Journal: Biomedicines

Article Title: Whole Exome Sequencing Identifies PHF14 Mutations in Neurocytoma and Predicts Responsivity to the PDGFR Inhibitor Sunitinib

doi: 10.3390/biomedicines10112842

Figure Lengend Snippet: PHF14 Depletion Enhances Cell Proliferation and Anchorage Independent Cell Growth. ( A ) A conserved 19-bp region in the human, mouse and rat PHF14 gene in Exon 10 (CGC ATG ATT CAA ATT CAG GAA) was selected as shRNA target to knockdown PHF14 expression. Five cell lines originating from human, mouse and rat were used to evaluate the biological effect of PHF14 depletion. PHF14 knockdown stable transfectants were established by puromycin selection, and the knockdown efficiency was determined by Western Blot using anti-PHF14 antibody. The densitometric analyses of the protein bands vs. the individual loading controls were shown under individual blot using the ImageQuant 5.2 software (GE Healthcare, Pittsburgh, PA). ( B ) Cell proliferation rate was enhanced in shRNA PHF4 knockdown transfectants compared to Nonsense controls as measured by CellTiter-Glo ® luminescent cell viability assay in a variety of cell lines. ( C ) Soft agar assay demonstrated that PHF14 knockdown induced an increase in colony size in human neuroblastoma SHSY-5Y cells. ( D ) A single guiding RNA (sgRNA) targeting a 20-bp region (TGG ATC GCA GCT CCA AGA GG) in PHF14 Exon 1 was designed for CRISPR-Cas9 mediated genetic editing. The knockout efficiency was confirmed by Western Blot. PHF14 knockout in human neuroblastoma SHSY-5Y cells promoted cell proliferation as measured by CellTiter-Glo ® luminescent cell viability assay. PHF14 knockout increased colony formation in soft agar assay. The results shown were representative of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: ShRNA PHF14 (TRCN0000312505) was purchased from Sigma-Aldrich Corp. sgRNA/Cas9 all-in-one expression clone targeting PHF14 (HCP223067-CG01-3-B) and scrambled sgRNA control were from GeneCopoeia, Inc. (Rockville, MD, USA).

Techniques: shRNA, Knockdown, Expressing, Selection, Western Blot, Software, Cell Viability Assay, Soft Agar Assay, CRISPR, Knock-Out

Journal: Cancer cell

Article Title: Overcoming Resistance to Dual Innate Immune and MEK Inhibition Downstream of KRAS

doi: 10.1016/j.ccell.2018.08.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: plentiGuide-puro-scramble sgRNA control , Broad Institute , N/A.

Techniques: Control, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Viability Assay, shRNA, Negative Control, Software, Binding Assay, Expressing

Journal: Translational Oncology

Article Title: Exogenous dihomo-γ-linolenic acid triggers ferroptosis via ACSL4-mediated lipid metabolic reprogramming in acute myeloid leukemia cells

doi: 10.1016/j.tranon.2024.102227

Figure Lengend Snippet: ACSL4 mediates DGLA-induced ferroptosis. ( a ) Western blot showing ACSL4 protein levels in Kasumi-1 cells expressing negative control sgRNA (sgNC) or ACSL4-targeting sgRNA (sgACSL4). ( b ) Cell viability of Kasumi-1 cells expressing sgNC or sgACSL4 treated with erastin for 24 h. ( c ) Cell viability of Kasumi-1 cells expressing sgNC or sgACSL4 treated with DGLA for 24 h. ( d - f )The levels of lipid ROS ( d ), MDA ( e ), and Fe 2+ ( f ) in Kasumi-1 cells expressing sgNC or sgACSL4 treated with DGLA (20 µM) for 24 h. ( g ) TEM images of Kasumi-1 cells expressing sgNC or sgACSL4 treated with DGLA (20 μM) for 24 h. Scale bars: upper panel, 1 μm; lower panel, 500 nm. White arrows indicate normal mitochondria, red arrows indicate abnormal mitochondrial structure, yellow arrows indicate lipid droplets. Data are shown as mean ± SD, n = 3 biologically independent experiments, ** p < 0.01, *** p <0.001.

Article Snippet: The corresponding sgRNAs were designed and cloned into the vector, and negative control sgRNAs (sgNC) were generated as controls (Genechem, China).

Techniques: Western Blot, Expressing, Negative Control

Journal: Translational Oncology

Article Title: Exogenous dihomo-γ-linolenic acid triggers ferroptosis via ACSL4-mediated lipid metabolic reprogramming in acute myeloid leukemia cells

doi: 10.1016/j.tranon.2024.102227

Figure Lengend Snippet: ACSL4 reprograms DGLA-associated lipids. ( a ) Lipids were analyzed by LC-MS/MS. Heatmap shows lipids fold-changes in Kasumi-1 cells expressing sgNC or sgACSL4 treated with DGLA (20 µM). ( b ) Distribution of fatty acid chains of species from lipids in ( a ) that were down-regulated upon DGLA treatment. ( c ) Pie chart, by PUFA chain subclass, showing proportions of each lipid species in the down-regulated lipids upon DGLA treatment. ( d ) Pie charts, by phospholipid subclass, showing proportions of lipid species containing DGLA that were identified in ( a ). Data are shown as mean ± SD, n = 3 biologically independent experiments.

Article Snippet: The corresponding sgRNAs were designed and cloned into the vector, and negative control sgRNAs (sgNC) were generated as controls (Genechem, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: Translational Oncology

Article Title: Exogenous dihomo-γ-linolenic acid triggers ferroptosis via ACSL4-mediated lipid metabolic reprogramming in acute myeloid leukemia cells

doi: 10.1016/j.tranon.2024.102227

Figure Lengend Snippet: Deletion of ACSL4 inhibits the anticancer activity of DGLA in vivo . ( a ) Hypodermic injection of Kasumi-1 cells stably transfected with sgNC or sgACSL4 into BALB/c nude mice and treated with DGLA or vehicle, established subcutaneous xenograft tumors (n = 6 mice/group). ( b ) Tumor images show the relative sizes of the xenograft tumors formed by Kasumi-1 cells expressing sgNC or sgACSL4 and treated with DGLA or vehicle on day 18. ( c ) Changes in tumor volumes over time for mice implanted with Kasumi-1 cells expressing sgNC or sgACSL4 and treated with DGLA or vehicle. ( d - e ) Measures of tumor volume ( d )and tumor weight ( e ) values of mice at the study endpoint. ( f - h ) The levels of lipid ROS ( f ), MDA ( g ), and Fe 2+ ( h ) in xenograft tumors treated with vehicle or DGLA for 18 days (n = 6 tumor samples from different animals per group). ( i ) TEM images of mitochondria ultrastructure in xenograft tumors treated with DGLA or vehicle for 18 days. Scale bars: upper panel, 1 μm; lower panel, 500 nm. White arrows indicate normal mitochondria, red arrows indicate abnormal mitochondrial structure, yellow arrows indicate lipid droplets. Data are shown as mean ± SD, ** p < 0.01, *** p <0.001.

Article Snippet: The corresponding sgRNAs were designed and cloned into the vector, and negative control sgRNAs (sgNC) were generated as controls (Genechem, China).

Techniques: Activity Assay, In Vivo, Injection, Stable Transfection, Transfection, Expressing

Journal: bioRxiv

Article Title: Deletion of the transcriptional regulator TFAP4 accelerates c-MYC-driven lymphomagenesis

doi: 10.1101/2022.12.19.520971

Figure Lengend Snippet: A, Schematic representation of hematopoietic reconstitution of lethally irradiated recipient mice with fetal liver derived donor HSPCs. Fetal liver cells from double transgenic Eμ-MYC/Cas9 E13.5 donor embryos (an abundant source of HSPCs) were lentivirally transduced with vectors encoding BFP or CFP as a marker and sgRNAs targeting Tfap4 (sgTfap4 ), a positive sgRNA targeting Trp53 (sgTrp53 ) or a negative non-targeting control sgRNA ( sgControl ). These transduced donor fetal liver cells were then transplanted into lethally irradiated C57BL/6-Ly5.1 recipient mice. Tumour-free survival of recipient mice was measured as days post-transplantation. Hematopoietic tissues and peripheral blood were harvested from tumour burdened recipient mice for further analysis. B, Kaplan-Meier survival curve showing tumour-free survival of mice transplanted with either of two vectors containing a sgTfap4 , a positive control sgTrp53 or a negative sgControl . N indicates number of sick mice/number of mice transplanted with sgRNA transduced HSPCs for each sgRNA. Median survival post transplantation in days is indicated in brackets. **** P < 0.0001.

Article Snippet: A vector constitutively expressing a negative control sgRNA targeting human NLRC5 (sgRNA-1 5’-GCTGCAGAAGTGTCAGCTCCAGG) and the BFP tag was also obtained from the Merck CRISPR glycerol stock arrayed whole mouse genome sgRNA library, and this vector was used for the pre-leukemic analyzes.

Techniques: Irradiation, Derivative Assay, Transgenic Assay, Transduction, Marker, Transplantation Assay, Positive Control

Journal: Theranostics

Article Title: EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells

doi: 10.7150/thno.48101

Figure Lengend Snippet: EZH2 knockdown reduces CSCs and inhibits the chemoresistance and tumorigenesis of ovarian cancer cells. (A) Western blot analysis of EZH2 expression in SKOV3, SK-1st, SK-2nd and SK-3rd cells (top). Representative images of spheres after 14 days of suspension culture (bottom). The scale bar is 50 µm. (B, C) qRT-PCR analysis of EZH2 mRNA expression in IGROV1/CD133+ (B) and SKOV3/ALDH1+ (C) cells compared to IGROV1/CD133- or SKOV3/ALDH1- cells. (D) Representative images and a statistical histogram of spheres generated by SKOV3 and OVCAR4 cells transfected with an overexpression plasmid DNA for EZH2 or a scrambled control. (E) Representative fluorescence images and a statistical histogram of spheres generated by SK-3rd cells transfected with a GFP-tagged lentivirus expressing shRNA targeting EZH2 (shEZH2) or nontargeting scrambled shRNA (shNC). (F) The proportion of ALDH1+ cells in SK-3rd cells decreased significantly after genetic knockdown of EZH2. (G) The 50% inhibitory concentration (IC50) of cisplatin in SK-3rd/shEZH2 and SK-3rd/shNC cells is shown. (H, I) Representative FACS images ( H ) and a statistical histogram ( I ) of the proportion of apoptosis/necrosis in GFP-labeled SK-3rd/shEZH2 cells compared with SK-3rd/shNC cells after 20 µM cisplatin treatment for 48 h. The proportion of apoptotic and necrotic cells (Annexin V+ and/or PI+) was compared between groups. (J) Effects of the EZH2 inhibitor (EPZ6438) on apoptosis induction in SK-3rd cells. The cells were treated with DMSO (control), cisplatin (20 µM) plus DMSO, or cisplatin in combination with EPZ6438 (5 µM) for 48 h. The proportion of apoptotic and necrotic cells (Annexin V+ and/or PI+) was compared between groups. (K, L) SK-3rd cells were exposed to cisplatin (5 or 10 µM), EPZ6438 (5 µM) or cisplatin in combination with EPZ6438 for 48 h. Representative phase contrast images of colony formation assays ( K ) and the proportion of cell viability ( L ) are shown. The data in B, C, D, E, F, G, I, J and L are the means ± SD of three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.001).

Article Snippet: The Cas9 lentivirus and lentiviral sgRNA vectors targeting EZH2 (sgEZH2) or scrambled control (sgNC) was purchased from Genechem (Shanghai, China).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Generated, Transfection, Over Expression, Plasmid Preparation, Fluorescence, shRNA, Concentration Assay, Labeling

Journal: Theranostics

Article Title: EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells

doi: 10.7150/thno.48101

Figure Lengend Snippet: CHK1 is an EZH2 target involved in CSC stemness. (A) Hierarchical clustering displays differential expression profiles for SK-3rd/shNC and SK-3rd/shEZH2 cells (N = 3 replicates). Rows represent individual samples, and columns represent genes. Each cell corresponds to the expression level of a particular gene in a given sample. A visual dual color code is used, with red and green indicating relatively high and low expression levels, respectively. A color scale is included to reflect gene expression. (B) Relative mRNA levels of CHK1 , LIN28A , IL8 and WEE1 in SK-3rd cells transfected with EZH2-targeted shRNA or scrambled shRNA. (C, D) qRT-PCR analysis of CHK1 mRNA expression in SKOV3/EZH2 (C) and SK-3rd/sgEZH2 cells (D) compared with scrambled control cells. Expression levels were normalized to β-actin. EZH2 was knocked out of SK-3rd cells using the CRISPR-Cas9 system. (E) Luciferase activity assays in SKOV3 and A2780 cells showing transactivation of the CHK1 promoter by EZH2 overexpression and repression of the CHK1 promoter by EZH2 silencing. (F-H) Schematic illustration of PCR-amplified fragments of the CHK1 promoter that was physically associated with EZH2 (F) . Chromatin immunoprecipitation (ChIP) assays were performed using an EZH2 antibody to screen EZH2-bound CHK1 promoter regions (region 1# (nucleotides -301 to -106) and region 2# (nucleotides +129 to +316)) in SKOV3 (G) and A2780 (H) cells. Human HOXA2 promoter primers were used as positive controls and α-satellite repeat primers were used as negative controls. Blue bars: incubation with negative control IgG; Red bars: incubation with EZH2 antibody. Each negative IgG control was normalized to the unit “1”. The data in B, C, D, E, G and H are the means ± SD of three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.001).

Article Snippet: The Cas9 lentivirus and lentiviral sgRNA vectors targeting EZH2 (sgEZH2) or scrambled control (sgNC) was purchased from Genechem (Shanghai, China).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, CRISPR, Luciferase, Activity Assay, Over Expression, Amplification, Chromatin Immunoprecipitation, Incubation, Negative Control

Journal: Theranostics

Article Title: EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells

doi: 10.7150/thno.48101

Figure Lengend Snippet: EZH2 regulates ovarian CSC properties through upregulation of CHK1. (A, B) Western blot analysis of EZH2 and CHK1 levels in SK-3rd/sgNC and SK-3rd/sgEZH2 cells transfected with an overexpression plasmid for CHK1 or a scrambled control (A) . Representative images and the number of spheres formed by 1,000 cells in each group are shown (B) . Scale bar: 50 µm. (C) The IC50s of cisplatin in SK-3rd, sgNC/NC, sgEZH2/NC and sgEZH2/CHK1 cells are presented as the means ± SD. (D) Colony formation assay showing the proportion of cell viability in SK-3rd, sgNC/NC, sgEZH2/NC and sgEZH2/CHK1 cells after cisplatin treatment at a concentration of 5 or 10 µM. (E, F) Representative FACS images (E) and a statistical histogram (F) of the proportions of the cell cycle distributions of SK-3rd, sgNC/NC, sgEZH2/NC and sgEZH2/CHK1 cells treated with PBS (control) or cisplatin (20 µM) for 48 h. The proportion of SK-3rd cells in the S and G2/M phases was compared between groups. (G, H) Representative FACS images (G) and a statistical histogram (H) of the proportions of apoptotic/necrotic cells among SK-3rd, sgNC/NC, sgEZH2/NC and sgEZH2/CHK1 cells treated with PBS (control) or cisplatin (20 µM) for 48 h. The proportion of apoptotic and necrotic cells (Annexin V+ and/or PI+) was compared between groups. (I) Western blot analysis for CHK1, pCHK1, pH2AX and p-CDC25C in SK-3rd, sgNC/NC, sgEZH2/NC and sgEZH2/CHK1 cells treated with PBS (control) or cisplatin (20 µM) for 48 h. The data in B, C, D, F and H are the means ± SD of three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.001).

Article Snippet: The Cas9 lentivirus and lentiviral sgRNA vectors targeting EZH2 (sgEZH2) or scrambled control (sgNC) was purchased from Genechem (Shanghai, China).

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Colony Assay, Concentration Assay

Journal: Theranostics

Article Title: EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells

doi: 10.7150/thno.48101

Figure Lengend Snippet: Overexpression of EZH2 is positively correlated with enhanced expression of pCHK1 in aggressive human ovarian tumors. (A) EZH2 and pCHK1 expression in EOC tissues taken from 44 patients was assessed by IHC. Representative IHC images of varying EZH2 (top) and pCHK1 (bottom) expression in human EOC are shown. Scale bar: 50 µm. (B) The expression of EZH2 or pCHK1 in platinum-resistant ovarian cancer tissues was significantly increased compared with that in platinum-sensitive ovarian cancer tissues. **P < 0.01. (C) Pearson's Chi-square test showing that EZH2 expression was associated with pCHK1 expression. (D) EZH2 high /pCHK1 high tumors were more likely to be platinum-resistant, whereas EZH2 low /pCHK1 low tumors were more likely to be platinum-sensitive. (E-H) Kaplan-Meier curves showing the PFS (E, F) and OS (G, H) of 44 EOC patients stratified by EZH2 or pCHK1 expression status. The log-rank test P values are shown.

Article Snippet: The Cas9 lentivirus and lentiviral sgRNA vectors targeting EZH2 (sgEZH2) or scrambled control (sgNC) was purchased from Genechem (Shanghai, China).

Techniques: Over Expression, Expressing