Journal: Arthritis and Rheumatism

Article Title: ADAMTS-4_v1 Is a Splice Variant of ADAMTS-4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

doi: 10.1002/art.38102

Figure Lengend Snippet: Proteolysis of ADAMTS-4_v1. A, Western blots probed with anti–FLAG M2 monoclonal antibody (mAb) or affinity-purified polyclonal goat antibodies to human ADAMTS-4 from purified ADAMTS-4_v1 that had been stored at −80°C during the course of the experiment (lane 0) or had been incubated with 2 units of furin at 37°C for 1, 2, or 3 hours (lanes 1, 2, and 3, respectively). B, Western blots probed with affinity-purified AltTS4.2, a rabbit antibody raised against a synthetic peptide representing a unique sequence in the C-terminus of ADAMTS-4_v1, or with affinity-purified goat antibodies to human ADAMTS-4 from purified ADAMTS-4_v1 that had been stored at −80°C during the course of the experiment (lane 0) or had been incubated at 37°C in the presence of 10 m M CaCl 2 for 1, 3, or 6 hours (lanes 1, 3, and 6, respectively). C, Western blot probed with anti–FLAG M2 mAb from purified ADAMTS-4_v1 that had been stored at −80°C during the course of the experiment (−80°C) or had been incubated for 6 hours at 37°C in the presence of 10 m M CaCl 2 (control) or 10 m M CaCl 2 plus 24 n M tissue inhibitor of metalloproteinases 1 (TIMP-1), 23 n M TIMP-2, or 5 m M EDTA.

Article Snippet: Bovine aggrecan (20 μg; Sigma) was incubated overnight at 37°C in the presence or absence of 2 units of furin (New England Biolabs), with purified ADAMTS-4, ADAMTS-4_v1, or anti-FLAG immunoprecipitates from cell lysates of untransfected HEK 293 cells, in TBS containing 10 m M CaCl 2 .

Techniques: Western Blot, Affinity Purification, Purification, Incubation, Sequencing

Journal: Arthritis and Rheumatism

Article Title: ADAMTS-4_v1 Is a Splice Variant of ADAMTS-4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

doi: 10.1002/art.38102

Figure Lengend Snippet: Aggrecanase substrate activity of ADAMTS-4_v1, as assessed by SensoLyte 520 aggrecanase 1 assay. A, Aggrecanase 1 activity of an anti–FLAG M2 immunoprecipitate from ADAMTS-4_v1 transfected with HEK 293 cells that had been predigested for 1 hour at 37°C with furin (▪) or an equal amount of undigested ADAMTS-4_v1 immunoprecipitate (□), as compared to that of the recombinant truncated ADAMTS-4 (•) (supplied in the assay kit). Enzyme activity (expressed as relative fluorescence units [RFU]) was measured every 5 minutes for 1 hour. B, Western blot probed with anti–FLAG M2 monoclonal antibody from anti–FLAG M2 immunoprecipitates from ADAMTS-4_v1–transfected HEK 293 cells used in A. Shown are undigested and furin-digested ADAMTS-4_v1 prior to their use in the assay (preassay) as well as undigested and furin-digested ADAMTS-4_v1 plus recombinant truncated ADAMTS-4 (rADAMTS-4) that was harvested from the assay plate after the 1-hour course of the aggrecanase 1 assay (postassay). C, Duplicate Western blot of B probed with affinity-purified goat anti-human ADAMTS-4 antibodies (BAF4307).

Article Snippet: Bovine aggrecan (20 μg; Sigma) was incubated overnight at 37°C in the presence or absence of 2 units of furin (New England Biolabs), with purified ADAMTS-4, ADAMTS-4_v1, or anti-FLAG immunoprecipitates from cell lysates of untransfected HEK 293 cells, in TBS containing 10 m M CaCl 2 .

Techniques: Activity Assay, Transfection, Recombinant, Fluorescence, Western Blot, Affinity Purification

Journal: Arthritis and Rheumatism

Article Title: ADAMTS-4_v1 Is a Splice Variant of ADAMTS-4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

doi: 10.1002/art.38102

Figure Lengend Snippet: Enzyme activity of ADAMTS-4_v1 against proteoglycan substrates. A, Western blot of bovine aggrecan digested with anti-FLAG immunoprecipitates from Triton X-100 extracts of untransfected HEK 293 cells (lanes 1 and 2), purified ADAMTS-4.1 (lanes 3 and 4), and ADAMTS-4_v1 (lanes 5 and 6), in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of furin. The blot was probed with neoepitope monoclonal antibody (mAb) BC-3 to detect cleavage at the interglobular domain aggrecanase cleavage site. B, Western blot of aggrecan (lanes 4 and 8) and aggrecan that had been deglycosylated with chondroitinase ABC (lanes 1 and 5), chondroitinase ABC plus keratanase I and keratanase II (lanes 2 and 6), or keratanase I plus keratanase II (lanes 3 and 7) digested with either ADAMTS-4.1 (lanes 1–4) or ADAMTS-4_v1 (lanes 5–8). Cleavage at the interglobular domain aggrecanase site was detected with BC-3. C and D, Western blots of an A3 extract of cartilage digested with anti-FLAG immunoprecipitates from Triton X-100 extracts of untransfected HEK 293 cells (lane 1), purified ADAMTS-4.1 (lane 2), or ADAMTS-4_v1 (lane 3) and then probed with mAb PR-1, which is specific for biglycan (C), or mAb 70.6, which is specific for decorin (D).

Article Snippet: Bovine aggrecan (20 μg; Sigma) was incubated overnight at 37°C in the presence or absence of 2 units of furin (New England Biolabs), with purified ADAMTS-4, ADAMTS-4_v1, or anti-FLAG immunoprecipitates from cell lysates of untransfected HEK 293 cells, in TBS containing 10 m M CaCl 2 .

Techniques: Activity Assay, Western Blot, Purification

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 1 Identification of potential genes implicated in colorectal cancer (CRC) and cancer metabolism-associated biological processes. (A) A screening procedure to find putative gene candidates. (B) Colorectal cancer (CRC) samples were found to differ from adjacent controls in terms of physiopathology and biological processes related to metabolism in a number of databases, including TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO) datasets (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549). (C) Following gene differential expression analysis, the total number of differentially expressed genes that crossed over into various databases was counted. (D) Six upregulated and four down regulated DEGs were identified based on a survival analysis of differentially expressed genes across six databases.In the databases of TCGA and ICGC, P < 0.05 was deemed statistically significant. (E) Six upregulated and four downregulated DEGs represent the molecular mechanisms impacting the onset of colorectal cancer and metabolic reprogramming. (F) Palmitoyltransferase ZDHHC6 expression in the ICGC and TCGA databases. (G) Pancarcinoma analysis using TCGA datasets to measure ZDHHC6 expression levels in various malignancies. (H) The overall survival (OS) of colorectal cancer patients in the TCGA and ICGC databases according to different ZDHHC6 expression levels. (I) After dividing the TCGA and ICGC samples’ ZDHHC6 expression levels into groups of high and low expression levels, the grouped samples underwent GSEA analysis. The data were expressed as the mean ± SEM. A P value less than 0.05 was considered statistically significant. ***P < 0.001

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Gene Expression, Quantitative Proteomics, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 2 Increased ZDHHC6 is positively associated with the development of human colorectal cancer (CRC). (A) ZDHHC6 mRNA expression levels in 73 pairs of CRC sample pairs (T) and their corresponding adjacent sample pairs (N). n = 73 pairs. (B) ZDHHC6 protein expression levels in sixteen pairs of similar adjacent tissues and colorectal cancer tissues selected at random. For each group, n = 3. (C) ZDHHC6 mRNA expression levels in relation to a range of CRC-associated cell lines, such as SNU-C2A, SW48, HT-29, LS1034, HCT116, and Caco-2, as well as the matching human normal colonic epithelial cell line (FHC), are displayed in qPCR analysis. For each group, n = 5. (D, E) ZDHHC6 protein expression in SNU-C2A, SW48, HT-29, LS1034, HCT116, Caco-2, and FHC cell line as demonstrated by western blotting (D) and immunofluorescence analysis (E). 200 μm; each group has n = 5. (F, G) qPCR analysis (F) and western blotting experiment (G) demonstrate the effect of the gradually increased dosage of 2-bromopalmitate (2-BP) on the relative ZDHHC6 mRNA and protein expression levels in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. For each group, n = 3. (H) An immunofluorescence assay demonstrating the co-expression of ZDHHC6 and Ki67 in response to 40 µM 2-bromopalmitate (2-BP) in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. 200 μm; each group has n = 3. Data are expressed as mean ± SEM. The relevant experiments presented in this section were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 4 ZDHHC6 facilitates lipid deposition and carcinogenesis in CRC cells. (A) A venn diagram shows the variations in metabolites produced by HCT116 cells with ZDHHC6 knockout (KO) and wild-type (WT) phenotypes. ZDHHC6 and fatty acid synthesis pathways have a significant association, according to pathway enrichment analysis of the 36 metabolites. Total peak area was used to correct the LC-MS-based untargeted metabolomic study and its findings. (B) Using these 36 differential metabolites, pathway analysis showed enhanced signaling pathways. (www.metaboanalyst.ca). (C) A heatmap showing how these 36 significantly altered metabolites changed. Student’s t-test, unpaired, two-tailed, P < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). (D, E) The ratios of various isotopic forms of FFA C16:0 (palmitate) in ZDHHC6 (KO) (D) and AdZDHHC6 (E) HCT116 cells after a brief exposure to glucose [U-13C]. When the cell density was around 85%, the media was changed to RPMI 1640 containing 2 g/L glucose tagged with [U-13C]. Following a 24-hour period, the PBS-rinsed cell culture plates were quickly frozen in liquid nitrogen and subjected to an LC-MS assay analysis (n = 4 per group). (F) Representative im munofluorescence pictures of HCT116 cells with ZDHHC6 (WT) and ZDHHC6 (KO) phenotypic, demonstrating ZDHHC6 expression, lipid accumulation (Bodipy staining), and corresponding intracellular triglyceride (TG) levels (n = 4 per group). (G, H) ZDHHC6 (WT) and ZDHHC6 (KO) HCT116 cells were injected into the right flanks of nude mice. Every two days, tumor volumes were measured. On day 22 following dissection, tumor pictures (G), growth curves, and weight (H) were recorded (n = 4 per group). Scale bars, 1 cm. (I) A heatmap utilizing untargeted metabolomic analysis comparing significantly changed metabolites between tumors originating from ZDHHC6 (KO) HCT116 cells and ZDHHC6 (WT) cell lines. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Produced, Knock-Out, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, Two Tailed Test, Cell Culture, Expressing, Staining, Injection, Dissection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 5 ZDHHC6 specifically binds to the lipid metabolism key transcription factor of PPARγ. (A) After 24 h of SFB-ZDHHC6 transfection in HCT116 cells, ZDHHC6-interacting proteins were identified by tandem affinity purification and mass spectrometry (MS). This was accomplished by removing S-protein, Flag, and streptavidin binding peptide (SFB). (B) ZDHHC6 or IgG antibodies were used to immunoprecipitate HCT116 cell lysates, and PPARγ, PPARα, PPARδ, SREBP1, and ZDHHC6 antibodies were used for western blotting experiments. (C) ZDHHC6 or IgG antibodies were used to immunoprecipitate cellular lysates of SNU-C2A, SW48, HT-29, LS1034, and Caco-2 cells, and ZDHHC6 or PPARγ antibodies were used for western blotting experiments. (D) GST pulldown assay using GST-PPARγ and purified His-ZDHHC6 in HCT116 cells. (E) Schematic of the experimental procedure showing the genes expression in HCT116, Caco-2, SNU-C2A and HT-29 after adenovirus-mediated ZDHHC6 overactivation (AdZDHHC6). The lower schematic diagram showing the inter section of the results from the proteomics and IP-MS analyses. (F) For a duration of 24 h, plasmids expressing Flag-PPARγ or Myc-ZDHHC6 individually or in combination were transfected into HCT116, Caco-2, SNU-C2A and HT-29 cells, respectively. His or Flag antibodies were used for immunoblotting after cellular lysates had been immunoprecipitated with Flag and/or His antibodies. (G) GST pulldown assay using GST-PPARγ and purified Flag-ZDHHC6 in Caco-2 and SNU-C2A cells, respectively. (H) Assay for immunofluorescence staining demonstrating ZDHHC6 and PPARγ co-expression in HCT116, Caco-2, and SNU-C2A cells. 20 μm. (I) In HCT116 cells, vectors containing the hinge-LBD domain, full length (FL), AF-1, DBD, and PPARγ were co-expressed with SFB-ZDHHC6. S-bead pulldown was used to immunoprecipitate cellular lysates. (J) Based on GSEA signaling pathway analysis, an assay of the TCGA-CRC and ICGC-CRC datasets showed a significant connection between ZDHHC6 and the PPARγ pathway in CRC. Data are expressed as mean ± SEM. The rel evant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Transfection, Affinity Purification, Mass Spectrometry, Binding Assay, Western Blot, GST Pulldown Assay, Purification, Expressing, Protein-Protein interactions, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 6 Identification of the palmitoylation site on PPARγ at evolutionarily conserved cysteine residues. (A) For a duration of 24 h, HCT116 cells were exposed to 60 µM 2-BP, 1 µM ABD957, 6 µM palmostatin B (Palm B), and 10 µM palmostatin M (Palm M) treatments. The slices that were fixed underwent immunofluorescence labeling using PPARγ (red) and pan-palmitoylation (green). 10 μm scale bars; n = 5 per group. (B) Schematic diagram of the Click-iT assay for palmitoylation measurement of PPARγ. HCT116 cells were treated with 100 µM Click-iT PA and azides for five hours. The resulting lysates were then submitted to Click-iT detection as per the product instructions, and PPARγ antibody western blotting analysis was performed. The indicated group’s expression of PPARγ is indicated by the western blotting bands on the right. (C) Using the GPS-Palm program (MacOS_20200219) (The CUCKOO Work group, http://gpspalm.biocuckoo.cn/) and the MDD-Palm algorithm (http://csb.cse.yzu.edu.tw/MDDPalm/), the palmitoylation site on PPARγ in Homo sapiens (upper) and Mus musculus (lower) is predicted to be located. PPARγ’s lower palmitoylation site contains conserved cysteine residues shared by Rattus norvegicus, Bos taurus, Canis familiaris, Mus musculus, and Homo sapiens. (D) After incubating Click-iT PA and azides for five hours on HCT116 cells overexpressing either PPARγ WT or PPARγ C313S mutant, the corresponding cellular lysates were obtained and Click-iT detection was performed in com pliance with the product’s instructions. After the palmitoylated proteins were added to the streptavidin-sepharose bead conjugate for pull-down detec tion, PPARγ and ACTIN antibodies were used in a western blotting examination. While PPARγ C313S was not palmitoylated in top gel, lane 6, or the control groups, it was for PPARγ WT in lane 5. Three separate runs of this experiment were conducted. (E) CHX was cultured with HCT116 cells overexpressing either the PPARγ WT or PPARγ C313S mutant for a specific amount of time. PPARγ and ACTIN antibodies were used in immunoblotting detection of the obtained cellular lysates. The relative PPARγ remaining ratio (n = 4 per group) is displayed in the right curve graph at the specified time point. (F) PPARγ WT or PPARγ C313S mutant overexpression was observed in the upper HCT116 cells. Pan-palmitoylation (green) and PPARγ (red) immunofluorescent label ing were applied to the cell sections. Lower, AdZDHHC6 + PPARγ C313S mutant or PPARγ C313S alone were overexpressed in HCT116 cells, respectively. The bar graph displays the intensity of PPARγ fluorescence in each of the indicated groups (n = 5 pictures; P < 0.05 vs. PPARγ C313S + AdControl or PPARγ WT). Scale bars, 20 μm. (G) In HCT116 cells, PPARγ-Flag and ZDHHC6-HA plasmids were transfected. Alk16 labeling was used to determine the palmi toylated PPARγ expression contents in the presence or absence of hydroxylamine therapy. (H) PPARγ-Flag was used to transfect SNU-C2A cells (WT) or ZDHHC6-deleted SNU-C2A cells, and Alk16 was used to label the cells. Subcellular fraction was extracted, and the levels of PPARγ protein were adjusted to verify that the input cells from the wild type and the knockout cell had the same quantity of PPARγ. Immunoblotting analysis was used to evaluate the palmitoylated PPARγ expression contents in the cell membrane (Mem.), cell cytoplasm (Cyto.), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Immunofluorescence, Labeling, Western Blot, Expressing, Mutagenesis, Control, Cell Culture, Over Expression, Fluorescence, Transfection, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 7 ZDHHC6-mediated palmitoylated PPARγ enhances its nucleus translocalization. (A) ZDHHC6 and PPARγ expression were examined in the ZDH HC6-deleted HCT116, SNU-C2A and SW48 cells, respectively (n = 3 per group). (B) ZDHHC6 and PPARγ co-expression in AdshZDHHC6-transfected HCT116 cells, along with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 vs. AdshRNA). The scale bars are 20 μm. (C) In ZDHHC6-deleted HCT116 or ZDHHC6-deleted SW48 cells, palmitoylation levels and PPARγ expression were analyzed using western blotting assay (n = 4 per group). (D) Western blotting assay using PPARγ, ACTIN, and HA antibodies, followed by PPARγ overexpressing the HA-tagged ZDHHC6 construct in various CRC cell lines (n = 3 per group). (E) Immunofluorescence pictures demonstrating the co-expression of PPARγ and ZDHHC6 in ZDHHC6-overex pressed HCT116 cells, together with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 compared to empty vector). The scale bars are 20 μm. (F) HCT116 cells underwent IP of HA after co-transfecting with PPARγ and HA-ZDHHC6. ZDHHC6 and PPARγ Mutual Co-IP shows that endogenous ZDHHC6 and PPARγ bind to each other in HCT116 cells. (G) Using various alkyl-labeled fatty acylation, such as alk-C14, alk- C16, alk-C18, and alk-C20, the palmitoylation of PPARγ in the indicated cells was detected. By using streptavidin bead pulldown to identify acylated PPARγ, an immunoblotting experiment using PPARγ and ACTIN antibodies (n = 6 per group) was performed. (H) To identify acylated PPARγ in SW48, LS1034, and HT-29 cells, the same methodology as in (G) was applied. Following that, the lysates (n = 6 per group) were subjected to western blotting analysis using PPARγ and ACTIN antibodies. (I) Using Click reaction-associated streptavidin pulldown, the palmitoylation levels of Flag-labeled PPARγ WT, PPARγ C313S, PPARγ C156S, PPARγ C176S, and PPARγ C159S mutants were examined. Three individuals per group underwent an immunoblotting experiment using Flag and ACTIN antibodies on the relevant lysates. (J) ZDHHC6-HA and PPARγ-Flag were the vectors used to transfect the HCT116 cells. Using alk-C16 labeling, higher, palmitoylated PPARγ levels were demonstrated in both the presence and absence of hydroxylamine therapy. The corresponding fluorescence density and ACLY and PPARγ co-expression in HCT116 WT or HCT116 ZDHHC6 (KO) cells are depicted in the lower representative immunofluorescence images, which were analyzed using Pearson’s method (n = 5 per group; P < 0.05 vs. WT). The scale bars are 20 μm. (K) After transfecting the HCT116 WT or HCT116 ZDHHC6 (KO) cells with PPARγ-Flag, the cells were labeled with alk-C16. To verify that the wild type and knockout cell components for input had the same quantity of PPARγ, subcellular fraction was obtained and PPARγ protein levels were adjusted. Western blotting analysis was used to assess palmitoylated PPARγ levels in the cell membrane (Mem.), cell cytoplasm (Cyto. ), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Transfection, Fluorescence, Western Blot, Construct, Immunofluorescence, Plasmid Preparation, Co-Immunoprecipitation Assay, Labeling, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 9 ZDHHC6-driven lipid biosynthesis contributes to CRC carcinogen esis by upregulating PPARγ. (A, B) In HCT116-related stable cells (Control, ZDHHC6, and ZDHHC6 + shPPARγ) (A) and HCT116-related stable cells (shControl, shZDHHC6, and shZDHHC6 + PPARγ) (B), the percentages of different isotopomers of FFA C16:0 following exposure to [U-13C] glucose are shown. Each group has n = 5. (C, D) The relative TG content and PPARγ expression abundance in the aforementioned cell lines from (A) and (B) are displayed in representative immunofluorescence pictures. Each group has n = 5. The scale bars are 20 μm. (E) In null mice, right flanks were in jected with ZDHHC6 + shPPARγ, ZDHHC6, and Control, stable cells related to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (F) The right flanks of null mice were injected with shCon trol, shZDHHC6, and shZDHHC6 + PPARγ, stable cells linked to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (G) Kaplan-Meier curves representing the survival analysis based on TCGA CRC prognostic data for ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-positive patients. (H) Based on the prognosis information from the ICGC CRC database, Kaplan-Meier curves were used to analyze the sur vival of ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-posi tive patients. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Control, Expressing, Immunofluorescence, Dissection, Injection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 10 Palmitoylation stabilizes PPARγ by ZDHHC6 via blocking its lysosomal degradation to promotes lipid biosynthesis-associated CRC development. As a palmitoyltransferase enzyme, ZDHHC6 regulates the synthesis of fatty acids. To be more precise, ZDHHC6 directly attaches palmitoyl groups to PPARγ, a protein that controls the expression of genes. By stabilizing PPARγ and blocking its lysosomal degradation, the palmitoylation mechanism triggers the production of ACLY and subsequently leads to the development of lipid buildup-related CRC carcinogenesis

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Blocking Assay, Expressing

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 copurifies with cyclin D1. (A) Lysates were fractionated by gel filtration chromatography, and 5% of each fraction was resolved by SDS-PAGE; elution of cyclin D1 (top panel) and CDK4 (bottom panel) was visualized by immunoblot analysis. The positions of eluting molecular weight standards are indicated at the top. (B) Fractions corresponding to lanes 4 to 8 in panel A were immunoprecipitated (IP) with a monoclonal antibody specific for cyclin D1 and either blotted for associated CDK4 (top) or assayed for their ability to phosphorylate recombinant GST-Rb (bottom). (C) Detection of cyclin D1 and cyclin D1-associated proteins by silver stain. Lane 1 contains molecular weight markers (sizes shown in kilodaltons), lane 2 contains proteins that nonspecifically bind to M2 beads from control NIH 3T3 lysates, and lane 3 contains cyclin D1 complexes isolated from FlagD1-T286A-3T3 lysates (an essentially identical pattern was recovered from Flag-D1-3T3 cells; data not shown). Proteins were eluted with excess Flag peptide. Positions of Flag-D1, CDK4, and p21Cip1 are indicated to the right, as is the position of Hsc70, along with peptides identified by mass spectrometry.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Filtration, Chromatography, SDS Page, Western Blot, Molecular Weight, Immunoprecipitation, Recombinant, Silver Staining, Control, Isolation, Mass Spectrometry

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Mitogen-dependent regulation of cyclin D1-Hsc70 binding. (A) Wild-type NIH 3T3 cells were used as an asynchronous culture (asyn) or synchronized by serum deprivation for 36 h and stimulated to reenter the cell cycle by addition of 10% fetal calf serum. Cell lysates were prepared at the indicated intervals (bottom) and immunoprecipitated (IP) with normal rabbit serum (NRS) or a cyclin D1-specific monoclonal antibody (D1). Precipitated proteins were then subjected to immunoblot analysis with antibodies specific for Hsc70 (top) or cyclin D1 (bottom). (B) D1-3T3 cells were synchronized as in panel A. Cell lysates were prepared at the indicated intervals (bottom) and precipitated with normal rabbit serum (NRS) or the M2 monoclonal antibody and subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel) or cyclin D1 (bottom panel). (C) Cell lysates prepared as for panel A were subjected to direct Western analysis with an Hsc70-specific antibody. (D) Whole-cell lysates prepared from asynchronous D1-3T3 cells or D1-3T3 cells cultured in medium containing 0.1% fetal calf serum for the indicated intervals were precipitated with normal rabbit antiserum (NRS) or with the M2 monoclonal antibody. Precipitates were immunoblotted with either Hsc70 (top panel) or cyclin D1 (lower panel) antibodies. The arrow indicates the point at which cells were placed in medium containing 0.1% serum.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Cell Culture

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 decreases cyclin D1 proteolysis. (A) NIH 3T3 cells were cotransfected with (lane 2) or without (lane 1) a vector encoding Hsc70 along with vectors encoding either cyclin D1 and CDK4 (top and bottom panels), cyclin D1-T286A and CDK4 (second panel), or Myc-tagged cyclin E and CDK2 (bottom panel). Levels of the indicated proteins were determined by direct Western blot analysis of total cell lysates prepared from cells transfected with the indicated vectors. (B) Whole-cell extracts were prepared from NIH 3T3 cells cotransfected with either Flag-D1 and CDK4 (lanes 1 and 2) or Flag-D1, CDK4, and Hsc70 and precipitated with either normal rabbit serum (lane 1) or the M2 monoclonal antibody (lanes 2 and 3). Cyclin D1 and coprecipitating CDK4 were detected by immunoblot analysis with antigen-specific antibodies. (C) NIH 3T3 cells were cotransfected with a plasmid encoding cyclin D1 with (lane 2) or without (lane 1) a vector encoding Hsc70K71E. Levels of cyclin D1 were monitored by immunoblot analysis. (D) p21/p27−/− MEFs were transfected with a plasmid encoding cyclin D1 without (lane 1) or with (lane 2) a plasmid encoding Hsc70. Levels of cyclin D1 were monitored by immunoblot analysis. (E and F) NIH 3T3 cells transfected with vectors encoding either Flag-D1 and CDK4 or Flag-D1, CDK4, and Hsc70 were treated with 50 μg of cycloheximide per ml, and lysates prepared from these cells at the intervals indicated at the bottom of each panel were subjected to immunoblot analysis for cyclin D1. The percentage of cyclin D1 remaining at each time point is indicated at the bottom of each lane.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Plasmid Preparation, Western Blot, Transfection

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Cyclin D1 and Hsc70 are components of multiple complexes. (A) Affinity-purified cyclin D1 complexes were resolved by gel filtration. Ten percent of each fraction was subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel), cyclin D1 (second panel), CDK4 (bottom panel), or p21Cip1 (bottom panel). (B and C) The fraction corresponding to lane 5 in panel A was precipitated with a p21-specific antiserum (B) or an Hsc70 monoclonal antibody (C). Associated proteins were monitored by immunoblot analysis with antibodies specific for the proteins indicated to the left of each panel. (D) The fraction corresponding to lane 5 of part A (158 kDa) was subjected to two rounds of immunodepletion with either normal rabbit antiserum (mock) or the Hsc70-specific monoclonal antibody. Codepletion of cyclin D1 (top panel), CDK4 (middle panel), and p21Cip1 (lower panel) was monitored by immunoblot analysis with the respective antibodies. (E) Asynchronous NIH 3T3 cells were pulse-labeled with [35S]methionine for 15 min (lanes 1 to 6) or pulse-labeled and subsequently chased for 10 min with excess unlabeled methionine for 10 min (lanes 7 to 12). Lysates prepared from these cells were resolved by gel filtration chromatography, and cyclin D1 was precipitated from fractions ranging from 670 kDa to 100 kDa (indicated at the top). Proteins were then analyzed by SDS-PAGE, and cyclin D1 was visualized by autoradiography.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Affinity Purification, Filtration, Western Blot, Immunodepletion, Labeling, Chromatography, SDS Page, Autoradiography

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Reconstitution of a 158-kDa cyclin-CDK complex. (A) Sf9 insect cells were infected with baculoviruses encoding cyclin D1, CDK4, and p21Cip1 with (lanes 1 to 6) or without (lanes 7 to 12) Hsc70. Lysates prepared from these cells were resolved by gel filtration chromatography and subjected to immunoblot analysis for Hsc70 (top panel) or cyclin D1 (bottom panel). The elution positions of molecular weight standards are indicated at the top of each panel. (B) Fractions corresponding to lanes 3 and 4 of part A were precipitated with the cyclin D1 monoclonal antibody. Coprecipitation of Hsc70 was monitored by immunoblotting with the Hsc70 monoclonal antibody. (C) Lysates prepared from Sf9 insect cells infected with baculoviruses encoding either cyclin D1 and CDK4 (lane 1), cyclin D1, CDK4, and Hsc70 (lane 2), cyclin D1, CDK4, and p21Cip1 (lane 3), or all four (lane 4) and metabolically labeled with [35S]methionine were precipitated with the cyclin D1 monoclonal antibody. Positions of Hsc70, cyclin D1, CDK4, and p21Cip1 are indicated to the left of the panel and were verified independently by immunoprecipitation with antigen-specific antibodies (data not shown). Labeled proteins were visualized by autoradiography.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Infection, Filtration, Chromatography, Western Blot, Molecular Weight, Metabolic Labelling, Labeling, Immunoprecipitation, Autoradiography

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 maintains cyclin D1-dependent cell cycle progression in the presence of inhibitory levels of p21Cip1. (A) NIH 3T3 cells were transfected with vectors encoding Flag-D1 and CDK4, Flag-D1, CDK4, and p21Cip1, or Flag-D1, CDK4, p21Cip1, and Hsc70; 24 h posttransfection, cells were labeled with bromodeoxyuridine (BrdU) for 20 h. Cells were fixed and stained with monoclonal M2 antibody (red), bromodeoxyuridine (green), and Hoechst dye (blue). (B) Quantitation of panel A, representing three independent experiments; error bars represent standard errors between experiments. (C) Whole-cell lysates prepared from cells transfected as for panel A were resolved by SDS-PAGE, and levels of p21Cip1 were monitored by immunoblot analysis with a p21-specific antibody.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Transfection, Labeling, Staining, Quantitation Assay, SDS Page, Western Blot

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Model depicting the participation of chaperones in the regulation of cyclin D1/CDK4 complex maturation (details in text).

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques:

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 3. Site 1 binds two distinct proteins and forms two DNA-protein complexes. A, sequences of the oligonucleotides used in Fig. 3 are shown. Only one strand is shown for each oligonucleotide. The numbering system uses translation start site as 11. B, an oligonucleotide spanning 268 to 239 of the Factor X gene (FXSite1) was labeled and incubated with 10 mg of nuclear extract (N.E.) from cell lines and rat tissues as indicated. Two DNA-protein complexes are designated complex A and complex B. The position of the free probe is marked as F. C, an oligonucleotide spanning site 1 was incubated with 12 mg of nuclear extract (N.E.) from human liver (lanes 1–12), 2 ml of in vitro translated HNF-4 in rabbit reticulocyte lysate (lane 13), or 2 ml of unprogrammed rabbit reticulocyte lysate (lane 14). Lanes 2–5 contain unlabeled site 1 oligonucleotide as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lanes 7–10 contain unlabeled APF-1 (a strong HNF-4 binding site derived from 287 to 266 of apolipoprotein C III gene) as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lane 12 contains 1 ml of HNF-4 antiserum. The positions of the antibody-HNF-4-DNA complexes are indicated by SS. D, probes containing the ACTTTG common motif from Factors VII, IX, X, and APF-1 were incubated with 12 mg of human liver nuclear extracts (N.E.) (lanes 1–8). One ml of HNF-4 antiserum was added in lanes 5–8. Positions of the immune complexes (supershift) are marked as SS. Two DNA-protein complexes specific to the APF-1 probe are marked by asterisks. E, the Factor X site 1 probe was incubated with 15 mg of nuclear extract (N.E.) from either HepG2 (lanes 1–3) or HeLa cells (lanes 4–6). In lanes 2 and 5, 200 3molar excess of unlabeled Sp1 consensus oligonucleotide was added. In lanes 3 and 6, 1 ml of Sp1 antibody was added. The position of a minor complex is denoted by an asterisk.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Labeling, Incubation, In Vitro, Binding Assay, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 4. Methylation interference analysis of the contact points of HNF-4 and Sp1 to site 1. A, On the left, Factor X site 1 was labeled on one strand (sense strand or antisense strand) and partially meth- ylated. The methylated probe was incubated with 100 mg of human liver nuclear extract and analyzed in regular electrophoretic mobility shift assay. The free probe fraction (F) and the complex B fraction (B, which contains HNF-4) were excised from the gel, cleaved with piperidine to reveal the G/A ladder, and resolved on a denaturing gel. On the right, the site 1 oligonucleotide was incubated with 10 footprint units of recombinant Sp1 and analyzed as described above. The free fraction (F) and bound fraction (B) were analyzed. The contact points revealed are marked as G. E indicates methylated positions that partially interfere with the binding. * indicates that methylation at these nucleotides enhances the protein binding. B, summary of results of methylation interference analysis at site 1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Methylation, Labeling, Incubation, Electrophoretic Mobility Shift Assay, Recombinant, Binding Assay, Protein Binding

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 5. Increasing amounts of Sp1 diminish the binding of HNF-4 to site 1. Labeled Factor X site 1 oligonucleotide was incubated with 12 mg of human liver nuclear extracts (N.E.) in the presence of 0, 0.2, 1, and 2 footprint units (lanes 1–4) of recombinant Sp1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Labeling, Incubation, Recombinant

Journal: Hypertension

Article Title: Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

doi: 10.1161/hypertensionaha.107.103275

Figure Lengend Snippet: Figure 1. Nox isoforms and NADPH oxidase subunits cofractionate in LRs in hRPT cells. A, Sucrose gradient analysis of LRs. The protein concentrations (mg/mL) from vehicle-treated cells are shown (top). Fractionated proteins (30 L per lane) were immunoblotted with monoclo- nal anti–caveolin-1 (Cav-1) and anti–flotillin-1 (Flo-1) antibodies (middle) and polyclonal anti-human D1 (hD1R) and anti-human D5 (hD5R) dopa- mine receptor antibodies (bottom). Low-density fractions (fractions 2 to 6) contain LRs, whereas the high-density fractions (fractions 7 to 12) contain non-LRs. The blots are 1 of 3 separate experiments. B through D, Distribution of Nox isoforms and oxidase subunits in LRs and non- LRs. Proteins from sucrose gradients, treated with vehicle (Con), fenoldopam (Fen, 1 mol/L/20 minutes), or CD (2%/1 hour) were probed with specific antibodies against Nox2, Nox4, p22phox, Rac1, and p67phox. The distributions of Nox2, Nox4, and p22phox are shown in B, and p67phox and Rac1 in C. The quantities of Nox2, Nox4, p22phox, and Rac1 in LRs are shown in D. Values are meansSEMs. n3 to 4, t test; *P0.01; Fen vs Con. E, Colocalization of LRs with Nox2 and p22phox in hRPT cells using double immunofluorescence labeling. The merged (yellow) confocal images show colocalization of immunoreactive LRs (green) and Nox2 or p22phox (red) in plasma membranes and in intracel- lular vesicles. The image is 1 of 3 to 5 separate experiments. Scale bar20 m.

Article Snippet: The sources of reagents were: monoclonal antibodies to human NADPH oxidase subunits (Nox2, p22phox, p67phox) (4); anti-Rac1, siRNA for Nox2 and Nox4 and control siRNA (Santa Cruz Biotechnology, Inc., CA); an affinity purified polyclonal anti-Nox4 antibody (5), polyclonal anti-D5R and anti-D1R antibodies generated from our laboratory, specificities of which have been reported (6-8); anti-caveolin-1 and anti-flotillin-1 (BD Biosciences-Transduction Labs., Lexington, KY); secondary antibodies for blotting and immunostaining (Jackson ImmunoReseach Labs. West Grove, PA); ECL western blotting detection reagents (Millipore Corporation, Billerca, MA); BCA protein assay reagent (PIERCE, Rockford, IL); protease inhibitor Cocktail set III, fenoldopam, SCH23390, 2-N-morpholino ethanesulfonic acid (Mes), methyl-β-cyclodextrin (βCD), triethanolamine, lucigenin, NADPH, and cholesterol (SIGMA- Aldrich, St. Louis, MO); cell culture reagents (Life Technologies -GIBCO/BRL, Rockville, MD); Amplex Red cholesterol assay kit and Vybrant LR labeling kit (Molecular Probes Inc., Eugene, OR 97402).

Techniques: Labeling, Clinical Proteomics

Journal: Hypertension

Article Title: Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

doi: 10.1161/hypertensionaha.107.103275

Figure Lengend Snippet: Figure 6. Effect of knockdown of Nox2 or Nox4 on the CD- induced increase in ROS production in hRPT cells. The cells were transfected with siRNA for Nox2 or Nox4 and assayed for ROS activity. Dynamic tracings of ROS activities (top) and the percentage changes in activity from control (bottom) are shown. Values are meansSEMs. n4 to 6, ANOVA (Newman-Keuls), *P0.001, #P0.01, CD vs Con, siRNA alone, or siRNACD.

Article Snippet: The sources of reagents were: monoclonal antibodies to human NADPH oxidase subunits (Nox2, p22phox, p67phox) (4); anti-Rac1, siRNA for Nox2 and Nox4 and control siRNA (Santa Cruz Biotechnology, Inc., CA); an affinity purified polyclonal anti-Nox4 antibody (5), polyclonal anti-D5R and anti-D1R antibodies generated from our laboratory, specificities of which have been reported (6-8); anti-caveolin-1 and anti-flotillin-1 (BD Biosciences-Transduction Labs., Lexington, KY); secondary antibodies for blotting and immunostaining (Jackson ImmunoReseach Labs. West Grove, PA); ECL western blotting detection reagents (Millipore Corporation, Billerca, MA); BCA protein assay reagent (PIERCE, Rockford, IL); protease inhibitor Cocktail set III, fenoldopam, SCH23390, 2-N-morpholino ethanesulfonic acid (Mes), methyl-β-cyclodextrin (βCD), triethanolamine, lucigenin, NADPH, and cholesterol (SIGMA- Aldrich, St. Louis, MO); cell culture reagents (Life Technologies -GIBCO/BRL, Rockville, MD); Amplex Red cholesterol assay kit and Vybrant LR labeling kit (Molecular Probes Inc., Eugene, OR 97402).

Techniques: Knockdown, Transfection, Activity Assay, Control

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 0, 4, 6, 8, or 18 hrs. Cells were stained with UBXN1 and ubiquitin and imaged (B) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 0, 8, or 18 hrs. Cells were stained with ubiquitin and perinuclear aggregate size was quantified using AggreCount. The mean area of the largest perinuclear aggregate for each time point (2μm 2 or 4μm 2 respectively) was used for all subsequent analysis to quantify perinuclear aggresomes. (C) Levels of p97 depletion in doxycycline-inducible shRNA Hela Flp-in T-Rex cell lines. Cells were treated with doxycycline for 72 hours. (D) sh-p97 cell lines were treated with 1 μM Btz for 18hrs and released into drug-free media for a further 24 hours in the presence or absence of p97 depletion. Cells were stained with ubiquitin and Hoechst. The number of cells containing ubiquitin-positive aggresomes was quantified using AggreCount. (E) Quantification of data in (D). The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show mean +/- standard deviation. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, shRNA, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 18 hrs. Cells were stained for p97, ubiquitin (FK2), and nuclei (Hoechst dye). (B) Hela Flp-in T-Rex cells were treated with 1 μM Btz, 2 μM CB-5083, or both for 8 hrs. Cells were released into drug-free media or media containing 1 μM CB-5083 for 24 hours. Cell lysates were probed for ubiquitin. (C) Cells were treated as in (B) and imaged for ubiquitin-positive aggregates and aggresomes. (D) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) were quantified using AggreCount for images in (C). (E) Perinuclear aggresomes (minimum size cutoff 2 μm 2 ) were quantified using AggreCount for the images in (C). (F) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) in the release samples were quantified using AggreCount for images in (C). (G) Perinuclear aggresomes in release samples were quantified using AggreCount for the images in (C). The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 18 hrs and cells were stained for UBXN1 and ubiquitin. Co-localization was determined by the Mander’s overlap coefficient for 25 cells in 3 replicate experiments. (B) NPL4, UFD1 and p47 localization to aggresomes labeled with ubiquitin. Co-localization was determined by the Mander’s overlap coefficient for 25 cells in 3 replicate experiments. (C) GFP UBXN1 co-localizes with aggresome markers: Proteostat, HDAC6, and 20S proteasomes in Btz treated cells. (D) Microtubules are required for GFP-UBXN1 localization to aggresomes. Nocodazole co-incubation in Btz treated cells prevents aggresome formation. Lower panel: The number of aggresomes was quantified. (E) Hela Flp-in T-Rex cells were treated with 0.1 mM sodium arsenite for 2 h. Cells were stained for stress granule marker G3BP1 and p97 adaptors (UBXN1 or p47, used here as a positive control. (F) Stable mCherry-Dcp1a cells (labeling P bodies) were stained with UBXN1. The indicated number of cells was analyzed from the three independent biological replicates. Graphs show the mean +/- s.e.m. ***: p<=0.001 as determined by unpaired Students t-test. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, Labeling, Incubation, Marker, Positive Control

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) HeLa Flp-in TRex cells were treated with 1 μM Btz for 18hrs. Cells were stained for p97 adaptors (UBXD1, UBXD2, FAF1, UBXD8, and ASPSCR1) and ubiquitin. Note that UBXD2 and UBXD8 are known to be ER-tethered adaptors and localize to both ER tubules at the cell periphery and ER sheets near the nuclear envelope. They do not co-localize with aggresomes and can be seen to occupy a greater cellular area (corresponding to ER sheets) than the aggresome. (B) Neuroblastoma cells SH-SY5Y were treated with 1 μM Btz for 18 hrs. Cells were stained for UBXN1 and ubiquitin. (C) Multiple Myeloma cell line MM1.S was treated with 1 μM Btz for 18 hrs. Cells were stained for UBXN1 and ubiquitin. (D) Hela Flp-in T-Rex cells were treated with 5 μg/ml of puromycin for 4 hrs. Cells were stained for UBXN1 and ubiquitin. ALIS: Aggresome-like induced structures. (E) Aggresomes formed by Btz treatment contain primarily newly synthesized ubiquitylated proteins. Co-treatment with the translational inhibitor cycloheximide results in the loss of aggresomes in Btz treated cells. Cells were stained for ubiquitin and nuclei (Hoechst dye). (F) Immunoblot of global ubiquitin conjugate levels corresponding to samples in (E). Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, Synthesized, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Immunoblot showing loss of UBXN1 in CRISPR-Cas9 generated knockout cells and re-expression of wildtype GFP-UBXN1 by doxycycline induction. (B) Wildtype (WT) and UBXN1 knock-out (KO) HeLa Flp-in TRex cell lines were treated with 1 μM Btz for 18 hrs. Cells were stained for ubiquitin. (C) Cell lysates from wildtype and KO celss treated with 1 μM Btz for 18 hrs were probed for ubiquitin. (D) GFP-UBXN1 expression in UBXN1 KO cells was induced by doxycycline. Cells were treated with Btz and stained for ubiquitin. Expression of GFP-UBXN1 reinstated aggresome formation in UBXN1 KO cells. (E) Quantification of data in panels (B and D). (F) WT and UBXN1 KO lines were treated with Btz for the indicated times and stained for ubiquitin. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05, as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Western Blot, CRISPR, Generated, Knock-Out, Expressing, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Parental or UBXN1 knock-out (KO) clone 6239 in HeLa Flp-in TRex cells were treated with 1 μM Btz for 18 hrs. Cells were stained for endogenous ubiquitin and nuclei. Bottom Panel: immunoblot showing loss of UBXN1 in CRISPR-Cas9 generated knockout clonal cell lines (Clone 9 is 6239 in panel A). (B) Quantification of aggresome formation upon transient depletion of UBXN1 with two separate siRNAs. Graphs show mean +/- standard deviation. Bottom Panel: immunoblot of transient depletion of UBXN1. The indicated number of cells was analyzed from the three independent biological replicates. *: p<=0.1, **: p<=0.05, as determined by unpaired students t-test. Scale bar: 10μm

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Knock-Out, Staining, Ubiquitin Proteomics, Western Blot, CRISPR, Generated, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Immunoblot validation of CRISPR-Cas9 gene-edited knockout (p47) or knockdown (NPL4) cell lines. (B) Aggresome formation in CRISPR-Cas9 gene-edited knockout cell lines for the indicated UBXD adaptors. (C) Quantification of data in (B). (D) HEK293T cells were transfected with the indicated FLAG or Myc-tagged constructs. NPL4 was affinity purified using FLAG magnetic beads and associated proteins were eluted with FLAG peptide. Endogenous p97 in the eluate was immunopurified and probed for UBXN1, NPL4, and ubiquitin. UBXN1 associates with the same p97 hexamer associated with NPL4. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Western Blot, Biomarker Discovery, CRISPR, Knock-Out, Knockdown, Transfection, Construct, Affinity Purification, Magnetic Beads, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Aggresome formation in Hela Flp-in T-Rex cells upon transient depletion of the indicated p97 adaptors. Cells were stained with anti-ubiquitin. (B) Levels of p97 adaptor depletion in Hela Flp-in TRex cell lines. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Domain organization of UBXN1 showing N-terminal ubiquitin associated domain (UBA), coiled coil domain (cc) and C-terminal ubiquitin X domain (UBX). (B) Expression of GFP-UBXN1, GFP-UBXN1-UBA mut and GFP-UBXN1-UBX mut in the UBXN1 KO cell line by the addition of doxycycline for 72 hrs. (C) UBXN1 KO cells expressing GFP-UBXN1 wildtype, GFP-UBXN1-UBA mut or GFP-UBXN1-UBX mut were treated with Btz for 18 hours and stained for ubiquitin. Re-expression of wildtype UBXN1 but not the UBX mut rescued aggresome formation. The UBA mut has smaller aggresomes but did not reach significance (D) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) were quantified for images in (C). (E) Perinuclear aggresomes were quantified for the images in (C). (F) Wildtype, UBXN1 KO (and indicated rescue lines), NPL4, and p97 depletion cell lines were plated in triplicate into 96-well plates and treated with 1 μM bortezomib for 18 hrs. Cell viability was measured and normalized to the value of untreated controls for each cell line. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Ubiquitin Proteomics, Expressing, Staining

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A and B) HEK-293T cells were transfected with the indicated Myc-tagged UBXN1 constructs. Cells were treated with Btz (A) and Myc affinity purifications were probed for endogenous ubiquitin (A) or p97 (B). (C) Cumulative frequency distribution of largest perinuclear aggregate areas in UBXN1 KO cells expressing GFP-UBXN1 wildtype, UBA, or UBX mutants. The left-ward shift of the curve indicates decrease in perinuclear aggregate area in the mutants (D) Wildtype, UBXN1 KO, or NPL4 KD cells were treated with Btz for 18 hrs and released into drug-free media to induce clearance of aggregates. The loss of UBXN1 or NPL4 did not impact aggregate clearance. (E) p97 interacts with HDAC6. HEK-293T cells were transfected with the indicated cDNAs and treated with Btz for 18 hrs, Myc affinity purifications were performed and probed for the indicated tagged proteins. (F) The depletion of HDAC6 inhibits aggresome formation and clearance in HeLa Flp-in TRex cells treated with Btz for 18 hrs. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Transfection, Construct, Ubiquitin Proteomics, Expressing

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in TRex cells were treated with the indicated concentration of the ubiquitin E1 inhibitor TAK273 or Btz for 8 hours. Cell lysates were probed for endogenous ubiquitin. (B) U2OS cells were treated with doxycycline to induce the expression of HTT Q91-mCherry. Cells were treated with 1 μM TAK273 for 8 hours, fixed and stained for ubiquitin. Inclusion bodies still form but are devoid of ubiquitin staining. (C) Quantification of inclusion bodies observed by imaging in cells treated with 1 μM TAK274, 1 μM Btz, and or 10 μM CB-5083 for 8 hours. (D) Depletion of UBXN1 leads to an increase in HTT Q91-mCherry inclusion bodies. Quantification is provided in . (E) PuLSA analysis of HTT Q91-mCherry aggregates in UBXN1 and p97 depleted cells. The gates show the distribution of polyQ aggregates of various sizes based on pulse height. The greater the pulse height, the larger the inclusion body and vice versa. Note that this population represents only the HTT aggregates and not the complete mCherry signal. Total mCherry signal is represented in . The indicated number of cells was quantified in three biological replicates shown by the black dot. Graphs show the mean and standard deviation. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Concentration Assay, Ubiquitin Proteomics, Expressing, Staining, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) U2OS cells were treated with doxycycline to induce the expression of HTT Q91-mCherry. Cells were fixed and stained for ubiquitin and UBXN1 to demonstrate co-localization with HTT Q91-mCherry. (B) UBXN1 was transiently depleted with siRNAs in U2OS HTT Q91-mCherry and imaged for inclusion body formation. The number of HTT Q91-mCherry inclusion bodies was quantified. (C) PuLSA analysis of HTT Q91-mCherry aggregates in UBXN1 and p97 depleted cells. (D and E) Representative fluorescent images of L4 larvae stage wild type, cdc-48.1(tm544) (D), ubxn-1(tm2759) and ubxn-4(ok3343) (E) loss-of-function mutants expressing polyQ40::YFP in body wall muscle. Images were taken of worms precisely age-matched at the L4.4 vulva developmental stage. Bottom panels in each show quantification of visible fluorescent aggregates in L4 larvae animals expressing polyQ40::YFP in either wildtype, cdc-48.1, ubxn-1 and ubxn-4 mutant animals. Quantification was only performed on worms at the L4.4 vulva development stage based on vulva morphology. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show mean +/- s.e.m. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Tukey (B) or Dunnett’s Test (D and E). Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Expressing, Staining, Ubiquitin Proteomics, Mutagenesis