo

doi: 10.1016/j.molmet.2020.101085

Figure Lengend Snippet: Hepatic FTO negatively regulates CUL4A in response to short-term DEN-induced liver damage. (A) Volcano plot of log2 ratio of livers of Ctrl and FTO L−KO at 0h, 12h, and 24h after ST-DEN injection vs the –log10 p value of a two-sided t-test (blue = significantly changed proteins (abs. log2 fold change >0.58 and p-value < 0.05), red = CUL4A). (B) Log2 LFQ intensities of CUL4A in Ctrl and FTO L−KO liver lysates at 0 h, 12 h, and 24 h after ST-DEN injection. (C) qPCR analysis of Cul4a expression in whole liver of ST-DEN injected Ctrl and FTO L−KO mice (n = 5 for each timepoint, two-way ANOVA). (D) Western blot analyses of CUL4A and FTO in whole liver lysates isolated from ST-DEN injected Ctrl and FTO L−KO mice and respective quantification (n = 4 for each timepoint, two-tailed unpaired multiple t-tests). CUL4A/β-ACTIN ratio normalized to 0 h Ctrl expression. (E) Methyl-RNA immunoprecipitation and IGG ctrl immunoprecipitation and subsequent qRT-PCR analysis of Cul4a mRNA pulldown in mRNA isolated from livers of 24 h ST-DEN injected Ctrl and FTO L−KO mice (n = 5, two-way ANOVA). p∗ 0.05. Data are means with SD (B–E) or SEM (H).

Article Snippet: The following TaqMan probes (Applied Biosystems, Darmstadt, Germany) were used for gene expression assays: Cul4a (Mm01220732_m1; Mm00461469_m1; Mm00461464_g1; Mm01220734_g1; Mm00461460_g1; Mm00461475_gH), Dgat1 (Mm00515643_m1), Dgat2 (Mm00499536_m1), Fasn (Mm00662319_m1), Fto (Mm00488755_m1), Pparg (Mm00440945_m1), Scd1 (Mm00772290_m1), Srebp1 (Mm00550338_m1), Srebp2 (Mm01306292_m1), Tbp (Mm00446973_m1).

Techniques: Injection, Expressing, Western Blot, Isolation, Two Tailed Test, RNA Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR

Journal: Molecular Metabolism

Article Title: Hepatic FTO is dispensable for the regulation of metabolism but counteracts HCC development in vivo

doi: 10.1016/j.molmet.2020.101085

Figure Lengend Snippet: CUL4A knockdown rescues elevated proliferation of livers of FTO L-KO mice. (A) Western blot analyses of CCNE1 in whole liver lysates isolated from ST-DEN injected Ctrl and FTO L−KO mice (n = 4 for each timepoint, two-way ANOVA). CCNE1/β-ACTIN ratio normalized to 0 h Ctrl expression. (B) Scheme of intravenous tail vain injections and different experimental mice groups. (C) qRT-PCR analysis of Cul4a and Fto expression in whole liver of 24 h ST-DEN injected Ctrl/AAV8 shRNA- scrmbl , Ctrl/AAV8 shRNA− Cul4a , FTO L−KO /AAV8 shRNA- scrmb , and FTO L−KO /AAV8 shRNA− Cul4a (n = 5, one-way ANOVA). (D) Western blot analysis of CUL4A and FTO in whole liver lysates isolated from 24 h ST-DEN-injected Ctrl/AAV8 shRNA- scrmbl , Ctrl/AAV8 shRNA− Cul4a , FTO L−KO /AAV8 shRNA- scrmb , and FTO L−KO /AAV8 shRNA− Cul4a (n = 5). (E) Ki67 ELISA with liver proteins isolated from 24 h ST-DEN-injected Ctrl/AAV8 shRNA- scrmbl , Ctrl/AAV8 shRNA− Cul4a , FTO L−KO /AAV8 shRNA- scrmb , and FTO L−KO /AAV8 shRNA− Cul4a (n = 4–5, one-way ANOVA). p∗ 0.05, p∗∗∗∗ 0.0001. Data are means with SD.

Article Snippet: The following TaqMan probes (Applied Biosystems, Darmstadt, Germany) were used for gene expression assays: Cul4a (Mm01220732_m1; Mm00461469_m1; Mm00461464_g1; Mm01220734_g1; Mm00461460_g1; Mm00461475_gH), Dgat1 (Mm00515643_m1), Dgat2 (Mm00499536_m1), Fasn (Mm00662319_m1), Fto (Mm00488755_m1), Pparg (Mm00440945_m1), Scd1 (Mm00772290_m1), Srebp1 (Mm00550338_m1), Srebp2 (Mm01306292_m1), Tbp (Mm00446973_m1).

Techniques: Knockdown, Western Blot, Isolation, Injection, Expressing, Quantitative RT-PCR, shRNA, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 1. Y211F conveys protein instability of PCNA through a CUL4A-dependent mechanism. A, expression of the FLAG-tagged Y211F mutant PCNA in the CUL4A-expressing MDA-MB-468 cells was reduced compared with the wild-type PCNA. Treatment with the proteasome inhibitor MG132 increased the expression level of the PCNA-Y211F protein. B, transfection of CUL4A-DN into the MDA-MB-468/Y211F cells resulted in enhanced expression of the PCNA- Y211F mutant. Quantitated result of data derived from three independent experiments is shown. *, p 0.05. C, depleting endogenous CUL4A by shRNA (shCUL4A) resulted in rescue of PCNA-Y211F expression. Levels of -tubulin are shown as the internal loading control. Quantitated result of data derived from threeindependentexperimentsisshown.*,p0.05.D,depletingexogenousCUL4BbyshRNA(shCUL4B)hadnoeffectonthelevelofPCNA-Y211F.E,silencing efficiency of shCUL4B was determined by quantitative RT-PCR. ***, p 0.005. Error bars, S.E.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Mutagenesis, Transfection, Derivative Assay, shRNA, Control, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 2. CUL4A interacts preferentially with Y211F PCNA. HEK293T cells were transfected with the indicated plasmids (FLAG-PCNA-WT, FLAG-PCNA- Y211F, HA-CUL4A, and the pcDNA3 vector alone). The ectopic PCNA was immunoprecipitated by an anti-FLAG antibody, and the precipitated FLAG- PCNA and HA-CUL4A were detected by the corresponding antibodies as indi- cated. The input lysates were examined for the expression of the transfected PCNA and CUL4A genes. Levels of -tubulin are shown as the internal loading control.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 4. Association between CUL4A and PCNA is negatively regulated by EGFR. A and B, MDA-MB-468 cells were serum-starved for 24 h and then stimulated by EGF (10 ng/ml) for 30 min. The cells were then lysed in NETN buffer and subjected to immunoprecipitation (IP) by the indicated antibodies. Immunoprecipitated PCNA (A) or CUL4A (B) was examined for the level of associated CUL4A or PCNA, respectively. C and D, MDA-MB-468 cells grown in normal conditions were treated with the EGFR kinase inhibitor AG1478 for 6 h. The cells were lysed, and the lysate was then subjected to immunoprecipitation for PCNA(C)orCUL4A(D).TheassociatedCUL4AandPCNAwasthenassessedbyWesternblottinganalysis.Averagebindingactivitiesbetweenthesetwoproteins from two independent experiments are shown. E, MDA-MB-468 cells grown in normal condition were mock-treated or treated with AG1478 as described in CandthenstainedwithprimaryantibodiesagainstPCNAorCUL4A.SecondaryantibodiesconjugatedwithrhodamineandFITCwereusedtodetectPCNA(red) and CUL4A (green), respectively. The slides were examined by fluorescence confocal microscopy, with photosections of 0.48 m each. Note that in the merged images, colocalization of PCNA and CUL4A appears as yellow in the nucleus. Nuclear colocalization of PCNA and CUL4A was quantitated using the ImageJ program. Right, Pearson’s correlation coefficients (Rp) are shown. A coefficient of 1 indicates complete colocalization. For details, see under “Experimental Procedures.” Error bars, S.E. *, p 0.05.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Immunoprecipitation, Fluorescence, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 5. CUL4A is responsible for the EGFR inhibition-mediated PCNA polyubiquitylation. Upper panel, MDA-MB-468 cells were treated with and without the EGFR inhibitor AG1478 (8 M) for 18 h in the presence of the proteasome inhibitor MG132 (5 M). Cell lysates were then immunoprecipi- tated (IP) with an anti-PCNA antibody (Santa Cruz Biotechnology). The blot was then probed with an anti-ubiquitin (Ub) antibody. The numbers indicate the intensity of the ubiquitylated ladder normalized by the intensity of total PCNA. Lower panel, the input levels of PCNA, phospho-EGFR (Y1068), total EGFR, and tubulin are shown. WB, Western blotting.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 6. CUL4A mediates polyubiquitylation preferentially on PCNA lacking Tyr-211 phosphorylation. A, right, HEK293T cells were transfected by the indicated plasmids, and the endogenous PCNA was immunoprecipitated (IP) by an anti-PCNA antibody, followed by immunoblotting (WB) with an anti-ubiquitin (Ub) antibody. Arrows denote the ubiquitylated PCNA (Ub-PCNA). Transfection devoid of ubiquitin significantly abolished ubiquitylation. Middle, average ratio of the ubiquitylated PCNA with total PCNA was quantitated based on results derived from two independent experiments. Error bar, S.E. Left, expression levels of the transfected wild-type CUL4A (by an anti-CUL4A antibody) and the CUL4A-DN mutant (by an anti-FLAG antibody) and the endogenous PCNA (by an anti-PCNA antibody) are shown by Western blotting, with -tubulin as the loading control. B, wild-type or Y211F PCNA (FLAG-tagged) and CUL4A were cotransfected into HEK293T cells with or without cotransfected ubiquitin cDNA. PCNA was immunoprecipitated by an anti-PCNA antibody, and the immunoblot was probed with an anti-FLAG antibody to detect the ubiquitylated PCNA molecules. Expression levels of the transfected wild-type CUL4A (by an anti-CUL4A antibody) and the transfected PCNA (by an anti-FLAG antibody) are shown by Western blotting, with -tubulin as the loading control.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Derivative Assay, Expressing, Mutagenesis, Control

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 7. CUL4A catalyzes ubiquitylation at the Lys-164 residue of PCNA. A, HEK293T cells were transfected with the indicated plasmids, and the lysates were subjected to immunoprecipitation (IP) with an anti-PCNA antibody. The Western blot (WB) was than probed with anti-FLAG anti- body to detect the ubiquitylated PCNA molecules. Expression levels of the transfected genes in cell lysates are shown. B, HEK293T cells were trans- fected with the indicated plasmids, and the expression levels of each PCNA construct were determined by an anti-FLAG antibody with -tubu- lin as the loading control.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Residue, Transfection, Immunoprecipitation, Western Blot, Expressing, Construct, Control

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 9. Depleting CUL4A sensitizes MDA-MB-468 cells to EGFR inhibi- tion. A, MDA-MB-468 cells freshly transduced with lentivirus harboring shCUL4A or the control shRNA were treated with AG1478 at the indicated concentration for 18 h, and the activities of BrdU incorporation were mea- sured. The data were normalized by a parallel experiment of 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B, MDA-MB-468 cells were transfected with a siRNA of PCNA to deplete the endogenous PCNA and thentransducedwitheitheracontroladenovirusoranadenovirusexpressing the wild-type or Y211F FLAG-tagged PCNA at 10 multiplicity of infection to reconstitute PCNA. The cells were then treated with AG1478 (5 M) or mock- treated for 48 h. Viable cells were measured for BrdU incorporation activities, which were compared after normalization with the control treatment for each viral transduction. Ratios of the treated with the mock-treated cells based on data derived from three independent experiments were plotted. **, p 0.01. Error bars, S.E.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Transduction, Control, shRNA, Concentration Assay, BrdU Incorporation Assay, Transfection, Infection, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

doi: 10.1074/jbc.m112.388843

Figure Lengend Snippet: FIGURE 10. Schematic representation of the EGFR-PCNA-CUL4A axis. We show in this study that CUL4A contributes to degradation of PCNA through Lys-164 polyubiquitylation. The ubiquitylation and PCNA degradation are enhanced when EGFR is inhibited. Tyr-211 phosphorylation of PCNA upon EGFRactivationleadstodissociationoftheCUL4-PCNAinteractionandthere- fore stabilizes PCNA on the chromatin.

Article Snippet: Plasmids and DNA Transfection—Expression plasmids of wild-type CUL4A (Addgene plasmid 19907) (32) and CUL4ADN (Addgene plasmid 15821) (33) were obtained from Addgene (Cambridge, MA).

Techniques: Phospho-proteomics