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: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 3. Enhancer activity compacts SOX9 promoter-enhancer hub in individual TNBC cells. (A and B) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (A) and SOX9.EC2, SOX9. EC3, or both (B) in MB157 (n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9.EC3 (magenta), and SOX9.EC1 (A, red) or SOX9.EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). (C and F) SOX9 enhancers inactiva- tion expands SOX9-EC1-EC3 and SOX9-EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9-EC1-EC3 (C) and SOX9-EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/SOX9.EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/SOX9.EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/SOX9.EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/SOX9.EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/SOX9.EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/SOX9.EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). (D and G) Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (D) and SOX9.EC1, SOX9. EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA (n = alleles). (E and H) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. (I) SOX9 promoter inactivation decreases SOX9-EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percent- ages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA (SOX9.P sgRNA) (n = alleles). Bottom-left: CDFs of SOX9-EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/SOX9.P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Activity Assay, Expressing, Control

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 6. SOX9 regulates oncogene MYC by positioning its enhancers. (A) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter- enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC.EC1 (magenta), MYC.EC2 (red), MYC.EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. (B) A total of 80% of differ- entially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. (C to E) SOX9 loss significantly in- creases 3D distances between the MYC promoter and SOX9-bound MYC.EC2 (C) or MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC.EC2 (C) and MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC.EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC.EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC.EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Represen- tative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 7. SOX9 loss decompacts MYC promoter-enhancer hubs. (A) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER+ MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC.EC1, SOX9-bound MYC.EC3, or both MYC.EC1 and MYC.EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in Fig. 6A top genome track (n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. (B and C) SOX9 loss expands MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC-EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC-EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm (n = cells). Middle: Allele per- centages with MYC promoter interacting (<350 nm) with MYC.EC1, MYC.EC2, or both MYC.EC1 and MYC.EC2 (B) and MYC.EC1, MYC.EC3, or both MYC.EC1 and MYC.EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Expressing

Journal: Experimental Biology and Medicine

Article Title: Nuclear factor E2-related factor 2 knockdown enhances glucose uptake and alters glucose metabolism in AML12 hepatocytes

doi: 10.1177/1535370217694435

Figure Lengend Snippet: Antibodies used for Western blot analysis

Article Snippet: The ratios of the mean values of protein level in AML12 cells between two selected groups are listed in Supplementary Table 2. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Company Reference Dilution Nrf2 Santa Cruz Sc-722 1:1000 HO-1 Abcam ab52947 1:1000 NQO1 Proteintech 11451-1-AP 1:1000 p-EIF2α S51 Millipore 04-342 1:1000 EIF2α Proteintech 11233-1-AP 1:1000 IL-1β Ruiying Biological RLT2322 1:1000 TNF-α Ruiying Biological RLM3477 1:1000 p-NF-κB p65 S276 Ruiying Biological RLP0187 1:1000 MMP2 Ruiying Biological RLT2798 1:1000 MMP9 Ruiying Biological RLT1892 1:1000 FGF21 Abcam ab171941 1:1000 AMPKα Ruiying Biological RLT0215 1:1000 Sirt1 Cell signaling Q96E86 1:1000 PGC-1α Abcam ab54481 1:1000 UCP1 Abcam ab23841 1:1000 Glut-4 Ruiying Biological RLT1930 1:1000 IGF-1R Ruiying Biological RLT2282 1:1000 FOXO1 Ruiying Biological RLT1757 1:1000 p-AKT S473 Ruiying Biological RLP0006 1:1000 AKT Ruiying Biological RLT0178 1:1000 GSK3α/β Proteintech 22104-1-AP 1:500 p-GSK3α/β Y279/Y216 Signalway 11002 1:500 Gapdh Santa Cruz Sc420485 1:1000 Open in a separate window Antibodies used for Western blot analysis Statistical analysis The data are presented as the mean ± SEM for the number of replicates indicated.

Techniques: Western Blot