Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPINs suppress the LPS-mediated NF-κB and IFN antiviral pathways. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 20 μg/ml LPS for the indicated period with HOIPINs. The cell lysates were immunoblotted with the indicated antibodies. b LPS-induced gene expression is suppressed by HOIPINs. BMDM cells were pre-treated with the indicated concentrations of HOIPINs for 30 min, and stimulated with 100 ng/ml LPS for 1 h. The mRNA levels were assessed by qPCR. c Suppression of IRF3 targets by HOIPIN-1. BMDM cells were stimulated with 100 ng/ml LPS for 8 h with HOIPIN-1, and interferon β ( n = 13) and Cxcl10 ( n = 10) were quantified by ELISA. d Suppression of antiviral signaling by HOIPIN-8. MEFs were stimulated with 10 μg/ml poly(I:C) for the indicated period with HOIPIN-8, and subjected to immunoblotting analysis. e Suppression of IRF3 targets by HOIPINs. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 10 μg/ml poly(I:C) for 2 h with HOIPINs. The mRNA levels were assessed by qPCR. f HOIPIN-1 inhibits ISRE-luciferase activity. MEF cells, transfected with the ISRE-luciferase reporter, were stimulated with 10 μg/ml poly(I:C) with or without HOIPIN-1 for 6 h, and the luciferase activities were analyzed. g LUBAC activity is indispensable for the IFN pathway. WT- and HOIP −/− -MEFs were treated with 10 μg/ml poly(I:C) and HOIPIN-1 for 2 h. Cell lysates were subjected to immunoblotting analysis. h HOIP is critical for the expression of IRF3-target genes. WT- and HOIP −/− -MEFs were treated as in g , and qPCR analyses were performed. i The Sendai virus (SeV)-induced antiviral response is suppressed by HOIPINs. MEFs were infected with SeV at a multiplicity of infection (MOI) of 10 for 8 h, and treated with the indicated concentrations of HOIPINs for 30 min. qPCR analyses were performed. In b , c , e , f , h , i , data are shown as mean ± SEM, n = 3 (sample numbers in c are indicated in the legend). NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Transfection, Expressing, Virus, Infection

Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPIN-1 shows potent toxicity to ABC-DLBCL cell lines. GCB-DLBCL cells (BJAB, SU-DHL-4, and HT) and ABC-DLBCL cells (TK, DLBCL2, and OYB) were cultured in the presence of DMSO or 10 μM HOIPIN-1. Taking the cell viabilities in the presence of DMSO as 100%, the relative cell viabilities of respective cell line in the presence of HOIPIN-1 were assessed by a CellTiter-Glo luminescent cell viability assay. b HOIPIN-8 enhanced the cell death of ABC-DLBCL cells than HOIPIN-1. DLBCL2 cells or BJAB cells were treated with DMSO, 10 μM HOIPIN-1, or 10 μM HOIPIN-8 for the indicated period, and the relative cell viabilities were assessed as in a . c Caspase activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated with or without 10 μM HOIPIN-8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. d Reduced NF-κB activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated and analyzed as in c . e HOIPIN-1 suppresses the expression of NF-κB target genes in ABC-DLBCL cells. Cells were treated with 10 μM HOIPIN-1 for 24 h, and qPCR analyses were performed. f Intracellular linear ubiquitin and IκBα phosphorylation in ABC-DLBCL cells are diminished by HOIPIN-1. HBL1 and BJAB cells were treated with 10 μM HOIPIN-1 for the indicated period, and cell lysates were immunoblotted with the indicated antibodies. g HOIPIN-8 has potent inhibitory effects on NF-κB activation and linear ubiquitination in ABC-DLBCL cells than those in HOIPIN-1. HBL1 cells were treated with the indicated concentrations of HOIPIN-1 or -8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. In b , e , data are shown as mean ± SEM, NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Cell Culture, Cell Viability Assay, Activation Assay, Expressing, Ubiquitin Proteomics, Phospho-proteomics

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: American Journal of Translational Research

Article Title: Upregulation of SPI1 during myocardial infarction aggravates cardiac tissue injury and disease progression through activation of the TLR4/NFκB axis

doi:

Figure Lengend Snippet: SPI1 siRNA reduced hypoxia-induced damage in cardiomyocytes. (A, B) mRNA (A) and protein (B) levels of SPI1 in HL-1 cells after different time of hypoxic treatment determined by RT-qPCR and western blot analysis; (C) Apoptosis rate in HL-1 cells after hypoxic treatment examined by TUNEL assay; (D) mRNA and protein levels of SPI1 in HL-1 cells after si-SPI1 transfection quantified by RT-qPCR and western blot analysis; (E) Apoptosis rate in HL-1 cells after SPI1 silencing determined by TUNEL assay; (F) Production of IL-6 and TNF-α in the supernatant of SPI1 cells examined using ELISA kits. All data are presented as mean ± SD. For cellular experiments, repetition = 3. Differences were analyzed by the unpaired t test (C-E), one-way ANOVA (A and B) or two-way ANOVA (F). *P < 0.05 vs. the control group; #P < 0.05 vs. the si-NC group.

Article Snippet: Antibodies for western blot analysis Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) A one-step TUNEL kit (Beyotime) was used to examine apoptosis of cardiomyocytes.

Techniques: Quantitative RT-PCR, Western Blot, TUNEL Assay, Transfection, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Upregulation of SPI1 during myocardial infarction aggravates cardiac tissue injury and disease progression through activation of the TLR4/NFκB axis

doi:

Figure Lengend Snippet: Overexpression of TLR4 activated the NFκB signaling pathway and blocked the protective roles of SPI1 siRNA. (A) SPI1 and TLR4 protein levels and the NFκB phosphorylation in HL-1 cells after si-SPI1 and oe-TLR4 transfection and hypoxia exposure determined using western blot analysis; (B) Apoptosis of HL-1 cells examined by the TUNEL assay; (C) Secretion of IL-6 and TNF-α in cells examined using ELISA kits; (D) Protein level of TLR4 and phosphorylation of NFκB in HL-1 cells after oe-TLR4 transfection and hypoxia exposure determined using western blot analysis; (E) Cell apoptosis examined by the TUNEL assay; (F) Secretion of the pro-inflammatory cytokines in cells examined using ELISA kits. For cellular experiments, repetition = 3. All data are presented as mean ± SD. Differences were analyzed by the unpaired t test (B and E) or two-way ANOVA (A, C, D, and F). *P < 0.05 vs. oe-NC; #P < 0.05 vs. the si-SPI1 + oe-NC group; @P < 0.05 vs. oe-NC.

Article Snippet: Antibodies for western blot analysis Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) A one-step TUNEL kit (Beyotime) was used to examine apoptosis of cardiomyocytes.

Techniques: Over Expression, Phospho-proteomics, Transfection, Western Blot, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 1. PWP1 Regulates Tissue Growth and Proliferation (A) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) leads to reduced compartment size. (B) Quantification of the ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas in (A). (C) Cell density ratio between posterior (P) (n = 10) and anterior (A) (n = 10) compartments in (A). (D) Pupation kinetics of control (n = 5) and dpwp1nclb1/2 (n = 5) larvae. dAEL, days after egg laying. (E) Representative images of control and dpwp1nclb1/2 pupae. (F) Quantification of pupal volumes of control (n = 4) and dpwp1nclb1/2 (n = 4) pupae. (G) Proliferation in Ctrl (Lac dsRNA) (n = 3) and dPWP1-specific dsRNA (n = 3) treated S2 cells. (H) Proliferation of HeLa cells after transfection with non-targeting (Ctrl) (n = 3) or PWP1-specific (n = 3) siRNAs. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S1.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Transfection

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 2. PWP1 Regulates Pol I-Mediated Ribosomal Gene Expression (A) dpwp1nclb1/2 mutants display short and thin bristles. (B) Pupation kinetics of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted larvae. dAEL, days after egg laying. (C) Representative images of control and dPWP1 fat-body (Cg-Gal4)-depleted pupae. (D) Quantification of volumes of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted pupae. (E) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with fibrillarin in fat bodies of early third instar larvae. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, and 28S rRNA (RNA polymerase I targets) expression in control larvae (n = 3) and dpwp1 mutants (n = 3). cdk7 was used as a reference gene. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S2.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Gene Expression, Control, Comparison, Quantitative RT-PCR, Expressing

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 4. PWP1 Functionally Cooperates with MYBBP1A and Nucleolin (A) Summary of dPWP1 interacting ribosome biogenesis regulators in S2 cells. (B) Summary of PWP1 interacting ribosome biogenesis regulators in HEK293 cells. (C) Co-purification of HA-tagged dPWP1 with dMYBBP1A upon pull-down of V5-tagged dMYBBP1A from S2 cells. Tubulin serves as a loading control. (D) Representative immunofluorescent images of PWP1 and Nucleolin localization in U2OS cells. Scale bar, 5 mm. (E) Representative immunofluorescent images of Nucleolin localization in U2OS cells followed by PWP1 depletion. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, 28S, and 5S rRNA expression in control larvae (n = 3) and dmybbp1a mutants (n = 3). cdk7 was used as a reference gene. (G) Representative images of control (w-) and mybbp1a mutant larvae at 96 hr after egg laying. (H) Representative immunofluorescent images of dPWP1 localization in fat bodies of early third instar larvae. Depletion of dMYBBP1A from the fat body (Fb-GAL4) leads to the dissociation of nucleolar dPWP1. Scale bar, 5 mm. (I) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (H). A total of 15 nuclei from 3 independent fat bodies were quantified. (J and K) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green or ethidium bromide staining) and newly transcribed RNA (upper panel, visualized by streptavidin-HRP detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min of 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S4.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Quantitative RT-PCR, Expressing, Mutagenesis, Nucleic Acid Electrophoresis, Staining, Transfection, Labeling, Cell Culture

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 5. mTORC1-Dependent Phosphorylation Regulates Nucle- olar PWP1 (A) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar fed and starved (6 hr) larvae. Scale bar, 5 mm. (B) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (A). A total of 15 nuclei from 3 independent fat bodies were quantified. (C) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar starved (4 hr) and re-fed (6 hr) larvae. Scale bar, 5 mm. (D) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (C). A total of 15 nuclei from 3 independent fat bodies were quantified. (E) Representative immunofluorescent images of dPWP1 localization in fat bodies dissected from early third instar larvae fed without or with rapamycin (14 hr). Scale bar, 5 mm. (F) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (E). A total of 15 nuclei from 3 independent fat bodies were quantified. (G) Immunoblot of S2 cell lysates expressing a V5-tagged form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin alone (10 min) or in combination with rapamycin (2 hr). Phospho-dPWP1 species (anti-V5) are indicated by arrowheads. (H) Immunoblot of lysates of S2 cell expressing a V5-tagged form of dPWP1 together with RNAi against LacZ (ctrl), dS6K, or dRaptor resolved on Phos-tag SDS-PAGE. Cells were treated without or with insulin (10 min). (I) Quantification of phospho-dPWP1 species of (H). (J) Immunoblot of S2 cell lysates expressing a V5-tagged wild-type or S384 alanine mutated form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin (10 min). (K) Quantification of phospho-dPWP1 species of (J). (L) Immunofluorescent analysis of fat body expressing the wild-type and S384A form of dPWP1 dissected from early third instar larvae. Scale bar, 5 mm. (M) Quantification of the nucleolus/nucleoplasm localization ratio in (L). A total of 15 nuclei from 3 independent fat bodies were quantified. **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S5.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Phospho-proteomics, Western Blot, Expressing, SDS Page

Journal: bioRxiv

Article Title: Neuroinflammatory Stress Preferentially Impacts Synaptic MAPK Signaling and Mitochondria in Excitatory Neurons

doi: 10.1101/2025.10.03.680087

Figure Lengend Snippet: (A) Experimental outline to achieve native-state proteomics of Camk2a excitatory neuron-specific crude synaptosomes. (B) Representative electron micrographs of P2 fractions confirmed that crude synaptosomes have sealed plasma membrane, filled with synaptic vesicles and intact mitochondria, synaptic cleft and postsynaptic terminals with electron-dense post-synaptic density. SVs, synaptic vehicles; PSD, post-synaptic density; Mt, mitochondria. (C) Representative immunofluorescence images of cortex from non-CIBOP negative controls (Camk2a-Cre Ert2 ) and Camk2a-CIBOP ( Rosa26 TurboID/wt /Camk2a-Cre Ert2 ) mice showing biotinylation (green streptavidin Alexa488) in relation to cell body (red neuron-specific β-III Tubulin). (D) Western blots of input and streptavidin affinity pulldown samples confirm strong protein biotinylation in Camk2a-CIBOP mice as compared to limited biotinylation in non-CIBOP negative controls. (E) Representative blots of the subcellular fractionation show enrichment of synaptic (SV2A, synaptophysin, synapsin-1, PSD95, complexin-1/2) and mitochondrial markers (HSP60, SDHA, VDAC) in crude synaptosome fractions (P2) compared to bulk brain homogenates (H), while depletion of cytosolic (α-tubulin) and nuclear (histone H3) markers, 4 μg of protein was loaded for each fraction. Volcano plots showing differentially enriched proteins in (F) bulk brain homogenate and (G) P2 fraction pulldowns comparing Camk2a-CIBOP mice vs negative controls. Red dots represent proteins biotinylated in Camk2a whole-neuron and Camk2a synaptosome proteomes (unpaired two-tailed t-test p <0.05). (H) Venn diagram showing the number of identified proteins and overlap between Camk2a whole-neuron and Camk2a synaptosome proteomes. Examples of unique enriched proteins in (I) Top enriched pathways for the 307 proteins identified exclusively in the Camk2a whole-neuron proteome. (J) Top enriched pathways for the 168 proteins identified exclusively in the Camk2a synaptosome proteome. Protein abundance is plotted as log2-transformed and normalized intensity values ( n =4 biologically independent samples). Also see Additional files 1 and 7 for related analyses and datasets, and Additional file 12 for Supplementary Fig. S1. Image was created using BioRender.com.

Article Snippet: Following our previously optimized protocol ( , ), biotinylated proteins were captured by streptavidin magnetic beads (ThermoFisher Scientific, 88817) incubating 41.5 μL beads per 500 μg of protein in 500 μL RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) for 1h at 4°C with rotation.

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Western Blot, Fractionation, Two Tailed Test, Quantitative Proteomics, Transformation Assay

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 1| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in the development and progression of unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated expression levels of CDK9 in the kidneys 6 h, 12 h, 2 days (d), 4d, and 7d after sham or UUO surgery. (b) Quantitation of relative signal intensities of CDK9/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group; #Po0.05, versus UUO 12-h group; $Po0.05, versus UUO 24-h group. (c–l) Confocal microscopy demonstrated the expression of CDK9 (green, d, h), α-smooth muscle actin (α-SMA, red, e, i), merged (f, j, k–m) and DAPI (4,6-diamidino-2-phenylindole; blue, c, g) in the mouse kidney with sham operation (c–f) and kidney with UUO (g–m). c–j, × 600. (k) Area in (j), × 1800. Arrows: samples of CDK9+/α-SMA+ myofibroblasts cells. (l) Quantitation of the percentages of CDK9+/α-SMA+ cells in total DAPI(+) cells. (m) Immunoprecipitation (IP)/WB demonstrated the interactions between Smad3 and Smad4, and Smad3 and CDK9 in the kidneys 6 h, 12 h, 2d, 4d, and 7d after sham or UUO surgery. (n) Quantitation of relative signal intensities of Smad4/Smad3 and CDK9/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay, Confocal Microscopy, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 3| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in unilateral ureteral obstruction (UUO). (a) Immunoprecipitation (IP)/western blotting (WB) demonstrated the interactions between CDK9 and Smad3 and CDK9 and Smad4 in the kidneys 7 days after sham or UUO surgery. Quantitation of relative signal intensities of (b) Smad3/CDK9 and (c) Smad4/CDK9 7 days after sham or UUO surgery. Data are mean ± s.d., n = 6. *Po0.05 versus WT sham or WT UUO. NS, P40.05 versus WT UUO. (d) IP/WB demonstrated interactions between Smad4 and CDK9 in the kidneys 7 days after sham or UUO surgery in Smad3 wild-type (WT) or Smad3 knockout (KO) mice. (e) WB demonstrated the expression levels of α-smooth muscle actin (α-SMA), fibronectin (FN), collagen I (Col. I) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. (f) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. Data are mean ± s.d., n = 6. *Po0.05; **Po0.01. (g) IP/WB demonstrated interaction between Smad3 and CDK9, phosphorylated Smad3 T179 (p-T179), and phosphorylated Smad3 C-terminus (p-Tail) in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO. (h) WB demonstrated the expression levels of α-SMA, FN, Col. I, and GAPDH in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. (i) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. Data are mean ± s.d., n = 6. *Po0.05; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Immunoprecipitation, Western Blot, Quantitation Assay, Knock-Out, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 4| Knockdown of cyclin-dependent kinase 9 (CDK9) decreases transforming growth factor-β1 (TGF-β1)-induced Smad3 linker phosphorylation and fibrotic response in renal fibroblasts. (a) Primary cultured mouse renal fibroblasts were transfected with CDK9 siRNA or a scrambled siRNA control. Two days after transfection, renal fibroblasts were stimulated with recombinant TGF-β1 for (a) 2 days or (b) 30 min. (a) Western blotting (WB) shows the expression of CDK9, α-smooth muscle actin (α-SMA), fibronectin, collagen I, and α-tubulin in renal fibroblasts. (b) Immunoprecipitation (IP)/WB or WB shows the interaction between Smad3 and Smad4, Smad3 p-Tail and nuclear p-T179, and the internal control Histone 2A in renal fibroblasts. Quantification of the relative signal intensities of (c) α-SMA/α-tubulin, fibronectin/α-tubulin, collagen I/α-tubulin, (d) Smad4/Smad3, and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 4. *Po0.05;***Po0.001; NS, not significant.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Knockdown, Phospho-proteomics, Cell Culture, Transfection, Control, Recombinant, Western Blot, Expressing, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 5| Cyclin-dependent kinase 9 (CDK9) promotes transforming growth factor-β1 (TGF-β1)-induced collagen I promoter activity. Western blotting (WB) demonstrated expression levels of (a) nuclear or (b) cytoplasm (Cyto) phosphorylated Smad3 T179 (p-T179) in the kidneys after sham or unilateral ureteral obstruction (UUO) surgery. (c) Immunoprecipitation (IP)/WB demonstrated interactions between CDK9 and Smad3 and CDK9 and Smad4 and the levels of p-T179 after TGF-β1 stimulation in mouse renal fibroblasts. (d) Collagen I promoter luciferase assay demonstrated the effects of CDK9 on Smad3, Smad4 and Smad3, and Smad4 enhancement on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by two- way analysis of variance for different vector transfections and with or without TGF-β1 treatment. Control versus TGF-β1, Po0.05; a–f represent different levels of collagen I promoter luciferase activity. (e) Collagen I luciferase activity assay demonstrated the effects of CDK9 on Smad3 WT or Smad3 linker-mutated enhancement on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01. (f–h) Collagen I lunciferase activity assay demonstrated the effects of double knockout of Smad3 and Smad4 (Smad3/4 / ) on CDK9 enhancement (f), kinase-dead CDK9 (dnCDK9) on Smad4 enhancement (g), and 3 serine at Smad3 C-terminus replaced with arginine (3S/A) on CDK9 enhancement (h) on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Luciferase, Plasmid Preparation, Transfection, Control, Double Knockout

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 6| Cyclin-dependent kinase 9 (CDK9) inhibitor inhibits transforming growth factor-β1 (TGF-β1)-induced fibrotic response in renal fibroblasts. (a) Collagen I promoter luciferase assay demonstrated effects of CDK9 inhibitor (CDK9i) and Smad3 inhibitor (SIS3) on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by one-way analysis of variance for different treatments. *Po0.05 versus control; #Po0.05 versus TGF-β1; $Po0.05 versus TGF-β1+CDK9i 50 nM. (b) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN), and internal control α-tubulin 3 days after different treatments in mouse renal fibroblasts. (c) WB demonstrated nuclear phosphorylated Smad3 T179 (Smad3 p-T179) and phosphorylated RNAPII Ser5 (RNAPII p-Ser5) for the indicated period of time of treatments in mouse renal fibroblasts.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Luciferase, Activity Assay, Control, Western Blot, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 7| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in 2-day unilateral ureteral obstruction (UUO) after administration of CDKi or/and SiS3. (a) Western blotting (WB) demonstrated the expression levels of nuclear phosphorylated Smad3 T-179 (p-T179) and Smad3 C-terminus (p-Tail Smad3) in the kidneys 2d after sham or UUO surgery. (b) Quantitation of relative signal intensities of p-T179/Histone 2A and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle or UUO CDK9i 1 μg/g/day group. (c) Immunoglobulin (IP)/WB demonstrated the interactions between Smad3 and Smad4, Smad3 and CDK9, and Smad4 and CDK9 in the kidneys 2d after sham or UUO surgery. (d) Quantitation of relative signal intensities of Smad4/Smad3, Smad3/CDK9, and Smad4/CDK9. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SIS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 8| The effects of cyclin-dependent kinase 9 inhibitor (CDK9i) and SiS3 on renal fibrosis and inflammation in unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN) and internal control α-tubulin in the kidneys 7d after sham or UUO surgery with different treatments. (b) Quantitation of relative signal intensities of α-SMA/α-tubulin, Col. I/α-tubulin, and FN/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group. (c) Quantitation of the number of F4/80+ macrophages in the kidneys 7d after sham or UUO surgery with different treatments. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Control, Quantitation Assay

Journal: Translational Psychiatry

Article Title: Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

doi: 10.1038/s41398-019-0647-7

Figure Lengend Snippet: Relative ADAMTS2 mRNA expression level in SK-N-SH cells incubated with clozapine (1 µM) ( N = 4), haloperidol (1 µM) ( N = 5), paliperidone (1 µM) ( N = 3) and aripiprazole (1 µM) ( N = 5) for the indicated times. Data are mean ± SEM; one-way ANOVA for multiple comparations: * p < 0.05, ** p < 0.01, *** p < 0.001 shows significance with respect to control (C; vehicle, grey bars).

Article Snippet: Aripiprazole, clozapine, haloperidol hydrochloride, H89 dihydrochloride, L 741,626, MDL 100907, paliperidone, risperidone, SKF 83822, SCH 39166, TCB-2, WAY 100635, 7-OH-DPAT and 8-OH-DPAT were purchased from Tocris Bioscience (Spain).

Techniques: Expressing, Incubation, Control

Journal: Translational Psychiatry

Article Title: Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

doi: 10.1038/s41398-019-0647-7

Figure Lengend Snippet: a ADAMTS2 mRNA levels by RT-qPCR in SK-N-SH cells incubated 1 h with the indicated selective receptor agonist (red bars): SKF 83822 (D 1 -class receptors) ( N = 5), 7-OH-DPAT (D 2 -class receptors) ( N = 3), 8-OH-DPAT (5-HT 1A receptor) ( N = 3) and TCB-2 (5-HT 2A/2C receptors) ( N = 5); and selective antagonist (blue bars): SCH 39165 (D 1 -class receptors) ( N = 3), L 741,626 (D 2 -class receptors) ( N = 4), WAY 100635 (5-HT 1A receptors) ( N = 3) and MDL 100907 (5-HT 2A receptors) ( N = 3) (Drug concentration 1 µM). b ADAMTS2 mRNA levels by RT-qPCR in cells incubated for 1 h with SKF 83822 ( N = 4) and pre-incubated also for 30 min with SCH 39166 ( N = 4), clozapine ( N = 3), haloperidol ( N = 4), paliperidone ( N = 4) or aripiprazole ( N = 4) (Drug concentration 1 µM). c CREB activity in cells transfected with CRE-Luc alongside pRL-Null: cells were pre-incubated for 1 h with the indicated APDs and then, incubated for 24 h with SKF 82833 (10 µM) ( N = 4). d ADAMTS2 mRNA levels by RT-qPCR: SK-N-SH cells were pre-incubated for 30 min with MAPK/ERK and cAMP-PKA inhibitors (selumetinib 1 µM N = 6 and H89 10 µM N = 4, respectively) and then, incubated for 1 h with SKF 82833 (1 µM) ( N = 5). e CREB activity in cells transfected with CRE-Luc alongside pRL-Null ( N = 4): SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 24 h with SKF 82833 (10 µM). f Western blottings showing relative phosphorylation levels of CREB and ERK1/2: SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 15 min with SKF 82833 (1 µM) ( N = 3). Blots are representative images of each western-blot. Data are mean ± SEM; Student’s t -test: * p < 0.05 and *** p < 0.001 vs. control condition (vehicle), and # p < 0.05, ## p < 0.01, ### p < 0.001 vs. SKF 83822 condition.

Article Snippet: Aripiprazole, clozapine, haloperidol hydrochloride, H89 dihydrochloride, L 741,626, MDL 100907, paliperidone, risperidone, SKF 83822, SCH 39166, TCB-2, WAY 100635, 7-OH-DPAT and 8-OH-DPAT were purchased from Tocris Bioscience (Spain).

Techniques: Quantitative RT-PCR, Incubation, Concentration Assay, Activity Assay, Transfection, Western Blot, Phospho-proteomics, Control

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: Enrichment of C‐type lectin domain family 4 member E (CLEC4E) in tumour‐associated macrophage (TAM) is correlated with unfavourable patient prognosis. (A) Volcano plot of gene enrichment in TAMs by RNA sequencing. (B) Heatmap of gene expressions in M0 and TAM by RNA sequencing, ranked by expression level in TAM. (C) Immunofluorescence of paired tumour and tumour adjacent tissues from melanoma patients. (D) Comparison of CLEC4E fluorescence intensity and macrophage count per field between paired tumour and tumour adjacent tissues. (E) CLEC4E fluorescence comparison between tumours from melanoma patients in stage I/II versus stage III/IV. (F) Overall survival analysis of patients with CLEC4E high and low expressions. Median CLEC4E fluorescence level was determined as the cutoff. (G) Overall survival analysis of patients with high CD68 + infiltration and high CLEC4E expression and patients with low CD68 + infiltration and low CLEC4E expression. Median CLEC4E fluorescence level and median CD68 + infiltration level were determined as the cutoffs.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Comparison, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) expression on tumour‐associated macrophage (TAM) promotes tumour growth in mouse models. (A) Workflow of melanoma mouse model of CLEC4E conditional knockout mice. (B) Tumour growth curve of B16F10 melanoma model. (C) Survival analysis of B16F10 melanoma model. Tumour volume exceeding 500 mm 3 was considered as the endpoint. (D) Pictures of melanoma tissues from CLEC4E knockout and control groups. (E) Body weight gain since intraperitoneal injection of ID8 cells in ovarian cancer model. (F) Survival analysis of ID8 ovarian cancer model. Body weight gain exceeding 4 g was considered as the endpoint. (G) Comparison of celiac tumour implantations in CLEC4E knockout and control groups. (H) Representative pictures of intestinal implantations. (I) Flow cytometry analysis of CD206 and CD68 from melanoma tissues at day 10. (J) Flow cytometry analysis of CD206 and CD68 from ovarian model ascites at week 8.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Knock-Out, Control, Injection, Comparison, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: Single‐cell RNA sequencing analysis of macrophages from melanoma tissues of C‐type lectin domain family 4 member E (CLEC4E) knockout and control mice. (A) Uniform manifold approximation and projection (UMAP) plot of total macrophages. (B) Bar chart showing proportions of macrophage clusters in CLEC4E knockout and control mice. (C) Pseudotime trajectory of macrophages. Cells were divided into five states. (D) Pseudotime trajectories and bar chart showing macrophage distributions of CLEC4E knockout and control mice. (E) Heatmap showing gene markers of each state and the proportion comparison between knockout and control mice. CLEC4E knockout mice had enriched macrophages in states 1 and 4, and decreased macrophages in states 2 and 5 compared to control mice.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: RNA Sequencing, Knock-Out, Control, Comparison

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) deletion suppresses tumour‐associated macrophage (TAM) proliferation and abundance in tumour microenvironment (TME). (A) Immunofluorescence of CD68 and Ki67 with B16F10 melanoma tumours from CLEC4E knockout and control mice. (B) Column charts comparing Ki67 + cells in TAM and macrophage count between control and CLEC4E knockout groups. (C) Macrophage sorting chart and qRT‐PCR comparing proliferation markers in macrophages from control and CLEC4E knockout mice. (D) qRT‐PCR of CLEC4E silencing efficiency and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis with CLEC4E silencing in TAM. (E) RT‐PCR verification of proliferation gene markers enriched in KEGG analysis. (F) Top 10 differentially expressed phosphorylated proteins between CLEC4E knockout and control macrophages. (G) Protein‒protein interaction (PPI) analysis of differentially expressed phosphorylated proteins in CLEC4E knockout versus control macrophages. (H) Western blot of PLC‐γ2, Syk and Erk phosphorylation with CLEC4E ligation in macrophages. (Trehalose‐6,6‐dibehenate) TDB concentrations were 0, 10, 25 and 50 µg/mL sequentially. (I) Cell counting kit‐8 assay of M0 and TAMs differentiated from RAW264.7 with TDB or Erk inhibitor (Erki). (j) Cell counting kit‐8 assay of peritoneal macrophages from CLEC4E knockout and control mice.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Immunofluorescence, Knock-Out, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Ligation, Cell Counting

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) knockout strengthens macrophage‒T cell interaction and T‐cell cytotoxicity. (A) Interactions of all cell clusters in control and CLEC4E knockout mice. (B) Bar charts showing the number and strength of total interactions. (C) Bar chart of interaction numbers of tumour‐associated macrophage (TAM)‒T cells. (D) Selective ligand‒receptor pair expressions between TAM and T cells. (E) Bar chart of RT‐PCR with sorted macrophages from mouse melanoma tissues. (F) Expressions of selective genes in T‐cell population. (G) Flow cytometry of granzyme B and CD8 in ovarian cancer ascites at week 18. (H) Flow cytometry of CD4 and CD8 in melanoma tissues at day 10. (I) Immunohistochemistry of granzyme B with intestinal implantation of ovarian cancer. (J) Immunofluorescence images of melanoma tumour tissues from two patients and the correlation between the area of mean fluorescence of CLEC4E and CD8 of 18 patients.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Knock-Out, Control, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: BET inhibitor strongly suppresses C‐type lectin domain family 4 member E (CLEC4E) expression on tumour‐associated macrophage (TAM). (A) Screening of 132 drugs for CLEC4E inhibition in RAW264.7 TAM induced by B16‐CM. (B) RT‐PCR of CLEC4E expression on BMDM with B16‐CM and NHWD‐870 treatment. (C) Western blot of CLEC4E expression on BMDM with B16‐CM and NHWD‐870 treatment. (D) Western blot of CLEC4E expression on THP‐1 TAM induced with SK28‐CM or A2780‐CM. (E) Immunofluorescence of Yumm1.7 melanoma tissues with BET inhibitor NHWD‐870 treatment.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: BET inhibitor downregulates C‐type lectin domain family 4 member E (CLEC4E) by targeting on BRD4/CEBPβ. (A) RT‐PCR showing the efficiency of RNA silencing of BRD2, BRD3 and BRD4 on BMDM‐TAM. (B) Western blot of CLEC4E with BRD2/3/4 silenced in BMDM‐TAM. (C) CHIP sequencing (CHIP‐seq) of A375 showing no BRD4 binding on CLEC4E. (D) Western blot of CLEC4E with CEBPβ silenced in BMDM‐TAM. (E) Western blot of CEBPβ with BRD2/3/4 silenced in BMDM‐TAM. (F) Western blot of CEBPβ with BRD4 silenced in THP‐1 tumour‐associated macrophage (TAM). (G) Western blot of CEBPβ with BET inhibitor NHWD‐870 (20 nM) treatment in THP‐1 TAM induced by indicated tumour conditioned medium. (H) Luciferase assay with 293T cells transfected with pGL3‐CEBPB or pLG3‐basic and BRD4 or NC plasmids. (I) CHIP‐seq of A375 showing BRD4 binding on the promoter of CEBPB.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, ChIP-sequencing, Binding Assay, Luciferase, Transfection

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , UMAP visualization comparing age distribution of subjects between the reference Velmeshev et al. postmortem control dataset (V19, left) and the complete integrated dataset from this study (right). Color gradient indicates subject age in years. b , Relative proportion of major cell types across individual samples. Cell types include glutamatergic neurons (GluN and GluL2-6), GABAergic interneurons (IN-MGE and IN-CGE), glial cells (astrocytes, oligodendrocytes, OPCs) and other cell types (microglia, endothelial cells). Abbreviations: Glu, glutamatergic; N, neurons; L, layer; CC, cortico-cortical projection neurons; IN-MGE/CGE, interneurons originating from the medial/caudal ganglionic eminence; OPC, oligodendrocyte precursor cells. c , Individual UMAP plots showing nucleus distribution for each patient and control. d , Quality metrics for snRNA-seq data across cell types and individuals: total count of unique molecular identifiers (UMIs) per nucleus (N counts), mean number of unique genes (N genes) detected per nucleus and percentage (%) of transcripts from mitochondrial genes.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Control

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Quantitative assessment of differentially expressed genes (DEGs) between patients and controls (log 2 (FC) > 0.4, p-value < 0.05, Fig. ). Left: Correlation between DEG count and total number of nuclei per cell type. Right: Correlation between DEG count and mean number of genes detected per nucleus in each cell type. Pearson correlation coefficients (R) and p-values are shown. Abbreviations: Astro, astrocytes; Glu, glutamatergic; N, neurons; L, layer; IN-MGE/CGE, interneurons originating from the medial/caudal ganglionic eminence; Micro, microglia; Oligo, oligodendrocytes; OPC, oligodendrocyte precursor cells. b-f , Gene Ontology (GO) pathway analysis of DEGs comparing focal FCDII patients to controls for interneurons ( b ), glutamatergic neurons ( c ), astrocytes ( d ), oligodendrocytes ( e ) and microglia ( f ). Top 10 significantly enriched GO terms with adjusted (adj.) p-value < 0.05 are shown per cell type.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques:

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Distribution of 808 genotyped nuclei in UMAP space: 117 were classified as Mut. (pt10 = 89, pt9 = 25, pt7 = 2, pt6 = 1) or as Ref. Right, Mut. nuclei percentages per cell type (top) and across cell types (bottom). b , Representative images of co-immunofluorescence staining on formalin-fixed paraffin-embedded sections ( n = 1 per patient) showing mTOR-hyperactive (pS6 + ) neurons (NEUN + , pt2), astrocytes (GFAP + , pt2), oligodendrocytes (OLIG2 + , pt2) and microglia (IBA1 + , pt10). Nuclei (in blue) are labeled with DAPI. Scale bars, 20 µm. All patients included in this experiment are detailed in Supplementary Table . c , Cytomegalic cells representing a minor fraction of mutated cells. Left, representative immunostaining of SMI311 + DNs and VIM + BCs on frozen brain tissue from pt5. Nuclei (in blue) are labeled with DAPI for total cell counting. Scale bar, 25 µm. Right, mutated cell percentage (inferred by the detected VAF) and proportion of DNs or BCs identified in each patient ( n = 1 section/patient/staining was analyzed). d , Schematic of the distribution of mutated cells across cell types and the fraction of mutated cytomegalic cells in pt10. Astro, astrocytes; Endo, endothelial cells; Hemi, hemispherical; Oligo, oligodendrocytes; Micro, microglia.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Immunofluorescence, Staining, Formalin-fixed Paraffin-Embedded, Labeling, Immunostaining, Cell Counting

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Expression analysis of cell-type-specific markers in mutation detected (Mut.) and reference detected (Ref.) nuclei. Dot size represents the proportion of nuclei expressing each marker; color intensity indicates average normalized expression level. Abbreviations: N, neurons; Glut.N, glutamatergic neurons; IN, interneurons; Astro, astrocytes; Oligo, oligodendrocytes; Micro, microglia. b , Fluorescence-activated nuclei sorting (FANS) gating strategy for cell population enrichment. Representative gating from pt9 is shown. Sequential gating begins with initial selection based on DAPI nuclear staining, followed by separation of neuronal (NEUN+) and non-neuronal populations. Cell-type-specific enrichment was then achieved using TBR1 for glutamatergic neurons, PAX6+/NEUN- for astrocytes, OLIG2+/NEUN- for oligodendrocytes, and PU.1 + /NEUN- for microglia. Note: TBR1 subpopulation analysis was only performed for pt9 due to tissue constraints. c , Quantification of somatic mutations across FANS-enriched cell populations was performed using two complementary approaches: ddPCR detection for MTOR and PIK3CA variants, and deep targeted amplicon sequencing (TAS) for the RHEB variant in pt9. Mutation-positive bulk brain DNA and mutation-negative blood DNA served as controls. ddPCR detection limits (LOD) were >3 FAM+ mutated droplets for MTOR , >6 FAM+ mutated droplets for PIK3CA in ddPCR analysis. Results are presented as mean ± SD where technical replication was feasible. Due to limited tissue availability, biological replicates could not be performed, and technical replicates were not possible for specific cell populations in patients pt4 (PAX6+/NEUN- and OLIG2+/NEUN-), pt9 (none), pt12 (PU.1+/NEUN-), and pt14 (PU.1+/NEUN-).

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Expressing, Mutagenesis, Marker, Fluorescence, Selection, Staining, Amplification, Sequencing, Variant Assay

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a-e , Validation of transcriptional changes (that is genes with absolute log 2 (FC) > 0.3) between mutation detected (Mut.) and reference detected (Ref.) glutamatergic neurons (GluN) and astrocytes (Astro). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. a-b , Differential expression analysis using 50 random subset comparisons of n = 29 GluN ( a ) or n = 17 astrocytes ( b ) amongst Mut. and Ref. nuclei of pt9 and pt10 compared to the rest of GluN and astrocytes, respectively. Left, proportion of shared dysregulated genes in ‘random’ vs ‘observed’ Mut. vs Ref. comparisons. Right, Jaccard similarity index. Wilcoxon signed rank test with continuity correction confirms significant differences between ‘random’ and ‘observed’ dysregulated genes in both GluN and astrocytes. c , Linear regression model using ‘random’ dysregulated genes from iteration n.1 of GluN and Astro cannot predict Mut. vs Ref. nuclei. Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. d , Linear regression model using observed dysregulated genes successfully discriminates Mut. from Ref. nuclei for both GluN and Astro, with potential false negatives identified in 14% (1/7) of pt9 and 9% (8/85) of pt10 Ref. GluN nuclei (indicated by black dotted box above red threshold line). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. e , Expression patterns of GluN and Astro ‘observed’ dysregulated genes across patients (pt9-10) and controls (ct1-3). Analysis restricted to samples with >10 Mut./Ref. nuclei. Statistical analysis of average gene expression in Mut. vs. Ref. GluN by individual using Kruskal-Wallis test shows significant mutation effects (genes upregulated in Mut.: H statistics = 340.50, FDR-adjusted p-value: 9.92×10 − 76; genes downregulated in Mut.: H statistics = 30.59, FDR-adjusted p-value: 3.19×10 − 8). No statistical test was performed for astrocytes since only one patient with > 10 Mut. nuclei was available. f , Top 10 GO terms for Mut. vs. Ref. dysregulated genes in astrocytes. g , Mean expression of GluN dysregulated genes between Ref. nuclei from patients vs. controls in Mut., Ref. and control GluN from pt9-10 and ct1-2. Left, all dysregulated genes. Right, epilepsy-related dysregulated genes. h , Top 10 GO terms for patient Ref. vs. control. nuclei dysregulated genes. Only significant GO terms (adjusted p-value < 0.05) are shown.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Biomarker Discovery, Mutagenesis, Quantitative Proteomics, Expressing, Gene Expression, Control

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Left, LCM–seq workflow for capturing pools of DNs, BCs and NNs from eight patients (pt1–5 and pt7–9). Right, heatmap of NEFM and VIM normalized expression with unsupervised hierarchical clustering. b , Label transfer of LCM–seq samples on to the snRNA-seq UMAP space showing NNs or DNs matching with GluNs and BCs with astrocytes. c , Left, NRGN and GFAP normalized expression heatmap with unsupervised hierarchical clustering. Right, co-immunofluorescence showing NRGN in pS6 + /SMI311 + DNs and GFAP in pS6 + /VIM + BCs (pt5) ( n = 1 section/patient/staining analyzed). GFAP-pS6 and VIM-pS6 double stainings were performed on two consecutive sections and the same BC was recognized in both sections. Nuclei (in blue) are labeled with DAPI. Scale bars, 50 µm. d , Visium spatial transcriptomics showing intermingled spots containing DNs and BCs across the tissue (pt5). Magnified images show representative DN- and BC-containing spots after hematoxylin and eosin staining ( n = 1 section per patient analyzed). Scale bars, 1.5 mm; insets = 55 µm. e , Top markers of DN- and BC-containing spots (pt5). Known histological markers for DNs ( NEFM ) and BCs ( CRYAB ) are enriched in spots with DNs and BCs. f , Spatial semi-supervised clustering of Visium spots showing clusters enriched in GluNs, astrocytes and oligodendrocytes (pt5) with top marker genes in parentheses. g , Distinct clusters for DNs, BCs, astrocytes (Astros) and GluNs from single cells (pt5 and pt9) of the MERSCOPE UMAP space. h , Heatmap of the top ten DN or BC markers with representative MERSCOPE images (pt5). DNs are identified as pS6 + /NEUN + and BCs as pS6 + /NEUN − ( n = 1 section per patient analyzed). Scale bars, 50 µm. i , Left, number of shared dysregulated genes across Mut. versus Ref. GluNs (snRNA-seq), DNs versus NNs (LCM–seq) and DN-containing spots (Visium). Right, top GO terms of DN upregulated genes. Ribo-nt., ribonucleotides; metab., metabolic; proc., process; Ribo-ns., ribonucleosides; RP., ribosomal proteins; rNTP, ribonucleoside triphosphates. j , Representative images of strong VDAC1 immunostaining in pS6 + DNs (pt2) ( n = 1 section/patient/staining analyzed). Scale bars, 50 µm. k , Electron microscopy of DNs (pt5) showing an accumulation of vesicular, swollen, damaged mitochondria (black circles) ( n = 1 section per patient analyzed). Scale bar, 2.5 µm. Detailed sample information for each experiment and analysis is provided in Supplementary Table . expr., expression; max., maximum; min., minimum.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Immunostaining, Electron Microscopy

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Semi-supervised clustering of Visium data from patients pt2, pt5 and pt9. Clusters (highlighted in red) are annotated based on predominant marker genes. To differentiate clusters enriched for the same cell type, the top expressed marker was added to the cluster annotation. Clusters lacking clear cell-type enrichment in pt9 are labeled ‘Unknown’. Abbreviations: N., neurons; IN, interneurons; NF high, cluster with high levels of neurofilament genes; Astro, astrocytes; Oligo, oligodendrocytes; Micro, microglia. On the right: corresponding hematoxylin and eosin (HE) -stained section. Scale bars = 1.5 mm. b , Spatial distribution of DN/BC-containing spots in pt2 and pt9 showing distribution across both anatomical space and transcriptionally-defined clusters. To differentiate clusters enriched for the same cell type, the top discriminating marker expressed was added to the cluster annotation. Scale bars = 1.5 mm. c , Expression analysis of cluster-defining markers in each Visium sample. Dot size indicates percentage of expressing spots; color intensity shows average normalized expression. Abbreviations: Pct. Expr., percentage of spots expressing the genes; Avg. Exprs., average normalized gene expression per group. d , Spatial distribution of cortical layer marker expression scores calculated using previously defined gene sets (Maynard et al., 2021) . Higher scores indicate increased expression of the gene set.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Marker, Labeling, Staining, Expressing, Gene Expression

Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Effects of anosmin-1 in tumor cell motility. (A) Serum-starved cells were treated with either SFM (negative control), 10 nM recombinant anosmin-1, or FBS (positive control). The average moving distance (μm) of 20 random cells tracked over 20 h are shown. Error bars indicate s.e.m. from five independent experiments. The P values calculated by two-way ANOVA between the SFM and anosmin-1-treated groups in each cell line are 0.0175 (LN229), 0.0037 (A172), and 0.0399 (U87MG), where * P ≤0.05 or ** P ≤0.01 is considered significant. (B) Effects of KAL1 knockdown on A172 cell motility. The P values obtained from three independent experiments are 0.0395 (for shRNA 673), 0.0253 (for shRNA 675), and 0.0015 (for shRNA 676) when compared with the nontargeting control shRNA. (C) As indicated, LN229 cells were pretreated with chemical inhibitors or specific antibodies for 30 min before addition of anosmin-1 (labeled A). Only anosmin-1 treatment alone or with nonspecific IgG resulted in a significant increase in motility. Error bars indicate s.e.m . from three independent experiments. (D) LN229, A172, and U87MG cells endogenously express anosmin-1, uPA, and FGFR1 proteins at variable levels. See Supplementary Table 3 for the mRNA levels of each gene. (E) KAL1 -shRNAs significantly knocked down the endogenous anosmin-1 protein as assessed by two different anti-anosmin-1 (mouse or rabbit polyclonal) antibodies. (F) Knockdown efficacy of each shRNA is indicated as the percentage of the remaining KAL1 mRNA assessed by qRT-PCR, compared with control shRNA, which was significant (*** P ≤0.0001) in all three shRNAs. qRT-PCR was performed in triplicates, from four independent experiments. Error bars indicate the s.e.m .

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Negative Control, Recombinant, Positive Control, Knockdown, shRNA, Control, Labeling, Quantitative RT-PCR

Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Interaction of anosmin-1 with β1 integrin activates downstream signal pathways. (A) Anosmin-1 co-immunoprecipitated with β1 integrin was identified by probing with anti-His or anti-GFP antibody in LN229 cells transfected with pHis-KAL, pKAL-GFP (+), or empty vector (−). Integrin β1 precipitated by anti-β1 antibody or nonspecific mouse IgG is shown as positive and negative control respectively. (B) Immunofluorescence staining of active integrin β1 (red) in LN229 cells expressing EGFP-tagged anosmin-1 (green). Nuclei were labeled with Hoechst (blue). The colocalization points are also shown (white). A display color-scatter plot is shown of red intensities (Ch1) vs green intensities (Ch2), with the pixels representing the actual color in the image and yellow indicating colocalization. Manders Overlap Coefficient was 0.83, where 1 represents perfect colocalization and 0 represents no colocalization ( Manders et al . 1992 ). On the right panel, an independent image demonstrating anosmin-1 localization at the leading edge of a polarized migrating cell. Scale bar is shown. (C) Induction of p-FAK, p-AKT, and p-ERK upon anosmin-1 treatment in serum-starved LN229 cells at the time points indicated. A172 lysate is included as a positive control for constitutive anosmin-1 expression. The ratio of phosphorylated vs total protein determined by densitometry is shown as fold induction compared with the control. All western blots were repeated twice. (D) Effects of FAK inhibitor (PF-228) on anosmin-1-induced motility. Anosmin-1 significantly increased LN229 cell motility (* P =0.0302 in anosmin-1 recombinant protein treated, and * P =0.0208 in HisKAL-transfected). The pretreatment with increasing concentrations of PF-228, but not with the solvent (DMSO), inhibited the effect of anosmin-1 in a dose-dependent manner. PF-228 alone reduced the basal level motility in LN229, as similarly reported in other cancer cell lines ( Slack-Davis et al . 2007 ). Error bars indicate s.e.m. from four independent experiments (**, P ≤0.01; ***, P ≤0.001).

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Immunofluorescence, Staining, Expressing, Labeling, Positive Control, Control, Western Blot, Recombinant, Solvent

Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Effects of anosmin-1 on cell adhesion and survival. (A) The percentage of cells adhered to the fibronectin-coated plates after 1 h at 37 °C was quantified and normalized to the nonspecific total adhesion on the poly- l -lysine-coated plate. LN229 His-KAL cells show 23% reduction (** P =0.0044) and U87MG KAL-GFP show 21% reduction (** P =0.0011) in cell adhesion, compared with the empty vector control. Experiments were performed in quintuplicate and repeated five times. Expression of the transfected anosmin-1 constructs is confirmed by anti-His or anti-GFP antibodies. (B) Effects of KAL1 knockdown on apoptosis. Caspase3/7 activity of A172 cells infected with shRNA was measured in relative light units and the average fold induction from four independent experiments is shown. P values are 0.0504 (for shRNA 673), 0.0086 (for shRNA 675), and 0.0002 (for shRNA 676). (C) Effects of KAL1 knockdown in phosphorylation status of FAK, AKT, and ERK in A172 cells. The relative ratio of phosphorylated vs total protein assessed by densitometry is shown as fold induction compared with the control shRNA, normalized to β-actin loading control. (D) Induction of PARP protein cleavage by KAL1 knockdown. The full length PARP protein is assessed by western blot in shRNA-infected A172 cells before (FBS) and after serum-starvation (SFM). The densitometry ratio is shown as fold induction compared with the control shRNA, normalized to the β-actin loading controls.

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Plasmid Preparation, Control, Expressing, Transfection, Construct, Knockdown, Activity Assay, Infection, shRNA, Phospho-proteomics, Western Blot