Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet:

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: Journal of Molecular Cell Biology

Article Title: AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX–MDM2 complex

doi: 10.1093/jmcb/mjw045

Figure Lengend Snippet: AXL induces MDMX Ser314 phosphorylation through the kinases CDK4/6 and p38. (A-i) Peptide count of the phosphosite Ser314 on MDMX in FLAG-MDMX and MDM2 versus FLAG-MDMX, MDM2, and AXL transfected 293T cells. (ii) MDMX phosphorylation map, indicating the main known phosphorylation sites and the novel Ser314 site. (iii) Scoring of CDK4/6 and p38 kinases, as predicted by the GPS (Xue et al., 2008) software. (B–D) 293T cells were transfected with various combinations of expression vectors for wt MDMX, MDMX(S314A), MDM2, and either AXL (B), p38 (C), or CDK4 (D). Western blot analysis was carried out with the indicated antibodies. (E) An in vitro kinase assay was carried out using immunopurified FLAG-tagged MDMX or MDMX(S314A) proteins incubated with cell lysates expressing constitutively active CDK4 or p38, or kinase-dead (K.D.) CDK4 or p38, in the presence or absence of adenosine triphosphate (ATP). The products were analysed by western blot using anti-phosphoserine antibody (upper panel) and anti-MDMX antibody (lower panel). (F) 293T cells were transfected with MDMX, MDM2, and AXL, and then treated with 0, 30, or 300 nM of Palbociclib (Palb) (i) or BIRB (ii) for 24 h. Western blot analysis was carried out with the indicated antibodies. (G) M14 cells were transfected with control plasmid, AXL, or CDK4 expression plasmids, and treated with 30 nM of Palbociclib for 8 h, followed by western blot analysis. (H) 293T cells were co-transfected with MDMX and MDM2, together with control or AXL expression plasmids, and then treated with either DMSO, LY294002 (LY, 40 μM), Foretinib (Foret, 1 μM), or R428 (1 μM) for 8 h. Western blot analysis was carried out with the indicated antibodies. (I) Schematic diagram of working hypothesis.

Article Snippet: Recombinant human Gas6 protein was purchased from R&D Systems and prepared in sterile water; R428, Foretinib, Palbociclib, BIRB 796, and Cisplatin were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Transfection, Software, Expressing, Western Blot, In Vitro, Kinase Assay, Incubation, Control, Plasmid Preparation

Journal: Journal of Molecular Cell Biology

Article Title: AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX–MDM2 complex

doi: 10.1093/jmcb/mjw045

Figure Lengend Snippet: AXL increases MDMX–MDM2 affinity and protects MDMX from MDM2-mediated degradation. (A) A Gal4-Luc mammalian two-hybrid reporter system expressing Gal4-MDM2 and VP16-MDMX alone or with different amounts of AXL expression plasmid was used in 293T cells. After transfection, cells were incubated for 24 h with either DMSO, Foretinib (1 μM), or Palbociclib (300 nM) before cells were harvested and analysed either with western blot (i) or using a Dual-Glo® Luciferase Assay System (Promega) (ii). E.V., empty vector. (B) 293T cells were co-transfected with various combinations of plasmids expressing AXL, MDM2, MDMX, MDMX(C462A), or MDMX(S314A), followed by western blot analysis with the indicated antibodies. (C) SK-MEL-28 cells were transfected with scrambled siRNA (siCtrl) or siRNA targeted against AXL (siAXL), combined with 1.5 or 3 μl of siRNA against MDM2 (siMDM2, 10 μM). (D) A375 cells were transfected with siCtrl or siAXL, and treated with 7.5 μg/ml of the proteasome inhibitor MG132 for 8 h. Cell lysates were harvested 48 h after transfection and subjected to western blot analysis. Relative amount of MDMX was quantified by ImageLab.

Article Snippet: Recombinant human Gas6 protein was purchased from R&D Systems and prepared in sterile water; R428, Foretinib, Palbociclib, BIRB 796, and Cisplatin were purchased from Selleck Chemicals.

Techniques: Expressing, Plasmid Preparation, Transfection, Incubation, Western Blot, Luciferase

Journal: Journal of Molecular Cell Biology

Article Title: AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX–MDM2 complex

doi: 10.1093/jmcb/mjw045

Figure Lengend Snippet: AXL inhibition enhances p53 pathway response to Cisplatin treatment. (A, C) A375 cells were transfected with scrambled siRNA (siCtrl) or siRNA targeted against AXL (siAXL) (A) or CDK4 (C) for 36 h, split into p35 dishes, cultured overnight, and then treated with 0, 10, or 15 μM of Cisplatin for 3 h. Cell lysates were harvested for western blot analysis with the indicated antibodies. (B) A375 cells were treated with DMSO, R428 (1 μM), or Foretinib (1 μM) for 8 h, and then treated with Cisplatin for 3 h before being harvested. Total RNA was extracted and subjected to qPCR analysis with the indicated primers. (D) A375 cells were treated with DMSO or Palbociclib (30 nM) for 0, 4, or 24 h, followed by treatment with Cisplatin for 3 h. Total RNA was extracted and subjected to qPCR analysis with the indicated primers. Gene expression was normalized to ACTB. Bars indicate SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Recombinant human Gas6 protein was purchased from R&D Systems and prepared in sterile water; R428, Foretinib, Palbociclib, BIRB 796, and Cisplatin were purchased from Selleck Chemicals.

Techniques: Inhibition, Transfection, Cell Culture, Western Blot, Gene Expression

Journal: Journal of Molecular Cell Biology

Article Title: AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX–MDM2 complex

doi: 10.1093/jmcb/mjw045

Figure Lengend Snippet: Knockdown of AXL increases the sensitivity of melanoma cells to Cisplatin and Vemurafenib. (A, B) A375 and SK-MEL-28 cells were transfected with scrambled siRNA (siCtrl) or siRNA targeted against AXL (siAXL). After 24 h, cells were split into 96-well plate for cell viability assay using AlamarBlue (A) or 6-well plate for colony formation assay (B). Cisplatin was used at increasing doses (50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM) for the cell viability assay and at the indicated doses for the colony formation assay. (C) p53 wild-type (A375-shCtrl) and p53 knockdown (A375-shp53) cells were seeded in 6-well plates and treated with Vemurafenib alone or combined with R428 or Foretinib at the indicated concentrations. SA-β-Gal staining was performed 10 days after treatment, and crystal violet staining was performed 8 days after treatment. Scale bar, 100 μm. (D) The intensity of crystal violet staining was measured by absorbance (i), and the relative difference of crystal violet staining between DMSO-treated and Foretinib- or R428-treated cells was calculated (ii, iii).

Article Snippet: Recombinant human Gas6 protein was purchased from R&D Systems and prepared in sterile water; R428, Foretinib, Palbociclib, BIRB 796, and Cisplatin were purchased from Selleck Chemicals.

Techniques: Knockdown, Transfection, Viability Assay, Colony Assay, Staining

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Photograph of the cut section of an ovarian sclerosing stromal tumor (SST; left) displaying classic SST appearance with yellow tissue at periphery and white, central fibrotic depression, and micrographs of hematoxylin & eosin stained representative section at low (top right) and high (bottom right) magnification. Scale bars, 1 cm (left), 200 μm (top right), 50 μm (bottom right). b Schematic representation of the FHL2-GLI2 fusion transcript including the exons and domains involved. The breakpoint of the 5′ and 3′ partner genes are represented as black vertical lines. Spanning reads are depicted and aligned to the predicted junction sequence. c Schematic representation showing the Reads Per Kilobase per Million (RPKM) mapped read counts of each GLI2 exon. The GLI2 fusion breakpoint is represented as a red dashed line. d Fluorescence in situ hybridization (FISH) of two representative SSTs using a three-color FHL2-GLI2 probe, with 5′ GLI2 (orange), 3′ GLI2 (red), and 5′ FHL2 (green), showing the presence of the FHL2-GLI2 fusion (white arrows). e Representative Sanger sequencing electropherograms of the genomic FHL2-GLI2 breakpoint. f RNA in situ hybridization (RNA-ISH) using custom FHL2-GLI2 probes (red) showing the chimeric FHL2-GLI2 mRNA expression in two representative SSTs harboring the FHL2-GLI2 fusion. g Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs from this study. h Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs and frequency of the FHL2-GLI2 fusion gene in 48 other ovarian sex cord-stromal tumors from this study. aGCT, adult-type granulosa cell tumor. i Frequency of FHL2-GLI2 fusion gene in 26 SSTs from this study and in 9950 tumors from 33 cancer types from The Cancer Genome Atlas (TCGA). AML acute myeloid leukemia, PCPG pheochromocytoma and paraganglioma.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Staining, Sequencing, Fluorescence, In Situ Hybridization, RNA In Situ Hybridization, Expressing

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY), and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. b Representative images of colony formation assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 5 mm; top). Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2 or FHL2-GLI2. The migratory effects/wound area was assessed at 0 and 24 h (Scale bar, 500 μm; top) and quantified (bottom). In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Quantitative assessment of CALB2 transcripts in immortalized mesenchymal stem cells (MSCs) and HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. b Representative western blot analysis of calretinin protein levels in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (bottom) of protein levels as compared to control. c Representative confocal micrographs of immunofluorescence analysis of calretinin (green) and 4–6-diamidino-2-phenylindole (DAPI, blue) in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 50 μm). Quantification (bottom) of calretinin intensity/cell relative to control. d Quantitative assessment of FOXL2 transcripts in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. In a – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Differential gene expression analysis of human sclerosing stromal tumors (SSTs) subjected to RNA-sequencing ( n = 8, this study) and high-grade serous ovarian carcinomas ( n = 16; The Cancer Genome Atlas) and other sex cord-stromal tumors (SCSTs, n = 11; this study). Gene expression fold-change is color-coded according to the legend. Only genes significantly differentially expressed ( P < 0.05; two-tailed unpaired t -test) are shown. CPM, count per million. b Expression levels of Sonic Hedgehog (SHH) pathway genes in human SSTs ( n = 11) and other sex-cord stromal tumors ( n = 9) as defined using NanoString. Expression levels and SHH enrichment scores are color-coded according to the legends. *** P < 0.001, Wilcoxon rank test. Hierarchical clustering was performed using complete linkage and Euclidian distance. c Quantitative assessment of the Sonic Hedgehog pathway PTCH1 and GLI1 transcripts in immortalized mesenchymal stem cells (MSCs), HEK-293 and medulloblastoma (DAOY) cells and of PTCH1 and CCND1 transcripts in human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. d Representative western blot analysis of PTCH1 and GLI1 protein expression in MSC, HEK-293 and DAOY cells and of PTCH1 and CCND1 in BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (below) of protein levels as compared to control. In c – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Representative confocal micrographs of immunofluorescence analysis of FLAG (red), 4–6-diamidino-2-phenylindole (DAPI, blue), and GFP (green) in HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Scale bars, 10 μm. b GLI response element (GLI-RE) luciferase reporter assay of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (top), and GLI-RE promoter activity in HEK-293 transiently transfected with control, FHL2-GLI2 and FHL2-GLI2 with R338A-K339A mutations in the Zinc Finger 5 of GLI2 required for DNA binding (FHL2-GLI2 Mutated; bottom). SV40-Renilla was used to normalize transfection efficiency. c Immunoprecipitation assay with SUFU antibody of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Western blot analysis using anti-FLAG and anti-SUFU antibodies, and tubulin as loading control (left). GLI-RE promoter activity in HEK-293 stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 transfected with SUFU or control (right). d Cell titer blue proliferation assay of HEK-293, DAOY and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 20 µM GANT61 or vehicle control (DMSO). GANT, GANT61. e FLAG Chromatin Immunoprecipitation (ChIP) assay of GLI1 and PTCH1 promoters (promoter 1 and 2) in MSC and HEK-293 cells stably expressing either control or FHL2-GLI2. GLI1 and PTCH1 gene body and MYOD1, gene promoters not under GLI regulation, were used as negative controls. In b – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; *P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Proliferation Assay, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY) and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2) or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). b Representative images of colony formation assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with Vismodegib 500 nM or vehicle control (DMSO). Scale bars, 5 mm. Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). The migratory effect/wound area was assessed at 0 and 24 h and quantified compared to DMSO (bottom). Vismo, Vismodegib. Scale bars, 500 μm. In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Genes & Development

Article Title: STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

doi: 10.1101/gad.189001.112

Figure Lengend Snippet: QKI is a GBM tumor suppressor gene. (A–C) QKI was knocked down by shRNAs, and 5000 human GBM cells or 20,000 mouse astrocytes were plated per well in six-well plates for soft agar colony assay. Experiments were performed in triplicates. U87 (p53-WT) (A) and Hs683 (p53-null) (B) human GBM cell lines, as well as Ink4a/Arf−/− Pten−/− primary mouse astrocytes (C) transduced with two independent shRNAs (shQKI-1 and shQKI-2) against QKI showed increased soft agar colony growth relative to shGFP and noneffective shQKI control (shQKI-3). A Western blot showing QKI protein levels upon shRNA knockdown is presented in A for the human system and in C for the mouse system. The soft agar colony result is presented in B; magnification 40×. (**) P < 0.01; (***) P < 0.001 compared with shGFP control. (D) We injected 2 × 106 Hs683 cells or Ink4a/Arf−/− Pten−/− mouse astrocytes with QKI knockdown by two independent shRNAs into each flank of immuno-compromised nude mice. shQKI cells in both cell types induced subcutaneous tumor formation in immuno-compromised nude mice but not the shGFP and noneffective shQKI controls. (E) Wild-type (WT) or RNA-binding-deficient (V157E) QKI isoforms were overexpressed individually or all together in U343 (p53-WT) and LN18 (p53-null) human GBM cells, and 5000 cells per well in six-well plates were plated for soft agar colony assay. Experiments were performed in triplicates. Overexpression of QKI-5, QKI-6, and QKI-7 isoforms in U343 and LN18 cells decreased soft agar colony formation. Overexpression of mutant QKI isoforms increased soft agar colony formation as a dominant-negative effect. QKI-5 and QKI-5 mutant migrate at ∼45 kDa, QKI-6 and QKI-6 mutant migrate at ∼38 kDa, QKI-7 and QKI-7 mutant migrate at ∼42 kDa. U343 cells were seeded in soft agar containing 5% serum; LN18 cells were in 1% serum. (**) P < 0.01; (***) P < 0.001 compared with vector control.

Article Snippet: The human glioma cell lines U343, LN18, U87, and Hs683 and the cell line HEK293T were purchased from American Type Culture Collection (ATCC).

Techniques: Colony Assay, Transduction, Control, Western Blot, shRNA, Knockdown, Injection, RNA Binding Assay, Over Expression, Mutagenesis, Dominant Negative Mutation, Plasmid Preparation

Journal: Genes & Development

Article Title: STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

doi: 10.1101/gad.189001.112

Figure Lengend Snippet: miR-20a partially carries out QKI tumor suppression. (A) Hs683-shGFP and Hs683-shQKI-2 were transiently transfected with miR-20a synthetic mimic or control mimic (miR-NT) and plated at 5000 cells per well in six-well plates for soft agar colony assay. Experiments were performed in duplicates. The miR-20a mimic decreased soft agar colony in shGFP cells relative to miR-NT control in shGFP cells. While shQKI and miR-NT double-transduced cells increased soft agar colony relative to shGFP and miR-NT double-transduced cells, miR-20a and shQKI double-transduced cells decreased soft agar colony relative to shQKI and miR-NT double-transduced cells. Furthermore, miR-20a and shQKI double-transduced cells exhibited decreased colonies relative to miR-NT and shGFP double-transduced cells. (**) P < 0.01; (***) P < 0.001 compared with miR-NT. (B) U87-shGFP and U87-shQKI-2 were stably transduced with miR-20a or miR-NT and plated for soft agar colony assay. While miR-20a decreased soft agar colony in shGFP cells, miR-20a also decreased soft agar colony in shQKI cells relative to miR-NT controls. Magnification, 40×. Experiments were performed in duplicates. (C) HCT116 colorectal cancer cells were stably transduced with miR-20a or miR-NT, and 5000 cells per well were plated in six-well plates for soft agar colony assay. Experiments were performed in duplicates. miR-20a-transduced HCT116 showed increased soft agar colony formation relative to miR-NT-transduced cells. (*) P < 0.05 compared with miR-NT. (D) We injected 2 × 106 shQki-2-Ink4a/Arf−/− Pten−/− astrocytes transduced with miR-20a or miR-NT subcutaneously into each flank of immuno-compromised nude mice, and Kaplan-Meier tumor-free survival curves were plotted. miR-20a-transduced shQki-2 cells showed significantly longer latencies in tumor formation relative to miR-NT-transduced shQki-2 cells (P = 0.03). (E) All QKI isoforms were stably overexpressed in U343 and LN18 cells together with miRZip-Ctrl or miR-Zip-miR-20a for miR-20a knockdown; 5000 cells were plated per well in six-well plates for soft agar colony assay, and experiments were performed in duplicates. QKI overexpression decreased soft agar colony formation in miRZip-Ctrl-transduced U343 and LN18 cells. QKI overexpression also rescued the increased soft agar colony generated by miRZip-20a. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (F) miR-20a levels were measured by qPCR in U343 and LN18 transduced with vector or QKI together with miRZip-Ctrl or miRZip-miR-20a for miR-20a knockdown. QKI overexpression increased miR-20a levels in miRZip-Ctrl-transduced U343 and LN18 cells, while QKI overexpression in miRZip-miR-20a cells rescued miR-20a expressions. (*) P < 0.05; (**) P < 0.01.

Article Snippet: The human glioma cell lines U343, LN18, U87, and Hs683 and the cell line HEK293T were purchased from American Type Culture Collection (ATCC).

Techniques: Transfection, Control, Colony Assay, Stable Transfection, Transduction, Injection, Knockdown, Over Expression, Generated, Plasmid Preparation

Journal: Genes & Development

Article Title: STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

doi: 10.1101/gad.189001.112

Figure Lengend Snippet: miR-20a carries out QKI tumor suppressor function partially via inhibiting TGFβR2. (A) Increasing concentration of siRNA against Qki progressively up-regulated Tgfβr2 protein levels in Ink4a/Arf−/− Pten−/− astrocytes. Relative protein levels for Qki and Tgfβr2 were quantified in the Western blot. (B) shQKI-2-transduced U87 and shQki-2-transduced Ink4a/Arf−/− Pten−/− astrocytes exhibited elevated TGFβR2 protein levels, as quantified by Western blot. Stable overexpression of miR-20a reversed the increased TGFβR2 protein levels in both cell types. (C) Hs683 with QKI knockdown increased soft agar colony formation. TGFβR2 knockdown by two independent shRNAs rescued the increased soft agar colony formation generated by QKI knockdown. Five-thousand cells were plated per well in six-well plates, and experiments were performed in duplicates. (*) P < 0.05; (**) p < 0.01. (D) U87 transduced with shQKI increased soft agar colony formation. TGFβR2 knockdown by two independent shRNAs rescued the increased soft agar colony formation generated by QKI knockdown. Magnification, 40×. Five-thousand cells were plated per well in six-well plates, and experiments were performed in duplicates. (E) We injected 2 × 106 shQki-2-Ink4a/Arf−/− Pten−/− mouse astrocytes transduced with shRNA against Tgfβr2 or GFP subcutaneously into each flank of immuno-compromised nude mice, and Kaplan-Meier tumor-free survival curves were plotted. While shQki-transduced cells formed tumors, shGFP-transduced cells did not form tumors. ShTgfβr2-transduced cells rescued the tumor formation enhanced by shQki-transduced cells (P = 0.02).

Article Snippet: The human glioma cell lines U343, LN18, U87, and Hs683 and the cell line HEK293T were purchased from American Type Culture Collection (ATCC).

Techniques: Concentration Assay, Western Blot, Over Expression, Knockdown, Generated, Transduction, Injection, shRNA

Journal: Scientific Reports

Article Title: CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation

doi: 10.1038/srep04261

Figure Lengend Snippet: (a) NB4 or HT93 APL cell lines were stably transduced with pLKO.1 lentiviral vectors expressing non-targeting or two independent CEBPA -targeting shRNAs. APL control and CEBPA knockdown cells were differentiated with 1 μM ATRA for 4 days. Knockdown efficiency in NB4 (top panels) or HT93 (bottom panels) APL cells was validated by qPCR. CEBPA mRNA expression was normalized to the housekeeping gene HMBS and is shown as n-fold changes compared to untreated SHC002 control cells. HK3 (b) and KLF5 (c) mRNA expression in NB4 or HT93 APL cells was determined by qPCR and analyzed as in 2a. Impairment in granulocytic differentiation of CEBPA knockdown cells was shown by a reduction of the neutrophil marker granulocyte colony-stimulating factor receptor (GCSFR or CSF3R) (d) Data represent the mean ± s.d. of at least three independent experiments. (e), (f) CEBPA Knockdown efficiency at the protein level in NB4 and HT93 APL cells was confirmed by western blotting. GAPDH is shown as a loading control. MWU: *p < 0.05, **p < 0.01 and ***p < 0.001.

Article Snippet: Data represent log 2 expression levels and the measured cycle threshold (Ct) values were normalized to the expression levels of the housekeeping genes HMBS and ABL1 .For quantification of KLF5, CEBPA, CEBPE and G-CSFR mRNA in human cells we used Taqman® Gene Expression Assays Hs00156145_m1, Hs00269972_s1, Hs00357657_m1, and Hs00167918_m1, (Applied Biosystems), respectively.

Techniques: Stable Transfection, Transduction, Expressing, Control, Knockdown, Marker, Western Blot

Journal: Nature Cancer

Article Title: Direct and selective pharmacological disruption of the YAP–TEAD interface by IAG933 inhibits Hippo-dependent and RAS–MAPK-altered cancers

doi: 10.1038/s43018-024-00754-9

Figure Lengend Snippet: a , Left, YAP and TAZ proteins mapped on the TEAD3 surface, as a result of structural alignments with the complex structures (PDB codes 5GN0 , 5OAQ ). The TEAD3 surface is shown in gray with the bound myristate in the buried LP drawn in dark gray. Middle/right, TEAD3–IAG933 cocrystal structure. Bound inhibitor is shown as a stick model with its surface in orange. IAG933 binds within the TEAD Ω-loop pocket to prevent coactivator binding by steric hindrance. The main hydrogen bonds and the salt bridge between protein, inhibitor and water are indicated with dotted green lines. b , Coimmunoprecipitation after a 4-h incubation of NCI-H2052 or MSTO-211H cells with DMSO (0 or –) or the indicated concentrations of IAG933 shows compound-induced inhibition of YAP and TAZ binding to TEAD isoforms. The blots are representative of two individual experiments; IP, immunoprecipitation. c , Dose-dependent inhibition of TEAD target gene expression in MSTO-211H and NCI-H226 cells treated for 24 h with IAG933. IC 50 values are between 11 and 26 nM. d , TEAD target gene inhibition kinetics (mean ± s.e.m., n = 4 of the three genes combined) in four mesothelioma cell lines treated with 300 nM IAG933. e , Antiproliferative activity of IAG933 (72-h treatment) in a panel of mesothelioma, Hippo-altered, non-Hippo-mutated or insensitive cell lines. The GI 50 in MSTO-211H cells was 73 nM. The results from one experiment or the mean of two experiments is shown; amp, amplification; Ex, exon; LoF, loss of function; WT, wild type. f , Real-time live-cell assessments of MSTO-211H and NCI-H226 cells treated with IAG933 or VT104; data show the mean of n = 2 replicates. g , Dose-dependent rescue of YTP activity in a CRISPR knock-in TEAD1 V406A/E408A -mutant YTP-resistant clone of (YAP-amplified) SF-268 glioma cells. These TEAD1 residues correspond to V415 and E417 of the TEAD3 protein. Two-tailed paired t -test P values are included in the graph. h , Correlation of pharmacological and genetic sensitivity profiles in 103 cancer cell lines. Each bar represents one cell line. The y axis shows cell survival values of averaged shRNA drop-out profiles for YAP, TAZ (WWTR1) and TEAD1. Bar colors stratify GI 50 for IAG933 (blue, maximum survival/refractory; red, minimum survival/sensitive). Data were analyzed by two-tailed Spearman correlation test between shRNA sensitivity and GI 50 ; P < 0.0001. i , In vitro pharmacological sensitivity of 283 cancer cell lines to IAG933 (GI 50 versus maximal response ( A max )). Color stratifies geometric mean expression of TEAD target genes CCN1 , CCN2 , ANKRD1 and AMOTL2 . Data were analyzed by two-tailed Spearman correlation test between gene signature and A max ( P < 0.0001) or between gene signature and GI 50 ( P < 0.0001).

Article Snippet: The following Taqman assays (Thermo Fisher Scientific) were used: human CCN1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.3413227.g; human ANKRD1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14671023; human CCN2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14485164.g; human AMOTL2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.39983582; human BCL2L1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.56a.14668121; human MCL1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.26560856; human DUSP6 (6-FAM/ZEN/3′ IBFQ): Hs04329643_s1; human SPYR4 (6-FAM/ZEN/3′ IBFQ): Hs01935412_s1; mouse Ccn1 (6-FAM/ZEN/3′ IBFQ): Mm00487499_g1; mouse Ankrd1 (6-FAM/ZEN/3′ IBFQ): Mm00496512_m1; mouse Gapdh (VIC-MGB): Mm99999915_g1; rat Ankrd1 (6-FAM/ZEN/3′ IBFQ): Rn00566329_m1; rat Ccn2 (6-FAM/ZEN/3′ IBFQ): Rn01537279_g1; rat Actb1 (VIC-MGB): Rn00667869_m1; dog Ccn1 (6-FAM/ZEN/3′ IBFQ): AJX01BF; dog Ankrd1 (6-FAM/ZEN/3′ IBFQ): Cf02662722_m1; dog Ccn2 (6-FAM/ZEN/3′ IBFQ): Cf02641589_m1; dog Gapdh (VIC-MGB): Cf02641589_m1.

Techniques: Binding Assay, Incubation, Inhibition, Immunoprecipitation, Targeted Gene Expression, Activity Assay, Amplification, CRISPR, Knock-In, Mutagenesis, Two Tailed Test, shRNA, In Vitro, Expressing

Journal: Nature Cancer

Article Title: Direct and selective pharmacological disruption of the YAP–TEAD interface by IAG933 inhibits Hippo-dependent and RAS–MAPK-altered cancers

doi: 10.1038/s43018-024-00754-9

Figure Lengend Snippet: a , Time-dependent effect in quantitative measurements of TEAD target gene expression in cultured mesothelioma cell lines MSTO-211H, NCI-H2052, NCI-H2452, and IST-MES2, with IAG933 PPI inhibitor and VT104 TEAD allosteric inhibitor. Data shown for four replicates, Mean ± SD. Two-tailed paired t test VT-104 [1.2 µM]” vs. “IAG933 [300 nM] using the results from all 3 genes: **p = 0.0002, *** $ , p = 0.0001***, p < 0.0001 for each cell line. b-f , Transcriptional modulation induced byIAG933 in cell lines from non-human species shows a similar range of activity after 24 h treatment. b , human, c , dog, d , rat, e , mouse cell lines were subjected to in vitro treatment with a concentration range of IAG933 for 16 h. mRNA levels of three YAP/TEAD target genes ( CYR61 , ANKRD1 and CTGF ), expressed as relative percentages vs the vehicle control (DMSO), are represented on the y-axis versus tested concentrations of IAG933 (μM) on the log-scaled x-axis. In some cell lines, one of the TEAD target genes could not be detected. f , Lists of the mean IC 50 values (n = 2–4) of IAG933, calculated using Fit models (203) from XLFit (Microsoft). g , Right: characterization of the SF-268 cell clones derived from lentivirus infection with a TEAD1 WT or TEAD1 V406A/E408A mutant. The sensitivity to IAG933 is impaired in the mutant clone. Mean ± SD, results of 6 individual experiments. Left: Rescue experiments that demonstrate TEAD-selectivity of IAG933 and YTPs in a colony formation assay. Cell colonies obtained using CRISPR knock-in engineered clones of the SF-268 YAP-amplified glioma cell line assess the rescue of YTP effects. This cell line mainly expresses and depends on the TEAD1 isoform. Wild-type and TEAD1 V406A/E408A mutant clones were treated with indicated doses of IAG933, YTP-17, or YTP-75. The picture is representative of 3 individual experiments.

Article Snippet: The following Taqman assays (Thermo Fisher Scientific) were used: human CCN1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.3413227.g; human ANKRD1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14671023; human CCN2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14485164.g; human AMOTL2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.39983582; human BCL2L1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.56a.14668121; human MCL1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.26560856; human DUSP6 (6-FAM/ZEN/3′ IBFQ): Hs04329643_s1; human SPYR4 (6-FAM/ZEN/3′ IBFQ): Hs01935412_s1; mouse Ccn1 (6-FAM/ZEN/3′ IBFQ): Mm00487499_g1; mouse Ankrd1 (6-FAM/ZEN/3′ IBFQ): Mm00496512_m1; mouse Gapdh (VIC-MGB): Mm99999915_g1; rat Ankrd1 (6-FAM/ZEN/3′ IBFQ): Rn00566329_m1; rat Ccn2 (6-FAM/ZEN/3′ IBFQ): Rn01537279_g1; rat Actb1 (VIC-MGB): Rn00667869_m1; dog Ccn1 (6-FAM/ZEN/3′ IBFQ): AJX01BF; dog Ankrd1 (6-FAM/ZEN/3′ IBFQ): Cf02662722_m1; dog Ccn2 (6-FAM/ZEN/3′ IBFQ): Cf02641589_m1; dog Gapdh (VIC-MGB): Cf02641589_m1.

Techniques: Targeted Gene Expression, Cell Culture, Two Tailed Test, Activity Assay, In Vitro, Concentration Assay, Control, Clone Assay, Derivative Assay, Infection, Mutagenesis, Colony Assay, CRISPR, Knock-In, Amplification

Journal: Nature Cancer

Article Title: Direct and selective pharmacological disruption of the YAP–TEAD interface by IAG933 inhibits Hippo-dependent and RAS–MAPK-altered cancers

doi: 10.1038/s43018-024-00754-9

Figure Lengend Snippet: a , Endpoint tumor responses of nine mesothelioma PDX models treated with 240 mg kg −1 YTP-75 once daily for 14–21 days. Data are shown as mean ± s.e.m.; n = 4, 5 or 6 mice per group depending on the model. Data were analyzed by two-tailed unpaired t -test (* P = 0.014, ** P = 0.002, ** # P = 0.0001 and *** P < 0.0001). Gene expression levels and genetic alterations across models retrieved from the Charles River database are displayed on the bottom. b , c , Antitumor efficacy of IAG933 or YTP-75 and change in body weight in two NF2 loss-of-function mouse xenograft models of non-mesothelioma cancers. Data are shown as mean ± s.e.m. and were analyzed by two-tailed paired t -tests (** P = 0.001, * P = 0.0146 and * # P = 0.0397); n = 5 per group (5938-HX triple-negative breast ductal carcinoma PDX model; b ) and n = 6 per group (NCI-H292 lung carcinoma CDX model; c ). d , Dose-dependent inhibition of the CCN2 TEAD target gene by YTP-75 (24-h treatment) in NIH-3T3 cells stably expressing YAP – MAML2 or TAZ – CAMTA1 fusion genes. Calculated IC 50 values are between 82 and 292 nM. e , Dose-dependent antitumor efficacy and change in body weight of IAG933 in subcutaneous NIH-3T3 xenograft tumors expressing TAZ–CAMTA1. Data are shown as mean ± s.e.m. and were analyzed by one-way ANOVA; n = 6 per group; * P = 0.0252.

Article Snippet: The following Taqman assays (Thermo Fisher Scientific) were used: human CCN1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.3413227.g; human ANKRD1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14671023; human CCN2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14485164.g; human AMOTL2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.39983582; human BCL2L1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.56a.14668121; human MCL1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.26560856; human DUSP6 (6-FAM/ZEN/3′ IBFQ): Hs04329643_s1; human SPYR4 (6-FAM/ZEN/3′ IBFQ): Hs01935412_s1; mouse Ccn1 (6-FAM/ZEN/3′ IBFQ): Mm00487499_g1; mouse Ankrd1 (6-FAM/ZEN/3′ IBFQ): Mm00496512_m1; mouse Gapdh (VIC-MGB): Mm99999915_g1; rat Ankrd1 (6-FAM/ZEN/3′ IBFQ): Rn00566329_m1; rat Ccn2 (6-FAM/ZEN/3′ IBFQ): Rn01537279_g1; rat Actb1 (VIC-MGB): Rn00667869_m1; dog Ccn1 (6-FAM/ZEN/3′ IBFQ): AJX01BF; dog Ankrd1 (6-FAM/ZEN/3′ IBFQ): Cf02662722_m1; dog Ccn2 (6-FAM/ZEN/3′ IBFQ): Cf02641589_m1; dog Gapdh (VIC-MGB): Cf02641589_m1.

Techniques: Two Tailed Test, Gene Expression, Inhibition, Stable Transfection, Expressing

Journal: Nature Cancer

Article Title: Direct and selective pharmacological disruption of the YAP–TEAD interface by IAG933 inhibits Hippo-dependent and RAS–MAPK-altered cancers

doi: 10.1038/s43018-024-00754-9

Figure Lengend Snippet: a , Antiproliferative activity dose matrices, 6-day readout; data represent the mean values of triplicates for IAG933 with dabrafenib, IAG933 with dabrafenib and LTT462 and IAG933 with dabrafenib and trametinib in HT-29 and MDST8 BRAF V600E -mutated CRC cells. b , CCN1 and CCN2 TEAD target gene expression in three cell lines after 24 h of treatment with dabrafenib (Dab) plus trametinib (Tram), YTP-10 or all three. Data are shown as mean ± s.e.m.; n = 4 for DMSO, n = 8 for YTP-10, n = 3 for dabrafenib/trametinib and n = 3 for the combination. Data were analyzed by one-way ANOVA, and comparisons to DMSO are shown (*** P < 0.001; other P values are indicated on graphs). c , Antitumor efficacy of IAG933, dabrafenib + LTT462 or all three therapeutic agents combined in mouse HT-29 xenografts. Data are shown as mean ± s.e.m.; n = 5 per group. Data were analyzed by one-way ANOVA, and comparisons to the vehicle group are shown (* P = 0.0255). 2QD indicates twice daily. d , Antitumor efficacy of dabrafenib + trametinib ± cetuximab or YTP-75 in the 5238-HX mouse PDX model. Data are shown as mean ± s.e.m.; n = 10 for the vehicle group, n = 18 for the dabrafenib/trametinib group, n = 32 for the dabrafenib/trametinib + cetuximab group and n = 6 for the dabrafenib/trametinib + YTP-75 group. Data were analyzed by one-way ANOVA, and triple combinations were compared (* P = 0.083) or body weights were compared to the vehicle group (** P = 0.0471 and *** P < 0.0002).

Article Snippet: The following Taqman assays (Thermo Fisher Scientific) were used: human CCN1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.3413227.g; human ANKRD1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14671023; human CCN2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.14485164.g; human AMOTL2 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.39983582; human BCL2L1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.56a.14668121; human MCL1 (6-FAM/ZEN/3′ IBFQ): Hs.PT.58.26560856; human DUSP6 (6-FAM/ZEN/3′ IBFQ): Hs04329643_s1; human SPYR4 (6-FAM/ZEN/3′ IBFQ): Hs01935412_s1; mouse Ccn1 (6-FAM/ZEN/3′ IBFQ): Mm00487499_g1; mouse Ankrd1 (6-FAM/ZEN/3′ IBFQ): Mm00496512_m1; mouse Gapdh (VIC-MGB): Mm99999915_g1; rat Ankrd1 (6-FAM/ZEN/3′ IBFQ): Rn00566329_m1; rat Ccn2 (6-FAM/ZEN/3′ IBFQ): Rn01537279_g1; rat Actb1 (VIC-MGB): Rn00667869_m1; dog Ccn1 (6-FAM/ZEN/3′ IBFQ): AJX01BF; dog Ankrd1 (6-FAM/ZEN/3′ IBFQ): Cf02662722_m1; dog Ccn2 (6-FAM/ZEN/3′ IBFQ): Cf02641589_m1; dog Gapdh (VIC-MGB): Cf02641589_m1.

Techniques: Activity Assay, Targeted Gene Expression

Journal: Frontiers in Microbiology

Article Title: Lacticaseibacillus casei Strain T21 Attenuates Clostridioides difficile Infection in a Murine Model Through Reduction of Inflammation and Gut Dysbiosis With Decreased Toxin Lethality and Enhanced Mucin Production

doi: 10.3389/fmicb.2021.745299

Figure Lengend Snippet: The Lacticaseibacillus casei -conditioned medium (LCM) of L. casei T21 attenuated C. difficile -induced IL-8 production, host gene expression, and increased transepithelial electrical resistance of C. difficile -stimulated colonic epithelial cells. The results are shown as IL-8 production in Caco-2 and HT-29 cells, respectively (A,B) ; IL-8 gene expression (relative to GAPDH ) in Caco-2 and HT-29 cells, respectively (C,D) ; the expression of SLC11A1 , HuR , and MUC-2 in Caco-2 cells, respectively (E–G) ; the expression of SLC11A1 , HuR , and MUC-2 in HT-29 cells, respectively (H–J) ; the transepithelial electrical resistance (TEER) values of Caco-2 cells (K) ; and the pH of cell culture medium (McCoy’s 5A modified medium for HT-29 cells) (L) . The results were from three independent experiments each in triplicate and expressed as the mean ± SEM. *p < 0.05.

Article Snippet: Colonic epithelial cells were then incubated with viable cells of C. difficile ATCC BAA1870 at multiplicity of infection (MOI) 1:300 either alone or with 5% (vol/vol) LCM for 24 h in 5% CO 2 at 37°C.

Techniques: Gene Expression, Expressing, Cell Culture, Modification