Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Construction of Non-viral Homology-Directed System for Correcting Mutated Canine Coagulation Factor IX (A) Schematic outline of the cFIX target locus. The top panel shows the cFIX gene, including the UTRs (black bars), introns (bright gray bars), and exons (light gray bars). Nucleotide and amino acid sequences of wild-type cFIX (WT cFIX) and a major disease causing cFIX mutation (G1477A, mutated cFIX) are shown (white letters in dark square). The bottom panel shows the mutated donor sequence (cFIXmod) used in this study (mutations are marked with an asterisk), which results in the identical amino acid sequence as WT cFIX. The gray horizontal bar schematically shows the guide RNA (gRNA)-binding site used in this study (gRNA-CRISPR/Cas9). Differences in the donor and mutated cFIX sequences are marked with stars. (B) Schematic outline of the DNA sequences contained in the Tet-on-inducible CRISPR/Cas9 for cutting the cFIX-mutated strand and the donor DNA that was transfected as PCR product (cFIXmod). NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activation. (C) CRISPR/Cas9 nuclease activity measured by T7E1 assay after transfection with the CRISPR/Cas9-encoding plasmid and the donor DNA. MW, molecular-weight size marker; NC, negative control referring to the mixture of untreated Huh7-cFIXmut, PLC/PRF/5-cFIXmut, and Hep3B-cFIXmut cells; 1, CRISPR/Cas9-treated Huh7-cFIXmut cells; 2, CRISPR/Cas9-treated PLC/PRF/5-cFIXmut cells; 3, CRISPR/Cas9-treated Hep3B-cFIXmut cells. (D) CRISPR/Cas9 nuclease off-target analysis of the top 5 predicted off-target sites. These were the histone deacetylase 7 gene (HDAC7), DNA-packaging protein Histone H3 (H3K27), the centrosomal protein of 192 kDa (CEP192), and the Zinc and Ring Finger 2 gene (ZNRF2), which were analyzed in Huh7-cFIXmut, PLC/PRF/5-cFIXmut, and Hep3B-cFIXmut cells by T7E1 assay after transfection with the CRISPR/Cas9-encoding plasmid.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Coagulation, Mutagenesis, Sequencing, Binding Assay, CRISPR, Transfection, Activation Assay, Activity Assay, Plasmid Preparation, Molecular Weight, Marker, Negative Control, Histone Deacetylase Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Design of Donor DNA Sequences for the Two-Vector and the Single-Vector Systems (A) Donor DNA used for the two-vector system. A PCR product of 208 bp (cFIXmod) covering the mutated cFIX location was used. (B) Schematic diagram of single vectors containing the Tet-on-inducible CRISPR/Cas9 for cutting the cFIX-mutated strand and the donor DNA. Three molecular setups of single vectors, which delivered all components for HDR, were designed: plasmid 1 (P1), plasmid 2 (P2), and plasmid 3 (P3). For P1 and P2, the donor is flanked by the gRNA recognition sequence. Note that the gRNA recognition sequences in P1 are in the opposite orientation while the gRNA recognition sequences of P2 flank the donor DNA in the identical orientation. The donor contained in plasmid P3 lacks the gRNA recognition sequence. NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activator.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Plasmid Preparation, CRISPR, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Gene Correction of Mutated cFIX through Naked DNA (A) The principle of the amplification-refractory mutation system qPCR (ARMS-qPCR) assay. Two qPCRs of each sample are performed. Primers of the reference PCR (Re-F,-R) amplify the upstream area of the HR region. The forward detection primer (Det-F) binds the upstream area of the HR region, and the reverse primer (Det-R) specifically binds to the HR cassette. (B–D) Naked DNA-mediated HDR events in mutated cFIX stable cell lines measured via ARMS-qPCR. Mod1%, Cas9 + donor, Cas9, Donor, and cell-cFIXmut display controls containing 1% modified template, cells transfected with CRISPR/Cas9 and optimized donor sequence, cells transfected with CRISPR/Cas9, cells transfected with optimized donor sequence, and cFIX stable cell lines Huh7-cFIXmut (B), PLC-PRF-5-cFIXmut (C), and Hep3B-cFICmut (D). (E) Indels measured by T7E1 assay after transfection with the non-viral homology-directed repair plasmids at the endogenous hFIX locus and the uncorrected cFIX transgene in Huh7-cFIXmut cells. MW, molecular weight markers; NC, negative control, PLC-cFIXmut or Hep3B-cFIXmut without treatment. P1, P2, P3, and Cas9 + donor display cells treated with plasmid 1, plasmid 2, plasmid 3, or Cas9 + donor. Data points represent SEM of three independent experiments performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Amplification, Mutagenesis, Stable Transfection, Modification, Transfection, CRISPR, Sequencing, Molecular Weight, Negative Control, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Gene Correction of Mutated cFIX through the Viral Vectors HCAdV5-Cas9 and ssAAV2-cFIX (A) Schematic outline of HCAdV5-Cas9 and ssAAV2-cFIX genomes. ITR, inverted terminal repeat; NLS, nuclear localization signal; PA, polyadenylation signal; TREG3, TRE3G promoter; EF1a, human elongation factor-1 alpha promoter; CAG, cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter; gRNA, guide RNA; Tet-on, tetracycline-controlled transcriptional activation. (B) HCAdV5-Cas9 nuclease activity measured by T7E1 assay. MW, molecular-weight size marker; ND, no detection.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Activation Assay, Activity Assay, Molecular Weight, Marker

Journal: Molecular Therapy. Nucleic Acids

Article Title: Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B

doi: 10.1016/j.omtn.2018.12.008

Figure Lengend Snippet: Genotypic and Phenotypic Corrections in Huh7-cFIXmut Cells after Co-infection with the Gene Correction Vectors HCAdV5-Cas9-cFIX or HCAdV5-Cas9 and ssAAV2-cFIX (A) Schematic outline of the experimental setup. Cellular genomic DNA was extracted at 72 h post-transduction for ARMS-qPCR assay, and cell media were collected at 48, 96, and 144 h post-transduction for the ELISA. (B) HCAdV5-Cas9-cFIX- or HCAdV5-Cas9- and ssAAV2-cFIX-mediated HDR events in mutated cFIX stable cells measured via ARMS-qPCR. Mod1%, controls containing 1% modified template; single vector, Huh7-cFIXmut cells transduced with HCAdV5-Cas9-cFIX; AdV + AAV (MOI 200), AdV + AAV (MOI 400),and AdV + AAV (MOI 600), Huh7-cFIXmut cells transduced with HCAdV5-Cas9 and ssAAV2-cFIX of different MOIs; Huh7-cFIXmut cells, mutated cFIX stable Huh7 cells; HCAdV5-Cas9, Huh7-cFIXmut cells transduced with HCAdV5-Cas9. (C) ELISA of cFIX concentration in the supernatant of Huh7-cFIXmut cells infected with HCAdV5-Cas9-cFIX or HCAdV-Cas9 and MOIs 200, 400, and 600 for ssAAV-cFIX. Data points represent SEM of three independent experiments performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The final vector was constructed by PCR amplifying the gRNA expression cassette and cloning into the pShV-TRE3G-Cas9-EF1α-Tet-ON3G plasmid via the restriction enzyme NheI-HF (New England Biolabs).

Techniques: Infection, Transduction, Enzyme-linked Immunosorbent Assay, Modification, Plasmid Preparation, Concentration Assay

Journal: bioRxiv

Article Title: dnaudit + pydnaweb: A lightweight text-based planning and documentation workflow for genetic cloning with automatic verification

doi: 10.1101/2025.05.31.657172

Figure Lengend Snippet: A . amplification of Fragment A by PCR; B . amplification of Fragment B containing the gRNA by PCR; C . amplification of Fragment C by fusion PCR; D . digestion of the Cas9 plasmid using the MssI restriction enzyme to generate the final Cas9 cassette; E . amplification of the dDNA cassette (GFP) by PCR; F . integration of the Cas9 cassette and Fragment C in the C. albicans HIS1 locus by homologous recombination.

Article Snippet: Finally, Fragment C was co-transformed into C. albicans SC5314 together with the appropriately digested CAS9 pADH99 plasmid (with MssI restriction enzyme; Addgene plasmid # 90979; ) and the donor DNA. dDNAs ( ) were amplified from the template CIp10–γmGFP (Addgene plasmid # 163119, 27) using the oligonucleotides dDNA_ATO1_GFP_Fw and dDNA_ATO1_GFP_Rv as forward and reverse primers, respectively ( Table S1 ).

Techniques: Amplification, Plasmid Preparation, Homologous Recombination

Journal: bioRxiv

Article Title: dnaudit + pydnaweb: A lightweight text-based planning and documentation workflow for genetic cloning with automatic verification

doi: 10.1101/2025.05.31.657172

Figure Lengend Snippet: The Cas9 endonuclease is guided by the gRNA to the target site within the ATO1 gene. The gRNA binds to the complementary DNA sequence, allowing Cas9 to introduce a double-strand break (DSB) at that locus (Figure S2). The donor DNA cassette containing the gene encoding GFP facilitates homology-directed repair (HDR), resulting in the insertion of the GFP sequence into the 3’ end of the ATO1 gene.

Article Snippet: Finally, Fragment C was co-transformed into C. albicans SC5314 together with the appropriately digested CAS9 pADH99 plasmid (with MssI restriction enzyme; Addgene plasmid # 90979; ) and the donor DNA. dDNAs ( ) were amplified from the template CIp10–γmGFP (Addgene plasmid # 163119, 27) using the oligonucleotides dDNA_ATO1_GFP_Fw and dDNA_ATO1_GFP_Rv as forward and reverse primers, respectively ( Table S1 ).

Techniques: Sequencing, Introduce

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: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A)GFP+ E. coli exhibit a sick colony morphologyafter infection with M13 phage carrying GFP-targeting (GFPT) CRISPR-Cas9. NT (non-targeting) or GFPT M13 were used to infect Sm R W1655 F+ sfgfp or Sm R W1655 F+ mcherry as a control. Cells were infected, diluted, and spotted onto media with selection for the vector; f1A or f1B indicates vector version. (B) CRISPR-Cas9 targeting the sfgfp gene can induce loss of fluorescence. Colonies arising from infection with NT-M13 or GFPT-M13 were subjected to several rounds of streak purification on selective media to ensure phenotypic homogeneity and clonality. The majority (11/16) of GFPT clones exhibited a loss of fluorescence. (C) Clones exhibiting loss of fluorescence either lack an sfgfp PCR amplicon or exhibit an amplicon of decreased size. Genomic DNA was isolated from streak-purified clones, and PCR was used to determine whether the sfgfp gene was present. PCR for the 16S rRNA gene was performed as a positive control. (D) Genome-sequencing results confirm that non-fluorescent clones have chromosomal deletions encompassing the targeted gene. Read depth surrounding sfgfp locus for a fluorescent control clone G9 (green line) and all non-fluorescent clones (gray lines). Deletion size is indicated in red; range indicates a deletion flanked by repetitive sequences. Black arrow and vertical line denote position of targeting. See also .

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Infection, CRISPR, Selection, Plasmid Preparation, Fluorescence, Purification, Clone Assay, Amplification, Isolation, Positive Control, Sequencing

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A) M13-delivered GFPT CRISPR-Cas9 leads to reduced competitive fitness of the GFP-marked strain. A co-culture of Sm R F+ sfgfp and Sm R F+ mcherry was incubated with NT-M13 or GFPT-M13 at a starting MOI of ~500. Carb was added to a final concentration of 100 μg/mL to select for phage infection. Co-cultures were sampled every 4 h over 24 h; cells were washed, serially diluted, and spotted onto non-selective media to assess targeting of the GFP-marked strain. (B) Carb in culture supernatants was not detectable within 4 h of growth using a Carb bioassay against indicator strain Bacillus subtilis 168; bioassay detection limit approximately 2.5 μg/mL. (C) Flow cytometry of co-cultures 8 h following the addition of phage and Carb show reduced GFP+ events in the GFPT versus NT condition. Representative flow plots show data from 1 of 3 biological replicates. Inset: bar graph quantifying percentage of GFP+ and mCherry+ events for 3 replicates (left); plating results for a single replicate on non-selective media (right). (D) GFPT CRISPR-Cas9 changes the shape of the distribution of GFP+ population. Histogram of mCherry+ and GFP+ events by intensity shows that a proportion of GFP+ cells in the GFPT condition have shifted to a state of lower fluorescence. Bars indicate the mean of 3 biological replicates; connected points are individual replicates. See also .

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: CRISPR, Co-Culture Assay, Incubation, Concentration Assay, Infection, Flow Cytometry, Fluorescence

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: (A) Sanger-sequencing results confirm the expected spacer present in phagemid DNA extracted from fluorescent yellow isolates (Y1) colonizing NT mice (M1, M4, M5, M6, M7, M8, and M10) and fluorescent red isolates (R1 and R2) colonizing GFPT mice (M13, M14, and M18). In contrast, 4 out of 5 fluorescent yellow isolates colonizing GFPT mice (M11, M13, M14, M16, and M18) were confirmed to have lost the spacer. No Sanger-sequence data were obtained for the last isolate (M13) with the report for failing being “no priming,” suggesting loss of a larger fragment from the phagemid. (B) Diagnostic digest of CRISPR-Cas9 phagemid DNA indicates loss of a portion of phagemid DNA for the phagemid extracted from M13 Y1. Expected fragment sizes from KpnI-XbaI double digest: 5,289, 3,285, and 2,573 bp. (C) Genome-sequencing data for M13 Y1 confirms loss of DNA from phagemid. Sequencing coverage across the GFPT phagemid reveals lack of reads corresponding to the cas9 gene and parts of the CRISPR array and tracrRNA.

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Sequencing, Diagnostic Assay, CRISPR

Journal: Cell reports

Article Title: Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

doi: 10.1016/j.celrep.2021.109930

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pCas9 (Low-copy vector carrying cas9 , tracrRNA, and CRISPR array; Cm R ) , , RRID: Addgene_42876.

Techniques: Clone Assay, Recombinant, Ligation, Sequencing, CRISPR, Plasmid Preparation, Expressing, Software

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

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

Journal: Stem Cells and Development

Article Title: A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration

doi: 10.1089/scd.2017.0234

Figure Lengend Snippet: NC cells transfected with TCOF1 siRNA impair regular migration of NC and MSC. (A) H9s-derived NC cells were transiently transfected with a siRNA to TCOF1. Left panel : Flow cytometry was performed for CD44 and P75 following 5 days of differentiation from NC to MSC, demonstrating that TCOF1 KD does not impair MSC differentiation. Right panel : Flow cytometric analysis of CD44 and P75 in NC transiently transfected with a siRNA to TCOF1. Red contour plots represent CD44+P75 double stained populations, and blue contour plots represent isotype control staining. (B) MTT cell proliferation assay was performed using TCOF1 KD NC cells (NC siRNA TCOF1) and siRNA scramble NC cells (NC siRNA SCRAMBLE) during 4 days. Results are presented as mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01. Two-sided Student's t -test. (C) Representative images of scratch wound assays of HESC-derived NC cells transiently transfected with Scramble siRNA or TCOF1 siRNA. Images were collected 4 days following transfection, at the indicated time points. Scale bar: 100 μm. (D) Box plot depicting the quantification of chemotaxis potential and migration of NC cells transfected with TCOF1 siRNA or Scramble siRNA during a 6 h CytoSelect Cell Migration Assay. FGF8B was used as a NC chemoattractant. DMEM/F12 + 10% fetal bovine serum (SERUM) was used as a positive control for cell migration. Data are expressed relative to the NC cells transfected with TCOF1 siRNA, maintained in FSB medium. * P < 0.05; ** P < 0.01. Two-sided Student's t -test. (E) Representative images of scratch wound assays of NC-derived MSC transiently transfected with Scramble siRNA or TCOF1 siRNA. Images were collected after 5 days of differentiation, at the indicated time points. Scale bar: 100 μm. KD, knockdown; MTT, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; siRNA, small interfering RNA. Color images available online at www.liebertpub.com/scd

Article Snippet: For gene targeting, 2.5 × 10 6 HIPSC were electroporated with 1 μg of generated TCOF1 targeting Cas9 plasmid in 100 μL of nucleofection mix from the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) using a 4D-nucleofector system (Lonza).

Techniques: Transfection, Migration, Derivative Assay, Flow Cytometry, Staining, MTT Cell Proliferation, Chemotaxis Assay, Cell Migration Assay, Positive Control, Small Interfering RNA

Journal: Stem Cells and Development

Article Title: A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration

doi: 10.1089/scd.2017.0234

Figure Lengend Snippet: Generation of TCOF1 heterozygous knockout HIPSC using CRISPR/Cas9. (A) Schematic of CRISPR/Cas9-mediated nonhomologous end joining strategy to generate INDELs leading to HIPSC TCOF1 knockout clones. (B) Representative genomic sequencing of HIPSC transfected with specific CRISPR TCOF1 gRNA. Heterozygous knockout clones showed the same deletion leading to a change in codon reading frame and the introduction of a premature STOP codon in TCOF1 Exon 1. (C) Immunoblot for Treacle protein demonstrating that NC derived from HIPSC TCOF1 +/− clones (C12 and C24) showed a reduction in Treacle compared with NC derived from HIPSC TCOF1 +/+ (C8) and NC derived from H9s (WT). (D) Time course MTT proliferation assay of TCOF1 +/+ NC derived from H9s (WT), HIPSC (C8), and NC derived from TCOF1 +/− HIPSC (C12 and C24), over a 4-day period. Proliferation rate decreased significantly in mutated cells compared with WT. Results are presented as mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001, two-sided Student's t -test. (E) Flow cytometric analysis of Annexin V staining depicting the apoptotic rate in TCOF1 +/+ NE-derived cells from H9s (WT), HIPSC (C8), and NE derived from TCOF1 +/− HIPSC (C12 and C24). Red histograms represent Annexin V staining, and blue histograms represent the unstained population. (F) Single cell analysis of cell migration in NC derived from TCOF1 +/+ HIPSC (C8) and TCOF1 +/− HIPSC (C24). TCOF1 +/− NC demonstrates impaired migration and reduced directionality of movement. Each dot and tail represent a single cell analyzed, with 30 cells analyzed from both conditions, over a 12-h period. gRNA, guide RNA; HIPSC, human induced pluripotent stem cell; INDELs, insertions or deletions; WT, wild type. Color images available online at www.liebertpub.com/scd

Article Snippet: For gene targeting, 2.5 × 10 6 HIPSC were electroporated with 1 μg of generated TCOF1 targeting Cas9 plasmid in 100 μL of nucleofection mix from the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) using a 4D-nucleofector system (Lonza).

Techniques: Knock-Out, CRISPR, Clone Assay, Genomic Sequencing, Transfection, Western Blot, Derivative Assay, Proliferation Assay, Staining, Single-cell Analysis, Migration