Journal: bioRxiv

Article Title: EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes

doi: 10.1101/2023.08.15.553204

Figure Lengend Snippet: Genes repressed by EHZ2-induced H3K27 trimethylation include CDKN2A , a key NC tumor suppressor. A. Time series histograms of log2 FC for genes with adjusted p-value < 0.05 from RNAseq performed on samples from the indicated time points. B. Volcano plots depicting DE genes comparing RNAseq from DMSO-treated (72h) with taz-treated cells (96h). C. Venn diagrams of DE genes identified in taz treated samples. D. Enrichment profiles in H3K27me3- and H3K27ac-associated chromatin at the transcriptional start site (TSS) of coding genes. E. Schematic of strategy to identify key genes mediating resistance to taz treatment using a CRISPR-CAS9 screen. F. Plot of log (fold change) (LFC) averaged for each gene vs. p-values from the CRISPR-taz-resistance screen of 10-15-cas9 cells. Shown are representative single biological replicates from duplicate experiments. pDNA, plasmid DNA.

Article Snippet: For negative control samples (e.g. rabbit α-mouse IgG) as well as H3K27me3 samples 1 μL of SNAP-CUTANA K-MetStat Panel (Epicypher #19-1002) was added to each sample.

Techniques: CRISPR, Plasmid Preparation

Journal: bioRxiv

Article Title: EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes

doi: 10.1101/2023.08.15.553204

Figure Lengend Snippet: H3K27me3 and H3K27ac domains do not co-localize. A. Confocal immunofluorescent images as indicated. Scale bars, 3µm. B. Line-scan profiles of confocal immunofluorescent images to left (generated from A). Scale bar, 5µm. Profiles were generated using ImageJ. C. Pearson correlation of H3K27ac and H3k27me3 localization. 24 nuclei per group were analyzed.

Article Snippet: For negative control samples (e.g. rabbit α-mouse IgG) as well as H3K27me3 samples 1 μL of SNAP-CUTANA K-MetStat Panel (Epicypher #19-1002) was added to each sample.

Techniques: Generated

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: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

Journal: Cell stem cell

Article Title: High-throughput automation enhances kidney organoid differentiation from human pluripotent stem cells and enables multidimensional phenotypic screening

doi: 10.1016/j.stem.2018.04.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: WA09 (H9) human ES cells , WiCell , WAe009-A.

Techniques: Plasmid Preparation, Recombinant, Titration, CRISPR, 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: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls ploidy in Y235T cells. A The presented bar graph illustrates the GJB3 mRNA amounts across various human tissues, with detailed information available in the Materials and Methods section. Urothelial cells (UC#1 and UC#2) were isolated from ureters from two separate patients who underwent nephrectomy at Ulm University. The mRNA levels were normalized to GAPDH . n = 3 independent experiments were performed. Error bars represent mean ± SEM. B The representative pictures display the HE, GJB3 and IgG staining in human ureter tissues (U#1 and U#2, respectively). C The representative Western blot result indicates GJB3 protein levels in Y235T cells with shGJB3. α-tubulin is used as a loading control. n = 3 independent experiments were performed. D The bar graphs depict the effectiveness of GJB3 knockdowns at the mRNA level in Y235T cells, with the measurements reference to the GAPDH mRNA level. n = 3 independent experiments were performed. Error bars represent mean ± SEM. E Representative pictures showing metaphase spreads of Y235T cells with shControl and shGJB3#2. Chromosomes are visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Control cells showing 46 chromosomes in most metaphase spreads. Exemplary pictures demonstrating the induction of aneuploidy in Y235T cells subsequent to GJB3 knockdown. The images show a metaphase spread of Y235T-shGJB3#2 cells with 51 chromosomes. F Chromosomes numbers of metaphase spreads from Y235T cells that were knockdown GJB3. n = numbers of (Each counting is indicated within the graph). Results are pooled from three independent sets of experiments. Mean ± SEM values are shown in the dot plot, and significance was determined by using Fisher’s exact test. G Representative pictures showing micronuclei of Y235T cells with shGJB3#1. Cell nuclei are stained with DAPI, and phalloidin Alexa Fluor 488 was used for F-actin visualization. White arrows indicate micronuclei. H Quantitation of cells with micronuclei upon knockdown of GJB3. Results from n = 3 separate series of experiments. The bar graph displays the mean ± SEM values, and the two-tailed Student's t-test was used to assess the significance. I Immunofluorescence results indicate the multinucleation of Y235T shGJB3#1 cell. Cell nuclei is visualized by DAPI, and F-actin is visualized by Alexa Fluor 488. J Quantitation of cells with multinucleation with knockdown of GJB3. Results from n = 3 independent sets of experiments. Mean ± SEM values are shown in the bar graph, and the significance was determined by two-tailed Student’s t -test. K Figures depict of mitotic abnormalities in metaphase and anaphase. DAPI (blue) indicates chromosomes, Cy5 (red) indicates α-tubulin, and Alexa Fluor 488 (green) labeling illustrates γ-tubulin. White arrows are used to indicate chromatid mislocation or multipolar centrosomes. L – M Quantitative evaluation of mitotic abnormalities. Results from n = 3 distinct experiments. The bar graph displays mean ± SEM data, and a two-tailed Student’s t -test was used to assess significance. Scale bars: 200 μm ( B main panels) 50 μm ( B insets) 20 μm ( E , G , I ) and 2 μm ( K ). Images are shot at total magnification of 100x ( B main panels), 630x ( B insets, E , G , I , K )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Isolation, Staining, Western Blot, Control, Knockdown, Quantitation Assay, Two Tailed Test, Immunofluorescence, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls spindle orientation and microtubule dynamics. A Exemplary pictures illustrating the disorientation of Y235T-shGJB3#1 cells. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. B Quantitative assessment of the spindle pole displacement factor (SPDF) in Y235T cells. n = 256 (Y235T-shScr), 253 (Y235T-shGJB3#1), 263 (Y235T-shGJB3#2), C , Representative images showing reorientation of UMUC3 cells with ectopic GJB3. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. D Quantitative assessment of the spindle pole displacement factor (SPDF) in UMUC3 cells. n = 155 (UMUC3-EV), and 140 (UMUC3-GJB3). Experiments present combined data from three separate sets of independent experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. To boost the proportion of prometaphase cells, cells were treated to dimerthylenastron for four hours prior to labeling. E Examples of images demonstrating microtubule growth in Y235T cells expressing shGJB3#2. F Rates of mitotic microtubule plus-end assembly in Y235T-shGJB3 cells. n = 60 cells are pooled from three independent sets of experiments. G Example of images demonstrating growth of microtubules in UMUC3-GJB3 cells. H Rates of mitotic microtubule plus-end assembly in UMUC3-GJB3 cells. n = 60 cells are combined from three separate sets of experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. GJB3 interacts with α-tubulin. The deletion of GJB3 in UROtsa cells using the CRISPR-cas9 method was detailed in the main article. I Western blot displaying GJB3 protein levels in UROtsa cells with a control guide RNAs directed against green flourescent protein or two distinct gRNAs targeting GJB3 (gGJB3#1 and GJB3#2). α-tubulin serves as loading control. n = 3 separate experiments were conducted. J Exemplary pictures displaying GJB3 and α-tubulin colocalization in UROtsa cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. K Quantitation of GJB3 and α-tubulin colocalization in UROtsa cells by Pearson’s correlation coefficient. n = 49 (UROtsa-gControl), 58 (UROtsa-gGJB3#1), 53(UROtsa-gGJB3#2) are pooled from three to four independent experiments. L GJB3 protein level in UMUC3 cells with ectopic GJB3 was detected by Western blot. α-tubulin is used as a loading control. n = 3 independent experiments were performed. M Representative images displaying the colocalization of GJB3 and α-tubulin in UMUC3 cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. N Quantitation of GJB3 and α-tubulin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 105 (UMUC3-EV), and 64 (UMUC3-GJB3) are pooled from three to four independent experiments. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test ( M , N ). O – P GJB3 bundle microtubule (MT) filament level was detected by Western blot. 5 × 10 11 MT/ml and 5–10 μm in length MTs were incubated with increasing concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . ( O ), Flag-GJB3, indicated by red arrowheads and ( P ), microtubules, indicated by red arrows, are visualized by specific antibodies. n = 3 independent experiments were performed. Scale bars: 5 μm ( A , C) and 1 μm ( E , G) and 2 μm ( J , M ). Images were captured at total magnification of 630x

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Two Tailed Test, Labeling, Expressing, CRISPR, Western Blot, Control, Quantitation Assay, Incubation, SDS Page, Centrifugation

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 as tumor suppressors in bladder cancer. A–B ( A ) RT-qPCR was used to measure the GJB3 mRNA expression , and ( B ) Western blot was employed to ascertain protein amounts. The relative mRNA expression was quantified with respect to GAPDH . The qPCR shows results of n = 4 technical repeats. Error bars represent Mean ± SEM. α-tubulin serves as loading control in Western blot. n = 3 independent experiments were performed. C Comparison of GJB3 mRNA levels in the CNUH (GSE13507) cohort of NMIBC ( n = 103) and MIBC ( n = 62) human bladder tumors. Statistical differences were defined by two-way Fisher’s ANOVA test * = p ≤ 0.05. D The IHC images represent the GJB3 staining (red arrows) in human normal bladder and bladder cancer tissues. E Quantitation of GJB3-IHC staining scores in human normal bladder and bladder cancer groups. F The IHC images display Gjb3 staining (red arrows) in mouse normal bladder and bladder cancer tissues during BBN-induced BC progression. G Quantitation of Gjb3-IHC staining scores in normal bladder and bladder cancer tissues during the BBN-induced BC progression in mice. The expression of Gjb3 gradually diminished after the BBN treatment, in contrast to the control mice (black dots), which were given water (orange dots). The bar graph displays mean ± SEM data, and a two-tailed Student's t-test was used to assess significance. Scale bars: 200 μm ( D and F , main panels) and 50 μm ( D and F insets). Images were captured at total magnification of 100 × ( D and F main panels), and 630 × ( D and F insets)

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Comparison, Staining, Quantitation Assay, Immunohistochemistry, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 inhibits cells invasion and migration. A – B Western blots displaying the GJB3 protein quantity assessments in RT4 and in UMUC3 cells with experimental modifications. The loading control was provided by α-tubulin levels. The Western blots were repeated for three times. C Cell migratory capacity in RT4 cell line with shGJB3#1 was detected by Wound healing/scratch. The exemplary imaged were captured at 24 and 120 h. D Normalized cell free area was used to quantify the impact of GJB3 knockdown on RT4 cells by Wound healing assay. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. E Cell migratory capacity in UMUC3 cell line with ectopic GJB3 was detected by Wound healing/scratch. The representative pictures captured at 12 and 24 h in case of UMUC3 cells. F Quantitation of normalized cell free area of UMUC3 cells with ectopic GJB3 performed by Wound healing assay n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. G The invasion capacity of RT4 cell line with shGJB3#1 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 144 h post-seeding. H Quantitation of invasive capacity of RT4 cells expressing the indicated shRNAs targeting GJB3. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. I The invasion capacity of UMUC3 cell line with ectopic of GJB3 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 48 h post-seeding. J, Quantitation of invasive capacity of UMUC3 cells with ectopic GJB3 expression. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. K Representative images of hematoxylin/eosin stainings showing the invasion capacity of RT4 cell line with shGJB3#2 by porcine bladder ex vivo organ culture method (the invasive capacity of BC cells in the ex vivo organ culture model was quantified as shown in Fig. S4). The cells were seeded on the surface of the de-epithelized porcine bladder for 21 days. L Quantitation graphs displaying the impact of GJB3 alteration on the invasive capacity of RT4 cells in ex vivo organ culture approach. n = 4 (RT4-shScr), n = 3 (RT4-shGJB3#1), n = 3 (RT4-shGJB3#2) M Representative images of hematoxylin/eosin stainings showing the invasion capacity of UMUC3 cell line with ectopic GJB3 by porcine bladder ex vivo organ culture approach. The cells were seeded on the surface of the de-epithelized porcine bladder for or 14 days. Insets: enlarged images of the areas shown by black boxes. Black arrows indicate the cells that have spread the farthest from the surface. n = 3 (UMUC3-EV), n = 4 (UMUC3-GJB3) independent experiments were performed. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. Scale bars: 200 μm ( C , E , G , I , K main panels, M main panels) and 100 μm ( K insets, M insets). Images were captured at total magnification of 50 × ( C , E ), 100 × ( G , I , K main panels, M main panels), and 200 × ( K insets, M insets). O - V Morphological changes and actin-enriched protrusions in UMUC3, and RT4 cells with altered GJB3 expression. Cells with actin-enriched protrusions are marked with white arrows. O - P Brightfield microscopy images depict cells exhibiting a transition towards a round morphology ( O ) of UMUC3 cells with ectopic GJB3 expression. P RT4 cells with GJB3 knockdown demonstrate an elongated shape. Magnifications indicate 100 × or 400 × , respectively. The scale bars refer to 100 μm (left), and 50 µm (right). Q - T Quantification of round or elongated morphology on fixed cells. A cell with elongated or round morphology is identified by the ratio of longest and shortest diameter of the cell from images captured randomly at 630 × magnification using a Zeiss TCS SP5 confocal microscope. Scale bars: 20 μm. The ratio is calculated as the longest diameter of the cell dividing by the shortest diameter of the cell. The ratio is calculated as longest diameter dividing by shortest diameter. A cells with Ratio ≤ 2 is identified as round morphology, while ratio > 2 is elongated morphology. Q , R Immunofluorescence staining photos illustrate the round morphology shifting of ( Q ) UMUC3 cells with GJB3 overexpression compared to cells transfected with empty vector (EV). R RT4 cells with GJB3 knockdown display a transition towards an elongated morphology compared to cells transfected with shScramble (shScr). Cell nuclei are stained with DAPI, and F-actin is labeled with Phalloidin-AF488. S , T The bar graphs reveals percentages of cells with different morphology in total in each group, shown above in Q and R . The percentage was calculated as number of cells with elongated (or rounded) morphology divide cell numbers in total. The percentage values in different groups are marked above or in the bars. Black bars indicate percentages of cells with elongated morphology, and gray bars indicate the percentage of cells with rounded morphology. The statistical significance is calculated by using chi-square statistic. U , V The graphs show the fraction of cells with actin-enriched protrusions in response to GJB3 alterations ( U ) in RT4 or ( V ) in UMUC3 cells. For each picture, the percentage of cells with actin-enriched protrusions is calculated by the number of the cells with actin-enriched protrusions divided by total number of cells. (n) indicates the number of pictures taken in the group

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Western Blot, Control, Knockdown, Wound Healing Assay, Two Tailed Test, Quantitation Assay, Staining, Expressing, Ex Vivo, Organ Culture, Microscopy, Immunofluorescence, Over Expression, Transfection, Plasmid Preparation, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 interactics with F-actin and influences invadopodia formation via actin dynamics. A Representative images demonstrating invadopodia formation of RT4 cells with shGJB3#2. B Quantitation of the invadopodia number was performed in RT4 cells. n = 959 (RT4-shScr), n = 718 (RT4-shGJB3#1), n = 481 (RT4-shGJB3#2). Cells are pooled from three independent sets of experiments. C, Representative pictures showing invadopodia formation of UMUC3 cells upon ectopic GJB3 expression. D Quantitation of the invadopodia number was performed in UMUC3 cells. n = 110 (UMUC3-EV), n = 137 (UMUC3-GJB3). Cells are pooled from three independent sets of experiments. F-actin is visualized by Alexa Fluor 488-phalloidin and Cortactin is visualized by Cy5, respectively. Yellow spots displaying cortactin and F-actin colocalization identify the invadopodia structures. The regions indicated by white boxes are magnified in the insets. The invadopodia are marked with white arrows. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. E Representative pictures indicate the gelatin degradation by RT4 cells upon GJB3 knockdown. F Gelatin degradation capacity of the cells was quantified by measuring the degradation area per RT4 cell. n = 2809 (RT4-shScr), n = 2447 (RT4-shGJB3#1), n = 3544 (RT4-shGJB3#2). G Representative pictures indicate the gelatin degradation by UMUC3-GJB3 cells. H Gelatin degradation capacity of the cells was quantified by measuring the degradation area per UMUC3 cell. n = 1048 (UMUC3-EV), and n = 1246 (UMUC3-GJB3) are pooled from three to four independent experiments. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. I The LifeAct–Ruby signal recovery duration in UMUC3 cells with or without GJB3 is depicted in representative images after LifeAct–Ruby signal bleaching. The white arrows indicate the areas of bleaching. J , The quantitation of bleaching recovery experiments with UMUC3 cells. n = 29 (UMUC3-EV), n = 31 (UMUC3-GJB3). Three different groups of separate experiments' cells are combined. The graphs' data points correspond to the mean ± SEM. P values were calculated using the two-tailed Student's t -test at t = 49 s. K Exemplary pictures displaying the colocalization of GJB3 with F-actin in control UROtsa cells. Insets: enlarged image of the areas shown by white box. L , Quantitation of GJB3 and F-actin colocalization in UROtsa by Pearson’s correlation coefficient. n = 161 (UROtsa-gControl), n = 206 (UROtsa-gGJB3#1), n = 276 (UROtsa-gGJB3#2). M Exemplary pictures displaying the GJB3/F-actin colocalization in UMUC3-GJB3 cells. Insets: enlarged image of the areas shown by white box. N Quantitation of GJB3 and F-actin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 672 (UMUC3-EV), and n = 831 (UMUC3-GJB3) are combined from 3 separate experiments. The two-tailed Student's t-test was used to establish significance, and the bar graph displays mean ± SEM results. Alexa Fluor 647 illustrates the F-actin, and Alexa Fluor 488 illustrates GJB3. Yellow highlights denote GJB3 and F-actin overlap. Insets: enlarged images of the colocalized areas shown by white boxes. O , P GJB3 binds bundle actin filaments in a dose-dependent manner by Western blot. Actin (2.5 mg/ml) concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . Red arrowheads indicate the GJB3, and the red arrow indicates actin filaments visualized by western blot with specific antibodies. n = 3 independent experiments were performed. Scale bars: 10 μm ( A , C , E , G , K and M main panels), 1 μm ( A , C insets), 2 μm ( E , G , K and M insets) and 1 μm ( I ). Images were captured at total magnification of 630 × ( A , C , I , K , M ) and 400 × ( E , G )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitation Assay, Expressing, Two Tailed Test, Knockdown, Control, Western Blot, SDS Page, Centrifugation

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: Cell reports

Article Title: Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions

doi: 10.1016/j.celrep.2020.107633

Figure Lengend Snippet: (A) Relative mRNA levels for IFIT1, MX1, RSAD2, IFI44L, and IFI27 in peripheral blood from a healthy control, P1,P2, P3, and P5, as assessed by qRT-qPCR. Bars represent the mean ± SD. (B) PBMCs from patients and healthy controls were immunophenotyped by CyTOF technology with a 40-marker panel. t-Stochastic neighbor embedding (t-SNE) plot of PBMCs from P1 and three healthy controls, showing SIGLEC1 (CD169) expression in the various immune populations. The monocyte compartment displays high levels of SIGLEC1 (CD169) expression in P1. (C) SIGLEC1 (CD169) expression in the various subtypes of monocytes (CD14 + CD16 − , CD14 + CD16 + , and CD14 − CD16 + ). (D) SIGLEC1 (CD169) expression in dendritic cells. (E) PBMCs from P1 and a control were analyzed by CyTOF. with a panel including several activated signaling markers. Heatmaps show the median expression levels of STAT1 and STAT3 in P1 relative to a healthy control, for the various immune cell subtypes. (F) PBMCs from P1 and a control were analyzed by CyTOF. with a panel including several activated signaling markers. Heatmaps show the median expression levels of pSTAT1, pSTAT3, pSTAT5, pSTAT6, pp38, pMAPKAP2, pERK, and pS6 in P1 relative to a healthy control, for the various immune cell subtypes.

Article Snippet: The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), ISG15 (Santa Cruz Biotechnology), β-actin (Cell Signaling Technology), and IFIT1 (Cell Signaling Technology).

Techniques: Control, Marker, Expressing

Journal: Cell reports

Article Title: Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions

doi: 10.1016/j.celrep.2020.107633

Figure Lengend Snippet:

Article Snippet: The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), ISG15 (Santa Cruz Biotechnology), β-actin (Cell Signaling Technology), and IFIT1 (Cell Signaling Technology).

Techniques: Virus, Molecular Cloning, Recombinant, Blocking Assay, Western Blot, Lysis, Extraction, Isolation, Mutagenesis, Reverse Transcription, Rapid Amplification of cDNA Ends, TA Cloning, Luminex, Transfection, Sequencing, CRISPR, Plasmid Preparation, Software

Journal: Shock

Article Title: ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells

doi: 10.1097/shk.0000000000001899

Figure Lengend Snippet: FIG. 4. ERG activation preserves VE-cadherin in endothelial adherens junctions in human lung microvascular endothelial cells. The group transfected with ERG CRISPR/Cas9 knockdown plasmid and the group treated with VEGF shows disruption of the adherens junction proteins VE-cadherin evidenced by discontinuity of their localization at the cell–cell junctions compared to their respective control groups (60). The group transfected with ERG CRISPR activation plasmid followed by VEGF treatment, shows relatively intact adherens junctions, evidenced by their continuous localization at the cell-cell junctions compared with VEGF alone group. Arrows indicate areas of cell-cell contacts where VE-cadherin is expected normally but not localized abundantly compared to other groups (n ¼ 4).

Article Snippet: VEGF was purchased from R&D Systems (Minneapolis, MN). b-Catenin and VE-cadherin primary antibodies and FITC-tagged secondary antibodies were obtained from Santa Cruz.

Techniques: Activation Assay, Transfection, CRISPR, Knockdown, Plasmid Preparation, Disruption, Control

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Derivative Assay, Sequencing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein (ZTA and RTA) expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by anti-YTHDF2 and anti-Myc antibodies. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p<0.001. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. Arrowhead denotes cleaved fragments. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) YTHDF2-depleted and control Akata (EBV+) cells were lytically induced with anti-IgG for 0 to 48 hrs. (B) YTHDF2-depleted and control P3HR1 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. (C) YTHDF2-depleted and control SNU-719 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, -8 and -6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of caspases, cleaved caspases, YTHDF2, PIAS1 and viral proteins (ZTA and RTA) was monitored by Western Blot using antibodies as indicated. Arrowheads denote cleaved fragments. See also - and Table S2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-C) Akata (EBV+) cells were lytically induced with anti-IgG for 0, 6, 12, 24 and 48 hrs (A). P3HR1 (B) and SNU-719 (C) cells were lytically induced with TPA and NaBu for 0 6, 12, 24 and 48 hrs. YTHDF2, EBV ZTA and RTA, cleaved caspase substrate (CASP sub.), cleaved PARP, cleaved CASP3 and cleaved CASP8 were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (D-F) Apoptotic induction by an intrinsic trigger promotes EBV reactivation. Akata (EBV+) (D), P3HR1 (E) and SNU-719 (F) cells were untreated or treated with increasing amount of Taxol for 48 hrs. YTHDF2, EBV ZTA and RTA, and cleaved caspase substrate (CASP sub.) were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata EBV(+) cells were transduced with lenti-vector control or Myc-CASP8 to establish stable cell lines. The cells were treated with anti-IgG for 0, 24 and 48 hrs. (A) CASP8, EBV ZTA and RTA were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (B) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. (C) Total RNA was extracted and then EBV lytic (ZTA and RTA) and latent (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 and LMP2) genes were analyzed by RT-qPCR. The value of vector control at 0 hr was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. (A-B) Total RNA was extracted and then EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. (C) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic Akata (EBV+) cells upon lytic induction. Akata (EBV+) cells were either untreated (control) or treated with anti-IgG antibody for 24 and 48 hrs as indicated. (A) The cells were stained with Propidium Iodide (PI) and then permeabilized for immnunostaining with anti-YTHDF2 antibody. (B) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. (C-D) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic SNU-719 cells upon lytic induction. SNU-719 cells were either untreated (control) or treated with TPA and NaBu for 24 and 48 hrs as indicated. (C) Cells were stained with Propidium Iodide (PI) and then permeabilized for immunostaining with anti-YTHDF2 antibody. (D) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. Scale bar, 20 μm

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The Akata (EBV+) (A), P3HR-1 (B) and SNU-719 (C) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then lytically induced with anti-IgG antibody or TPA/NaBu as indicated for 48 hrs. Western Blot showing the protein levels of RIP, phospho-RIP (p-RIP) and phospho-RIP3 (p-RIP3) using antibodies as indicated. β-actin blots were included as controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Arrowheads denote cleaved fragments. Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001. (D) Schematic representation of 5 YTHDF2 cleavage-mimicking fragments. These fragments were cloned into a lentiviral vector with a C-terminal Myc-tag. (E) SNU-719 cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. (F) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (G) Caspase-mediated cleavage impairs YTHDF2 binding to CNOT1. Halo-V5-tagged WT YTHDF2 and the individual fragments were co-transfected with HA-tagged CNOT1 SH domain into 293T cells as indicated. Co-immunoprecipitation (Co-IP) experiments were performed using anti-V5 antibody-conjugated magnetic beads. The immunoprecipitated samples and total cell lysates (Input) were analyzed by Western Blot with antibodies as indicated. (H) Model showing the functional consequences of YTHDF2 cleavage in CNOT1 binding and the targeting of m 6 A-modified RNA. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: CRISPR, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Transduction, Stable Transfection, Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Functional Assay, Modification

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) SNU-719 cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines (see ). RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. The value of vector control was set as 1. (B) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines . RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. The value of vector control at 24 hrs was set as 1. (C) Akata (EBV+) cells were transduced with lentiviruses carrying vector control, Halo-tag, WT YTHDF2 or individual YTHDF2 fragment to establish stable cell lines. Western Blot analysis showing Halo, YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (D) Total mRNA was extracted from cells treated in panel (C). EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. The value of vector control at 24 hrs was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. N.S., not significant; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( , , ) (B-C) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated (B). CASP8 mRNA levels were analyzed by RT-qPCR using CASP8 primers (C). The value of vector control at 0 hr was set as 1. (D-E) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (D). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (E). The value of vector control at 0 hr was set as 1. (F-G) Caspase-8 depletion suppresses EBV replication in YTHDF2-depleted cells. YTHDF2-depleted Akata (EBV+) cells were transduced with lentivirus carrying control sgRNA or CASP8-sg1 to establish cell lines and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (F). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (G). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Activation Assay, Activity Assay, Derivative Assay, Expressing, Construct, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Inhibition, Transduction

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic induction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.. The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001. See also , and Table S3.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Expressing, Activation Assay, Modification, Binding Assay, Western Blot, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Modification, Western Blot, Inhibition, Incubation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A and B), METTL14 (C and D), WTAP (E and F), VIRMA (G and H) and YTHDF3 (I and J) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation for 24 or 48 hrs. EBV ZTA and RTA mRNA expression levels were monitored by RT-qPCR (A, C, E, G and I). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (B, D, F, H and J). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation