Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 1. BLU-VIPR allows for optogenetic CRISPR using light-induced expression of gRNA. ( A ) The RGR design consists of an HH ribozyme followed by the gRNA and an HDV ribozyme. After precise self-cleavage of the ribozymes, a functional gRNA is released. ( B ) Design of construct for VPR-EL222-dependent activation of C120 promoter transcription, allowing for simultaneous expression of mCherry and gRNA after exposure to blue light (470 nm). ( C ) Comparison of mCherry reporter expression in HEK293T cells after transfection with VPR-EL222 or VP16-EL222 constructs f ollo w ed b y e xposure to blue light f or 24 h. T he e xperiment w as perf ormed three times and one representativ e image is sho wn. Scale bars are 100 μm. ( D ) Out-of-frame Cas9 reporter where the expression of tdTomato is restored after Cas9-mediated indels. ( E ) HEK293T cells were transfected with BLU-VIPR (without mCherry) containing a gRNA targeting the out-of-frame sequence in tdTomato and Cas9. After 48 h of light exposure, the cells were cultured for an additional 72 h before detection of tdTomato demonstrating successful optogenetic induction of indels by Cas9. Scale bars are 55 μm. T he e xperiment w as perf ormed three times and one representativ e image is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: CRISPR, Expressing, Functional Assay, Construct, Activation Assay, Comparison, Transfection, Sequencing, Cell Culture

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 4. Optogenetic gene editing of primary mouse Cas9 + T lymphocytes in vitro . ( A ) Design of the retroviral MSCV-BLU-VIPR constructs for blue-light induced expression of gRNA after transduction of primary mouse T lymphocytes. ( B ) Cas9 + splenic mouse T lymphocytes (Thy1.2 + ) were transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA, f ollo w ed b y e xposure to blue light and analy sis b y flo w cytometry. ( C ) After 48 h of light exposure, followed by a 72-h dark period, the T lymphocytes were stained for Thy1.2, gated for singlets and viability, and then analyzed for T h y1.2 e xpression. R epresentativ e result from tw o e xperiments is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vitro, Retroviral, Construct, Expressing, Transduction, Cytometry, Staining

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 5. BLU-VIPR allows for optogenetic gene editing of Cas9 + T lymphocytes in vivo . ( A ) Optogenetic setup for illumination of lymph nodes with blue light (470 nm). ( B ) Illumination of a single iLN. ( C ) Cas9 + splenic mouse T lymphocytes (CD45.1 + T h y1.2 + ) w ere transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA. T he transduced cells w ere adoptiv ely transferred to TCR β−/ −CD45.2 + mice. After reconstitution of the T cell lymphocyte pool, we illuminated an iLN with blue light to induce gene editing of Thy1.2. ( D ) Gating strategy to determine levels of Thy1.2 by flow cytometry. ( E ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from illuminated iLNs were determined by flow cytometry. ( F ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from nonilluminated mice w ere determined b y flo w cytometry. ( G ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) in blood bef ore and after illumination of an iLN w ere determined b y flo w cytometry.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vivo, Transduction, Flow Cytometry, Cytometry

Journal: Experimental Biology and Medicine

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

doi: 10.1177/1535370217694435

Figure Lengend Snippet: Antibodies used for Western blot analysis

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

Techniques: Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 1 Identification of potential genes implicated in colorectal cancer (CRC) and cancer metabolism-associated biological processes. (A) A screening procedure to find putative gene candidates. (B) Colorectal cancer (CRC) samples were found to differ from adjacent controls in terms of physiopathology and biological processes related to metabolism in a number of databases, including TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO) datasets (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549). (C) Following gene differential expression analysis, the total number of differentially expressed genes that crossed over into various databases was counted. (D) Six upregulated and four down regulated DEGs were identified based on a survival analysis of differentially expressed genes across six databases.In the databases of TCGA and ICGC, P < 0.05 was deemed statistically significant. (E) Six upregulated and four downregulated DEGs represent the molecular mechanisms impacting the onset of colorectal cancer and metabolic reprogramming. (F) Palmitoyltransferase ZDHHC6 expression in the ICGC and TCGA databases. (G) Pancarcinoma analysis using TCGA datasets to measure ZDHHC6 expression levels in various malignancies. (H) The overall survival (OS) of colorectal cancer patients in the TCGA and ICGC databases according to different ZDHHC6 expression levels. (I) After dividing the TCGA and ICGC samples’ ZDHHC6 expression levels into groups of high and low expression levels, the grouped samples underwent GSEA analysis. The data were expressed as the mean ± SEM. A P value less than 0.05 was considered statistically significant. ***P < 0.001

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Gene Expression, Quantitative Proteomics, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 2 Increased ZDHHC6 is positively associated with the development of human colorectal cancer (CRC). (A) ZDHHC6 mRNA expression levels in 73 pairs of CRC sample pairs (T) and their corresponding adjacent sample pairs (N). n = 73 pairs. (B) ZDHHC6 protein expression levels in sixteen pairs of similar adjacent tissues and colorectal cancer tissues selected at random. For each group, n = 3. (C) ZDHHC6 mRNA expression levels in relation to a range of CRC-associated cell lines, such as SNU-C2A, SW48, HT-29, LS1034, HCT116, and Caco-2, as well as the matching human normal colonic epithelial cell line (FHC), are displayed in qPCR analysis. For each group, n = 5. (D, E) ZDHHC6 protein expression in SNU-C2A, SW48, HT-29, LS1034, HCT116, Caco-2, and FHC cell line as demonstrated by western blotting (D) and immunofluorescence analysis (E). 200 μm; each group has n = 5. (F, G) qPCR analysis (F) and western blotting experiment (G) demonstrate the effect of the gradually increased dosage of 2-bromopalmitate (2-BP) on the relative ZDHHC6 mRNA and protein expression levels in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. For each group, n = 3. (H) An immunofluorescence assay demonstrating the co-expression of ZDHHC6 and Ki67 in response to 40 µM 2-bromopalmitate (2-BP) in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. 200 μm; each group has n = 3. Data are expressed as mean ± SEM. The relevant experiments presented in this section were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 4 ZDHHC6 facilitates lipid deposition and carcinogenesis in CRC cells. (A) A venn diagram shows the variations in metabolites produced by HCT116 cells with ZDHHC6 knockout (KO) and wild-type (WT) phenotypes. ZDHHC6 and fatty acid synthesis pathways have a significant association, according to pathway enrichment analysis of the 36 metabolites. Total peak area was used to correct the LC-MS-based untargeted metabolomic study and its findings. (B) Using these 36 differential metabolites, pathway analysis showed enhanced signaling pathways. (www.metaboanalyst.ca). (C) A heatmap showing how these 36 significantly altered metabolites changed. Student’s t-test, unpaired, two-tailed, P < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). (D, E) The ratios of various isotopic forms of FFA C16:0 (palmitate) in ZDHHC6 (KO) (D) and AdZDHHC6 (E) HCT116 cells after a brief exposure to glucose [U-13C]. When the cell density was around 85%, the media was changed to RPMI 1640 containing 2 g/L glucose tagged with [U-13C]. Following a 24-hour period, the PBS-rinsed cell culture plates were quickly frozen in liquid nitrogen and subjected to an LC-MS assay analysis (n = 4 per group). (F) Representative im munofluorescence pictures of HCT116 cells with ZDHHC6 (WT) and ZDHHC6 (KO) phenotypic, demonstrating ZDHHC6 expression, lipid accumulation (Bodipy staining), and corresponding intracellular triglyceride (TG) levels (n = 4 per group). (G, H) ZDHHC6 (WT) and ZDHHC6 (KO) HCT116 cells were injected into the right flanks of nude mice. Every two days, tumor volumes were measured. On day 22 following dissection, tumor pictures (G), growth curves, and weight (H) were recorded (n = 4 per group). Scale bars, 1 cm. (I) A heatmap utilizing untargeted metabolomic analysis comparing significantly changed metabolites between tumors originating from ZDHHC6 (KO) HCT116 cells and ZDHHC6 (WT) cell lines. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Produced, Knock-Out, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, Two Tailed Test, Cell Culture, Expressing, Staining, Injection, Dissection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 5 ZDHHC6 specifically binds to the lipid metabolism key transcription factor of PPARγ. (A) After 24 h of SFB-ZDHHC6 transfection in HCT116 cells, ZDHHC6-interacting proteins were identified by tandem affinity purification and mass spectrometry (MS). This was accomplished by removing S-protein, Flag, and streptavidin binding peptide (SFB). (B) ZDHHC6 or IgG antibodies were used to immunoprecipitate HCT116 cell lysates, and PPARγ, PPARα, PPARδ, SREBP1, and ZDHHC6 antibodies were used for western blotting experiments. (C) ZDHHC6 or IgG antibodies were used to immunoprecipitate cellular lysates of SNU-C2A, SW48, HT-29, LS1034, and Caco-2 cells, and ZDHHC6 or PPARγ antibodies were used for western blotting experiments. (D) GST pulldown assay using GST-PPARγ and purified His-ZDHHC6 in HCT116 cells. (E) Schematic of the experimental procedure showing the genes expression in HCT116, Caco-2, SNU-C2A and HT-29 after adenovirus-mediated ZDHHC6 overactivation (AdZDHHC6). The lower schematic diagram showing the inter section of the results from the proteomics and IP-MS analyses. (F) For a duration of 24 h, plasmids expressing Flag-PPARγ or Myc-ZDHHC6 individually or in combination were transfected into HCT116, Caco-2, SNU-C2A and HT-29 cells, respectively. His or Flag antibodies were used for immunoblotting after cellular lysates had been immunoprecipitated with Flag and/or His antibodies. (G) GST pulldown assay using GST-PPARγ and purified Flag-ZDHHC6 in Caco-2 and SNU-C2A cells, respectively. (H) Assay for immunofluorescence staining demonstrating ZDHHC6 and PPARγ co-expression in HCT116, Caco-2, and SNU-C2A cells. 20 μm. (I) In HCT116 cells, vectors containing the hinge-LBD domain, full length (FL), AF-1, DBD, and PPARγ were co-expressed with SFB-ZDHHC6. S-bead pulldown was used to immunoprecipitate cellular lysates. (J) Based on GSEA signaling pathway analysis, an assay of the TCGA-CRC and ICGC-CRC datasets showed a significant connection between ZDHHC6 and the PPARγ pathway in CRC. Data are expressed as mean ± SEM. The rel evant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Transfection, Affinity Purification, Mass Spectrometry, Binding Assay, Western Blot, GST Pulldown Assay, Purification, Expressing, Protein-Protein interactions, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 6 Identification of the palmitoylation site on PPARγ at evolutionarily conserved cysteine residues. (A) For a duration of 24 h, HCT116 cells were exposed to 60 µM 2-BP, 1 µM ABD957, 6 µM palmostatin B (Palm B), and 10 µM palmostatin M (Palm M) treatments. The slices that were fixed underwent immunofluorescence labeling using PPARγ (red) and pan-palmitoylation (green). 10 μm scale bars; n = 5 per group. (B) Schematic diagram of the Click-iT assay for palmitoylation measurement of PPARγ. HCT116 cells were treated with 100 µM Click-iT PA and azides for five hours. The resulting lysates were then submitted to Click-iT detection as per the product instructions, and PPARγ antibody western blotting analysis was performed. The indicated group’s expression of PPARγ is indicated by the western blotting bands on the right. (C) Using the GPS-Palm program (MacOS_20200219) (The CUCKOO Work group, http://gpspalm.biocuckoo.cn/) and the MDD-Palm algorithm (http://csb.cse.yzu.edu.tw/MDDPalm/), the palmitoylation site on PPARγ in Homo sapiens (upper) and Mus musculus (lower) is predicted to be located. PPARγ’s lower palmitoylation site contains conserved cysteine residues shared by Rattus norvegicus, Bos taurus, Canis familiaris, Mus musculus, and Homo sapiens. (D) After incubating Click-iT PA and azides for five hours on HCT116 cells overexpressing either PPARγ WT or PPARγ C313S mutant, the corresponding cellular lysates were obtained and Click-iT detection was performed in com pliance with the product’s instructions. After the palmitoylated proteins were added to the streptavidin-sepharose bead conjugate for pull-down detec tion, PPARγ and ACTIN antibodies were used in a western blotting examination. While PPARγ C313S was not palmitoylated in top gel, lane 6, or the control groups, it was for PPARγ WT in lane 5. Three separate runs of this experiment were conducted. (E) CHX was cultured with HCT116 cells overexpressing either the PPARγ WT or PPARγ C313S mutant for a specific amount of time. PPARγ and ACTIN antibodies were used in immunoblotting detection of the obtained cellular lysates. The relative PPARγ remaining ratio (n = 4 per group) is displayed in the right curve graph at the specified time point. (F) PPARγ WT or PPARγ C313S mutant overexpression was observed in the upper HCT116 cells. Pan-palmitoylation (green) and PPARγ (red) immunofluorescent label ing were applied to the cell sections. Lower, AdZDHHC6 + PPARγ C313S mutant or PPARγ C313S alone were overexpressed in HCT116 cells, respectively. The bar graph displays the intensity of PPARγ fluorescence in each of the indicated groups (n = 5 pictures; P < 0.05 vs. PPARγ C313S + AdControl or PPARγ WT). Scale bars, 20 μm. (G) In HCT116 cells, PPARγ-Flag and ZDHHC6-HA plasmids were transfected. Alk16 labeling was used to determine the palmi toylated PPARγ expression contents in the presence or absence of hydroxylamine therapy. (H) PPARγ-Flag was used to transfect SNU-C2A cells (WT) or ZDHHC6-deleted SNU-C2A cells, and Alk16 was used to label the cells. Subcellular fraction was extracted, and the levels of PPARγ protein were adjusted to verify that the input cells from the wild type and the knockout cell had the same quantity of PPARγ. Immunoblotting analysis was used to evaluate the palmitoylated PPARγ expression contents in the cell membrane (Mem.), cell cytoplasm (Cyto.), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Immunofluorescence, Labeling, Western Blot, Expressing, Mutagenesis, Control, Cell Culture, Over Expression, Fluorescence, Transfection, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 7 ZDHHC6-mediated palmitoylated PPARγ enhances its nucleus translocalization. (A) ZDHHC6 and PPARγ expression were examined in the ZDH HC6-deleted HCT116, SNU-C2A and SW48 cells, respectively (n = 3 per group). (B) ZDHHC6 and PPARγ co-expression in AdshZDHHC6-transfected HCT116 cells, along with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 vs. AdshRNA). The scale bars are 20 μm. (C) In ZDHHC6-deleted HCT116 or ZDHHC6-deleted SW48 cells, palmitoylation levels and PPARγ expression were analyzed using western blotting assay (n = 4 per group). (D) Western blotting assay using PPARγ, ACTIN, and HA antibodies, followed by PPARγ overexpressing the HA-tagged ZDHHC6 construct in various CRC cell lines (n = 3 per group). (E) Immunofluorescence pictures demonstrating the co-expression of PPARγ and ZDHHC6 in ZDHHC6-overex pressed HCT116 cells, together with the matching fluorescence density as determined by Pearson’s analysis (n = 4 per group; P < 0.05 compared to empty vector). The scale bars are 20 μm. (F) HCT116 cells underwent IP of HA after co-transfecting with PPARγ and HA-ZDHHC6. ZDHHC6 and PPARγ Mutual Co-IP shows that endogenous ZDHHC6 and PPARγ bind to each other in HCT116 cells. (G) Using various alkyl-labeled fatty acylation, such as alk-C14, alk- C16, alk-C18, and alk-C20, the palmitoylation of PPARγ in the indicated cells was detected. By using streptavidin bead pulldown to identify acylated PPARγ, an immunoblotting experiment using PPARγ and ACTIN antibodies (n = 6 per group) was performed. (H) To identify acylated PPARγ in SW48, LS1034, and HT-29 cells, the same methodology as in (G) was applied. Following that, the lysates (n = 6 per group) were subjected to western blotting analysis using PPARγ and ACTIN antibodies. (I) Using Click reaction-associated streptavidin pulldown, the palmitoylation levels of Flag-labeled PPARγ WT, PPARγ C313S, PPARγ C156S, PPARγ C176S, and PPARγ C159S mutants were examined. Three individuals per group underwent an immunoblotting experiment using Flag and ACTIN antibodies on the relevant lysates. (J) ZDHHC6-HA and PPARγ-Flag were the vectors used to transfect the HCT116 cells. Using alk-C16 labeling, higher, palmitoylated PPARγ levels were demonstrated in both the presence and absence of hydroxylamine therapy. The corresponding fluorescence density and ACLY and PPARγ co-expression in HCT116 WT or HCT116 ZDHHC6 (KO) cells are depicted in the lower representative immunofluorescence images, which were analyzed using Pearson’s method (n = 5 per group; P < 0.05 vs. WT). The scale bars are 20 μm. (K) After transfecting the HCT116 WT or HCT116 ZDHHC6 (KO) cells with PPARγ-Flag, the cells were labeled with alk-C16. To verify that the wild type and knockout cell components for input had the same quantity of PPARγ, subcellular fraction was obtained and PPARγ protein levels were adjusted. Western blotting analysis was used to assess palmitoylated PPARγ levels in the cell membrane (Mem.), cell cytoplasm (Cyto. ), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Transfection, Fluorescence, Western Blot, Construct, Immunofluorescence, Plasmid Preparation, Co-Immunoprecipitation Assay, Labeling, Knock-Out, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 9 ZDHHC6-driven lipid biosynthesis contributes to CRC carcinogen esis by upregulating PPARγ. (A, B) In HCT116-related stable cells (Control, ZDHHC6, and ZDHHC6 + shPPARγ) (A) and HCT116-related stable cells (shControl, shZDHHC6, and shZDHHC6 + PPARγ) (B), the percentages of different isotopomers of FFA C16:0 following exposure to [U-13C] glucose are shown. Each group has n = 5. (C, D) The relative TG content and PPARγ expression abundance in the aforementioned cell lines from (A) and (B) are displayed in representative immunofluorescence pictures. Each group has n = 5. The scale bars are 20 μm. (E) In null mice, right flanks were in jected with ZDHHC6 + shPPARγ, ZDHHC6, and Control, stable cells related to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (F) The right flanks of null mice were injected with shCon trol, shZDHHC6, and shZDHHC6 + PPARγ, stable cells linked to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n = 5. (G) Kaplan-Meier curves representing the survival analysis based on TCGA CRC prognostic data for ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-positive patients. (H) Based on the prognosis information from the ICGC CRC database, Kaplan-Meier curves were used to analyze the sur vival of ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-posi tive patients. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Control, Expressing, Immunofluorescence, Dissection, Injection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming.

doi: 10.1186/s13046-024-03154-0

Figure Lengend Snippet: Fig. 10 Palmitoylation stabilizes PPARγ by ZDHHC6 via blocking its lysosomal degradation to promotes lipid biosynthesis-associated CRC development. As a palmitoyltransferase enzyme, ZDHHC6 regulates the synthesis of fatty acids. To be more precise, ZDHHC6 directly attaches palmitoyl groups to PPARγ, a protein that controls the expression of genes. By stabilizing PPARγ and blocking its lysosomal degradation, the palmitoylation mechanism triggers the production of ACLY and subsequently leads to the development of lipid buildup-related CRC carcinogenesis

Article Snippet: The readymade CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc.

Techniques: Blocking Assay, Expressing

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Up-regulated Hallmark and Gene Ontology (GO) pathways in ID8 cell single-guide SMARCA4 (sg SMARCA4 ) compared to those in sgNTC. Ribodeplete RNA sequencing was performed. Statistical analysis was based on hypergeometric test and performed using ClusterProfiler. IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; TNFA, tumor necrosis factor–α; FDR, false discovery rate. ( B ) Gene expression heatmap of type I IFN pathway–related genes in ID8 cells. Reads per kilobase of transcript per million mapped reads values were scaled to z -score for visualization. Gene expression fold change of sg SMARCA4 versus sgNTC cells is color coded according to the legend. ( C ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation results for IFN genes in ID8 cells (sgNTC and four sg SMARCA4 clones). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control (sgNTC). n = 3 independent experiments. ( D ) MHC1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. ( E ) PD-L1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. MFI, median fluorescence intensity. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; n.s., not significant. Error bars represent ± SEM. Samples in duplicates [(A) and (B)] and triplicates [(D) and (E)]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Clone Assay, Expressing, Control, Flow Cytometry, Fluorescence, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Changes in genomic site accessibility in sg SMARCA4 (KO1) versus sgNTC cells. Log 2 fold change and FDR-adjusted P value (Wald test P values from DESeq2 with Benjamini-Hochberg correction). ( B ) Gene set enrichment analysis (GSEA) of immune pathways with increased accessibility in sg SMARCA4 versus sgNTC cells. Kolmogorov-Smirnov statistic with Benjamini-Hochberg correction. Exact q values indicated in each panel. ( C ) Changes in chromatin accessibility at transcription start sites (TSSs) of ISGs (CXCL10 and CCL2) in sg SMARCA4 versus sgNTC cells. ( D ) Motifs enriched in open chromosomal regions affected by SMARCA4 deficiency. P value and binomial test were performed using Homer2. Experiments performed in duplicates. GO, Gene Ontology; KO, knockout; KO_1, target exon 14 clone 1; NTC, non-target control; TF, transcription factor.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) IFNAR-1 neutralizing assay in ID8 single-guide NTC (sgNTC) compared to that in sg SMARCA4 cells (four sg SMARCA4 clones). Cells were treated with Mock or IFNAR-1 antibody for 48 hours at 10 μg/ml. ( B ) IRF3 expression levels in sg SMARCA4 versus sgNTC cells by qRT-PCR. ( C ) IRF3 expression in doxycycline-inducible short hairpin NTC (shNTC) and shIRF3-transfected sg SMARCA4 cells by qRT-PCR. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC and sg SMARCA4 cells. The latter were transfected with shIRF3 or shNTC (with and without POLYI:C). ( E ) Enriched IRF motif at TSSs of ISG locus from ATAC-seq analysis of ID8 sgNTC versus sg SMARCA4 cells. Experiments performed in duplicates. ( F ) Analysis of publicly available ChIP-Atlas data of IRF3 DNA binding sites on ISGs in murine immune cell lines. Statistical analysis was performed using two-tailored unpaired t test [(A) to (D)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. n = 3 independent experiments in (A) to (D). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sgNTC_1 in (A), (B), and (D) and sg SMARCA4 transfected with shNTC in (C), as indicated]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; LPS, lipopolysaccharide stimulated, NTC, non-target control; NTC_1 = NTC clone 1; POLYI:C, polyinosinic-polycytidylic acid stimulated.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Neutralizing Assay, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Volcano plots for differential expression of TEs in ID8 cells. ( B ) Heatmap of long terminal repeats (LTR) with adjusted P < 0.05. ( C ) Representative pictures (top) of double-stranded RNA (dsRNA) identification by immunofluorescence (IF) in sg SMARCA4 and sgNTC ID8 cells with quantification (bottom). RNAse, ribonuclease. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC cells and sg SMARCA4 cells transfected with shMAVS or shNTC. ( E ) MAVS expression in doxycycline-inducible shNTC and shMAVS-transfected sg SMARCA4 cells by qRT-PCR. Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sg SMARCA4 transfected with shNTC in (C) and sgNTC_1 in (D), as indicated]. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. Samples in duplicates [for (A) and (B)]. n = 3 independent experiments [in (C) and (D)]. Red line in (A) represents a cutoff of an adjusted P value of <0.05. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Immunofluorescence, Quantitative RT-PCR, Transfection, Expressing, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the OC tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated intraperitoneally with 10 million ID8 sgNTC or sg SMARCA4 ( KO_4 ) tumor cells, and spectral flow cytometry was performed 21 days later on harvested ascites samples. ip, intraperitoneal. ( B ) Frequency of tumor PD1 + CD4 + T cells and PD1 + CD8 + T cells. ( C ) Frequency of NK1.1 + cells expressing Granzyme B + . ( D ) Frequency of tumor dendritic cells (of CD45 + cells) and MHCII-expressing dendritic cells. ( E ) Frequency of tumor macrophages (of CD45 + cells) and PD-L1–expressing macrophages. ( F ) In vivo bioluminescence imaging of tumor burden in sg SMARCA4 versus sgNTC tumors (unpaired t test of area under the curve). Statistical analysis was performed using two-tailored unpaired t test [(B) to (F)]. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Error bars represent ± SEM. n = 10 mice per group. KO, knockout; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, In Vivo, Imaging, Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the B16-F10 tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated subcutaneously with 150,000 shNTC, sgEV, sh SMARCA4_1 , sh SMARCA4_2 , or sg SMARCA4 tumor cells, and spectral flow cytometry was performed 21 days later on harvested tumor samples. ( B ) Frequency of tumor CD8 + T cells and MFI of ICOS + -expressing CD8 + T cells in B16-F10 model. ( C ) Frequency of NK1.1 + cells in B16-F10 knockout and knockdown models (of CD45 + cells). ( D ) MFI of MHCII in macrophages in B16-F10 SMARCA4 knockout and knockdown models. ( E ) Tumor volume of sh SMARCA4 versus shNTC B16 tumors (unpaired t test of final time points). n = 5 mice per group, three biological replicates. ( F ) Frequency of NK1.1 + cells in sh SMARCA4 versus shNTC tumors (of live cells) in RAG2 −/− mice. ( G ) MFI of MHCII-expressing macrophages in B16-F10 sh SMARCA4 versus shNTC tumors in RAG2 −/− mice. ( H ) Tumor volumes of sh SMARCA4 versus shNTC B16 tumors in RAG2 −/− mice (unpaired t test of final time points). n = 3 to 5 mice per group, three biological replicates. Statistical analysis was performed using two-tailored unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent ± SEM. Dox, doxycycline; EV, empty vector; NTC, non-target control; SC, subcutaneous.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Knock-Out, Knockdown, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) mRNA abundance (TPM, transcripts per million) in WT β cells (left), DESeq2-adjusted P values from differential expression analysis in WT versus Bmal1 -/- β cells (middle), and presence or absence of an annotated BMAL1 binding site near genes of putative IVM targets (right). (B) Rhythmic expression of P2ry1 gene in synchronized pseudoislets from WT Beta-TC-6 cells as assessed by quantitative real-time PCR (n=3) (FDR adjusted P value < 0.05).

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Quantitative Proteomics, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) Venn diagram of BMAL1 binding sites identified by ChIP-sequencing overlapping with differentially-expressed genes identified by RNA-sequencing in Bmal1 -/- β -cell line compared to control cell line ( top ). Browser tracks showing decreased expression of P2ry1 gene in Bmal1 -/- cells compared to controls. BMAL1 binding sites upstream of the P2ry1 gene are also indicated ( bottom ). (B) Bioluminescence from WT insulin-NanoLuc pseudoislets in response to 10 µM IVM and/or 10 µM of the P2Y1 antagonist MRS2179 (n=4-8 experiments, 3-8 repeats per experiment). (C) Ratiometric determination of intracellular Ca 2+ using Fura2-AM dye in WT Beta-TC-6 cells stimulated in the presence or absence of 10µM IVM (n=3-8 experiments, 4-12 repeats per experiment). (D) Insulin secretion by ELISA in pseudoislets from P2ry1 KOs and control WT and Bmal1 -/- Beta-TC-6 cells (n=4/genotype/condition). Benjamini and Hochberg FDR-adjusted P values were computed for multiple comparisons following two-way ANOVA. (E) First two principal components (PC1 and PC2) following unbiased principal component analysis (PCA) of DESeq2 normalized counts in WT, WT + IVM, P2yr1 KO, and P2yr1 KO cells (n=4 per group). (F) Mean log 2 -transformed DESeq2-normalized counts in WT, WT + IVM, P2yr1 KO, and P2yr1 KO cells (n=4 per group) at differentially-expressed (1.5 fold, adjusted P value < 0.05) transcripts identified between WT and WT + IVM treated cells). All values represent mean + SEM. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Binding Assay, ChIP-sequencing, RNA Sequencing, Control, Expressing, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: bioRxiv

Article Title: Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

doi: 10.1101/2021.11.05.467499

Figure Lengend Snippet: (A) Quantitative real-time PCR screening for disruption of P2ry1 gene expression (n=3-4/genotype) ( top ). Decreased P2Y1 receptor protein expression by Western blot in WT and Bmal1 -/- Beta-TC-6 cells after genetic disruption ( bottom ). (B) Loss of effect of IVM on gene expression in P2ry1 mutant β cells identified by RNA-sequencing (n=4/genotype/condition). Dots represent values that exceed 1.5-fold of the interquartile range. All values represent mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Cells were co-transfected with guide RNA, P2Y1 CRISPR/Cas9 KO, and P2Y1 HDR plasmids (Santa Cruz Biotechnology, Dallas, TX) by Lipofectamine 2000 (Thermo Fisher Scientific, Amarillo, TX).

Techniques: Real-time Polymerase Chain Reaction, Disruption, Gene Expression, Expressing, Western Blot, Mutagenesis, RNA Sequencing

Journal: International Journal of Biological Sciences

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

doi: 10.7150/ijbs.69281

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

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

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

Journal: International Journal of Biological Sciences

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

doi: 10.7150/ijbs.69281

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

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

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

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) GLI1 mRNA levels according to TCGA data for patients with the seven deadliest cancers in the United States in 2014. The data are medians with the 5th and 95th percentiles and standard deviations (error bars).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques:

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) In vitro methylation assay with PRMT1 and wild-type (WT) or R597K-mutant Gli1. Left panel, Coomassie Blue staining. Right panel, fluorography.

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: In Vitro, Methylation, Mutagenesis, Staining

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Left panel, Western blot analysis of meGli1 and total Gli1 in MIA PaCa-2 luciferase cells stably transfected with an empty vector (Vec), wild-type Gli1 (Gli1WT), or R597K-mutant Gli1 (Gli1RK). Right panel, mRNA expression levels, measured by quantitative real-time PCR, of Gli1 target genes in Vec-, Gli1WT (WT)-, and Gli1RK (RK)-transfected MIA PaCa-2 cells. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01 (paired two-tailed Student’s t-test).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Western Blot, Luciferase, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Responses of MIA PaCa-2 stable clones to gemcitabine with or without PRMT1 depletion. shCtrl: control shRNA; shPRMT1: PRMT1 shRNA; Vec: MIA PaCa-2 stable clone with empty vector; WT: stable clone with wild-type Gli1; RK: stable clone with Gli1R597 mutant. Error bars represent SD (n = 4).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Clone Assay, Control, shRNA, Stable Transfection, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A–D) Quantitative polymerase chain reaction (qPCR) analyzing the gene transcript fold changes of Opa-1 and MICOS across aging: (A) Opa1 transcripts, (B) Mitofilin transcripts, (C) Chchd3 transcript, and (D) Chchd6 transcripts. (E) Western Blot of OPA1, mitochondrial dynamic proteins, and MICOS protein expression. For all panels, error bars indicate SEM, and Mann–Whitney tests were used for statistical analysis. Each dot represents an individual qPCR run (n=4). Significance values indicate ***P ≤ 0.001 and ****P ≤ 0.0001. For all western blotting experiments, n = 4.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A-E) Individual knockout (KO) of Opa1 , Mitofilin , Chchd3 , and Chchd6 and representative transmission electron micrographs. (F–H) quantification upon KO state of each MICOS gene and Opa1 (n = 10 cells) was performed in 3-D reconstruction: (F) average single mitochondrion area, (G) average single mitochondrion perimeter, (H) average single mitochondrion circularity index, and (I) average single mitochondrion length across individual MICOS KO. (J) 4′,6-diamidino-2-phenylindole (DAPI) staining, MitoPY1 (5 uM, 45 min at 370 c magnification of 60x), and merge channels in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (K) 4′,6-diamidino-2-phenylindole (DAPI) staining, MitoBright Deep Red (10 uM, 30 min at 37 0 c), DCFDA (10 uM, 30 min at 37 0 c, magnification of 60x), and merge channels in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (L) Plate reader-based reactive oxygen species (ROS) quantification. (M) Microscopy-based ROS quantification of MitoPY1 orange, (N) DCFDA, and (O) MitoSox Deep Red. For all statistical tests, a one-way ANOVA statistical test was performed with Dunnett’s multiple comparisons test. For 3D microscopy, each dot represents a mitochondrion, with their number varied between control (n=81), Opa1 KO (n=153), Chchd3 KO (n=139), Chchd6 KO (n=180), and Mitofilin KO (n=156). Significance values indicate *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001; ns, not significant.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Knock-Out, Transmission Assay, Staining, Control, Transfection, Microscopy

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A) Representative traces of mitochondrial calcium uptake in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (B) Percentage of mCa2+ uptake rate calculated from (C) representative traces of mitochondrial calcium retention capacity in control, MITOFILIN KD, and CHCHD6 KD HEK293 cells. The number of boluses of calcium taken up by cells is shown in circles. (D) Percentage change in mitochondrial calcium retention capacity calculated from representative traces of mitochondrial calcium retention capacity. (E) Western blot showing siRNA-mediated KD of CHCHD6 /CHCHD6 in HEK293 cells. (F) Western blot showing siRNA-mediated KD of MITOFILIN /MIC60 in HEK293 cells. (G) Serial block face scanning electron microscopy obtained representative images of mitochondria endoplasmic reticulum contact site morphology overlaid on orthoslice and (H) isolated in three dimensions in three-month and (I-J) 2-year samples. For all statistical tests, one-way ANOVA statistical test was performed with Dunnett’s multiple comparisons test. N=3-5 for all calcium experiments, as run in triplicates. Significance values indicate **P ≤ 0.01.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Control, Transfection, Western Blot, Blocking Assay, Electron Microscopy, Isolation