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: International Journal of Biological Sciences

Article Title: TET1s deficiency exacerbates oscillatory shear flow-induced atherosclerosis

doi: 10.7150/ijbs.69281

Figure Lengend Snippet: CX40 mediates TET1s-induced endothelial barrier reinforcement. (A) Heatmap of the top 20 selected upregulated genes by RNA sequencing. (B) RT-qPCR was used to test the mRNA levels of the top 5 upregulated genes from RNA-seq and three hemodynamic-sensitive genes. (C) The CX40 protein expression level was quantified by WB (n=6 per group). (D-L) Stable CX40 -/- p-HUVECs were generated by transfecting human connexin 40-specific CRISPR/Cas9 KO plasmids. Then, TET1s-adenovirus was used to transfect CX40 -/- and CX40 +/+ p-HUVECs to generate CX40 +/+ +NC, CX40 +/+ +OE, CX40 -/- +NC and CX40 -/- +OE p-HUVECs. (D) The fluorescence intensity of the lower chamber medium was tested as described in Fig. C (n>6 per group). (E, H) Immunofluorescence staining for F-actin and VE-cadherin. The green dotted line indicates the intercellular space area. (F-G) Quantitative analysis of single-cell F-actin length and intercellular space area to image E (n>10 per group). (I-K) Quantitative analysis of VE-cadherin discontinuity, intercellular space area and ratio of VE-cadherin in several morphological categories to image H (n>10 per group). All data were presented as the mean ± SD.

Article Snippet: P-HUVECs were transfected at 60-70% confluence with connexin 40 (CX40) CRISPR/Cas9 KO plasmids (h) (sc-401031, Santa Cruz Biotechnology) and CX40 HDR (sc-401031-HDR, Santa Cruz Biotechnology) using UltraCruz® Transfection Reagent (sc-395739, Santa Cruz Biotechnology) according to the manufacturer's protocol.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Generated, CRISPR, Fluorescence, Immunofluorescence, Staining, Quantitative Single Cell

Journal: International Journal of Biological Sciences

Article Title: TET1s deficiency exacerbates oscillatory shear flow-induced atherosclerosis

doi: 10.7150/ijbs.69281

Figure Lengend Snippet: TET1s increases CX40 expression by inhibiting histone deacetylation on the promoter of CX40. (A-B, D-E) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus and further tested after 48 h. (A) The global protein levels of ac-H3K27 and H3K27 in p-HUVECs were tested by Western blot (n=6 per group). (B) Sin3a interaction with TET1s and TET1-FL was analyzed by Co-IP (n=3 per group). (C) Schematic of human CX40 promoter and CHIP-qPCR products. TS indicates transcriptional start; P1-P5 indicates primer 1-primer 5; F indicates forward primer, R indicates reversed primer. (D-E) ChIP-qPCR was used to test Sin3a and ac-H3K27 enrichment in the CX40 promoter (-550 bp to +43 bp) (n=4 per group). (F-G) p-HUVECs were transfected with TET1s-overexpressing adenovirus and negative control adenovirus for 48 h and added HATI2 to media. (F) ChIP-qPCR was used to test ac-H3K27 enrichment in the CX40 promoter. (G) The CX40 mRNA levels were tested by RT-qPCR (n=4 per group). All data were shown as the mean ± SD.

Article Snippet: P-HUVECs were transfected at 60-70% confluence with connexin 40 (CX40) CRISPR/Cas9 KO plasmids (h) (sc-401031, Santa Cruz Biotechnology) and CX40 HDR (sc-401031-HDR, Santa Cruz Biotechnology) using UltraCruz® Transfection Reagent (sc-395739, Santa Cruz Biotechnology) according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Negative Control, Western Blot, Co-Immunoprecipitation Assay, ChIP-qPCR, Quantitative RT-PCR

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Heatmaps show how varying the cancer cell proteolysis value (x axis) impacts on different metrics in the absence of fibroblasts. WT indicates the ‘wild-type’ value based on experimental parameterisation using A431 cancer cells. ( b ) Heatmaps show the differential values resulting from the inclusion of fibroblasts (effectively a comparison of and Figure 3—figure supplement 1a). Red indicates an increase when fibroblasts are present, dark blue a reduction when in the presence of fibroblasts. ( c ) Images show simulation output initiated with a spheroid, no fibroblasts, a uniform chemotactic cue, and varying cancer cell proteolysis. Left panel – day 7output in the absence of permissive track, right panel – day 5 output in the presence of permissive track. ( d ) Heatmaps show how varying the distribution of extracellular matrix (ECM) density in organotypic simulations impacts on different metrics when fibroblasts are included in all simulations. Parametrisation and colourmap as in ( a ). ‘Aligned’ refers to alternating tracks of high and low ECM density parallel to direction of invasion. ‘Chessboard’ refers to three-dimensional (3D) chessboard distribution of high and low ECM density values. ( e ) Heatmaps show how varying the cancer cell proteolysis value (x axis) impacts on different metrics when cancer-cell proliferation rate is halved, and fibroblasts are included in all simulations. Parametrisation and colourmap as in ( a ). ( f ) Western blots of MMP14, alpha-catenin, vimentin, fibronectin, and β-actin in A431 cells engineered using Crispr/Cas9 to delete MMP14 or CTNNA1, or to over-express MMP14. ( g ) Images show F-actin (magenta) and degraded collagen I represented by fluorescence of DQ collagen I (green) in 3D culture of A431 cells genetically engineered as indicated. ( h ) Plot shows the quantification of strand width in spheroid invasion assay of A431 WT or MMP14 over-expressing cells, which are pre-treated with mitomycin C. Unpaired t-test was performed. Error bars indicate 95% confidence intervals, one dot represents one strand. For comparison, light blue lines show the same metrics in the absence of mitomycin C (data from ). Figure 3—figure supplement 1—source data 1. Quantification of invading strand width in A431 WT and MMP14 OE cells pretreated with mitomycin C. Figure 3—figure supplement 1—source data 2. Uncropped western blot images of WT, MMP14 KO, MMP14 OE, CTNNA1 KO, MMP14 KO/CTNNA1 KO, and MMP14 OE/CTNNA1 KO A431 lysates stained for MMP14, alpha-catenin, vimentin, fibronectin, or β-actin.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Comparison, Western Blot, CRISPR, Fluorescence, Invasion Assay, Expressing, Staining

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Principal component analysis plots show the metrics derived from over 2000 simulations in the presence of fibroblasts covering variation in cancer cell–cancer cell adhesion with values indicated by the intensity of magenta, cancer cell proteolysis (not colour coded), and cancer cell–matrix adhesion (not colour coded). ( b ) Heatmaps show how varying the cancer cell–cancer cell adhesion value (x axis) impacts on different metrics when fibroblasts are included in all simulations. WT indicates the ‘wild-type’ value based on experimental parameterisation using A431 cancer cells. Yellow indicates a high value, dark blue a low value. ( c ) Images show the effect of modulating cancer cell-cell adhesion via Crispr KO of CTNNA1 in cancer cells (green) in both organotypic and spheroid assays including fibroblasts (magenta). Scale bar = 100 μm. ( d ) Quantification of three biological replicates of the experiment shown in panel (c) with strand length, strand width, and tapering shown – 1 unit is equivalent to 0.52 μm. Unpaired t-test was performed. Error bars indicate 95% confidence intervals, one dot represents one strand. ( e ) Plots show the track invasion score with varying cancer cell–cancer cell adhesion in simulations lacking fibroblasts but with a single permissive track favouring invasion. Cartoons indicate the initial set up of cell positions and the directional cue in the simulation. Figure 5—source data 1. Quantification of invading strand length, width, and tapering in A431 cells with/without CTNNA1 manipulation.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Derivative Assay, CRISPR

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Images show the β-catenin (magenta), F-actin (orange), DNA (blue), and active myosin (pS19-MLC - green) networks in control A431 and CTNNA1 KO A431 cells.( b ) Images β-catenin (magenta), F-actin (orange), DNA (blue), and active myosin (pS19-MLC - green) networks in control A431- and 10-μM Y27632-treated cells. Scale bar = 20 μm. ( c ) Images show β-catenin (magenta), F-actin (orange), DNA (blue), and active myosin (pS19-MLC - green) networks in control A431 ROCK:ER- and 4-OHT-treated cells. Scale bar = 20 μm. ( d ) Images show organotypic killing assays using control or MMP14 over-expressing A431 cells in the presence or absence of 10 μM Y27632. Scale bar = 100 μm. Plot shows the quantification of strand width from three biological replicates – 1 unit is equivalent to 0.52 μm. One-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals, one dot represents one strand. ( e ) Images show organotypic invasion assays using MMP14 over-expressing A431 cells additionally engineered to contain ROCK:ER in the presence or absence of 4-OHT. Scale bar = 100 μm. Plot shows the quantification of strand width from three biological replicates. Unpaired t-test was performed. Error bars indicate 95% confidence intervals, one dot represents one strand. Figure 6—source data 1. Quantification of invading strand width in A431 cells with/without manipulation of actomyosin contractility.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Control, Expressing

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Plots show the quantifications of relative intensity of pMLC in A431 WT, CTNNA1 KO, A431 WT cells treated with Y27632 and ROCK:ER expressing A431 ± 4(O)HT at the edge or cell-cell junction of the cells. Mean, quartiles, and extremes are shown, data from 3 independent experiments. ( b ) Images show the F-actin (magenta) and myosin (MYH9/MHCIIa - green) networks in control A431- and 10-μM Y27632-treated cells. Scale bar = 20 μm. ( c ) Images show the F-actin (magenta) and myosin (MYH9/MHCIIa - green) networks in control A431 ROCK:ER with/without 4-OHT treatment. Scale bar = 20 μm. ( d ) Images show the F-actin (magenta), DNA (DAPI; blue), and MYH9/MHCIIA (green) staining in human squamous cell carcinoma tissue. ‘t’ indicates tumour clusters, arrows point to supra-cellular actomyosin network, scale bar is 50 microns. Figure 6—figure supplement 1—source data 1. Quantification of pMLC intensity in A431 WT, CTNNA1 KO, and cells with actomyosin manipulation.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Expressing, Control, Staining

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Heatmaps show how varying the matrix proteolysis (x-axis) and cancer cell–cancer cell adhesion value (y axis) impacts on different metrics when fibroblasts are included in all simulations. WT indicates the ‘wild-type’ value based on experimental parameterisation using A431 cancer cells. Yellow indicates a high value, dark blue a low value. ( b ) Images show the effect of combinatorial modulation of matrix proteolysis and cancer cell-cell adhesion via Crispr KO of CTNNA1 and/or MMP14 and/or MMP14 over-expression in cancer cells (green) in both organotypic assays including fibroblasts (magenta). Scale bar = 100 μm. ( c ) Quantification of three biological replicates of the experiment shown in panel (b) with strand length and strand width shown – 1 unit is equivalent to 0.52 μm. One-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence interval, one dot represents one strand. ( d ) Images show the effect of combinatorial modulation of matrix proteolysis and cancer cell-cell adhesion via Crispr KO of CTNNA1 and/or MMP14 and/or MMP14 over-expression in cancer cells (green) in both spheroid assays including fibroblasts (magenta). ( e ) Quantification of three biological replicates of the experiment shown in panel (d) with strand length and strand width shown. Scale bar = 100 μm. One-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence interval, one dot represents one strand. Figure 7—source data 1. Quantification of invading strand width and length in A431 cells with/without manipulation of MMP14 and/or CTNNA1.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: CRISPR, Over Expression

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Images show EdU-labeled proliferating cells (green) and DNA (blue) in spheroid invasion assay with A431 WT, MMP14 KO, MMP14 OE, or CTNNA1 KO (magenta). ( b ) Plot shows the quantification of EdU-labeled cells shown in (a). One-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals, n=3 biological replicates. ( c ) Plot shows quantification of growth of A431 cells with the indicated manipulations of MMP14 and CTNNA1 in two-dimensional cell culture. Two-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals, n=3 biological replicates. ( b ) Phase contrast images show the growth of A431 ROCK:ER cancer cell colonies in the presence or absence of 4-OHT. Scale bar = 50 μm. ( c ) Plot shows quantification of the growth assay shown in (b). Data from three biological replicates. Two-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals, n=3 biological replicates. Figure 8—figure supplement 1—source data 1. Quantification of proliferation of WT, MMP14, CTNNA1, and/or ROCKER manipulated A431 in 2D and 3D culture.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Labeling, Invasion Assay, Cell Culture, Growth Assay

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) Heatmaps show how varying the matrix proteolysis (left) or cancer cell–cancer cell adhesion value (right) impacts on predicted cell growth in the presence or absence of fibroblasts. WT indicates the ‘wild-type’ value based on experimental parameterisation using A431 cancer cells. Yellow indicates a high value, dark blue a low value. ( b ) Phase contrast images show the growth of cancer cell colonies with the indicated manipulations of MMP14 and CTNNA1 after 8 days surrounded by matrix. Scale bar = 50 μm. ( c ) Plot shows quantification of the growth assay shown in (b). Two-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals. Data from three biological replicates. ( d ) Fluorescent image shows reflectance of collagen fibre (cyan) and cell membrane of A431 WT cells in three-dimensional (3D) culture. ( e ) Fluorescent image shows reflectance of collagen fibres around A431 WT cells in 3D culture at two time points. t=0 min: magenta, t=100 min: green. ( f ) Fluorescent images show reflectance of collagen fibres (cyan) and cell membrane of A431 WT, CRNNA1 KO, or MMP14 over expressing cells (red) in 3D culture. White arrows highlight the formation and motion of collagen bundles adjacent to the cell clusters, yellow arrows highlight gaps. Figure 8—source data 1. Quantification of cancer cell proliferation in 3D culture.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Growth Assay, Membrane, Expressing

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet: ( a ) H&E images are shown on tumours growing in the ears of mice with the indicated manipulations of MMP14 and CTNNA1. Scale bar = 50 μm. ( b ) Plot shows quantification of A431 tumour growth with the indicated manipulations of MMP14 and CTNNA1. ( c ) Table shows quantification of mice with primary tumours and mice with lymph node metastases when injected with A431 cells with the indicated manipulations of MMP14 and CTNNA1. The total number of mice for each condition also applies to the data plotted in (b). Two-way ANOVA with post-hoc multiple comparisons was performed. Error bars indicate 95% confidence intervals. Figure 9—source data 1. Tumour size and number of metastasis in WT and MMP14 and/or CTNNA1 manipulated tumour-bearing mice.

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Injection

Journal: eLife

Article Title: Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

doi: 10.7554/eLife.76520

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo-sapiens ) , px458 CTNNA1 gRNA , Santa Cruz , sc-419475 , .

Techniques: Transfection, Construct, Sequencing, Control, Generated, Membrane, Imaging