Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured in low or high density. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without serum. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without aphidicolin. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. cGAS in nuclear/cytosolic fractions of indicated cell types. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Western Blot, Cell Culture

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Fluorescence images of GFP‐hcGAS, GFP‐hcGASΔcGAMP, GFP‐hcGASΔDNA, and GFP‐hcGASΔOligo in HEK293 cells cultured with or without aphidicolin. Scale bar: 20 μm. B Corresponding quantification of (A). The nuclear cGAS/total cGAS was calculated from 6 different fields with n > 50 cells. C, D A nuclear export signaling (NES) is not sufficient to dislodge chromatin‐bound cGAS from the nucleus. (C) Fluorescence images of GFP‐hcGAS, GFP‐hcGAS‐NLS, and GFP‐hcGAS‐NES in HEK293 cells. Scale bar: 10 μm. (D) Immunoblots of subcellular fractions of GFP‐hCGAS‐, GFP‐hCGAS‐NLS‐, and GFP‐hCGAS‐NES‐expressing HEK293 cells. Data information: Data are presented as means ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05 and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Fluorescence, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Micronuclei (indicated by arrowhead) in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells before (0 h) or 24 h after γ‐irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show mean ± SEM ( n = 3 independent experiments) representing six different microscopic fields with over 200 cells. C IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n = 3 independent experiments. D Experimental outline for micronucleus generation and cell death after γ‐irradiation. E Micronucleus (indicated by arrowhead) and cGAS staining in WT and cGAS −/− BMDMos exposed to γ‐irradiation (10 Gy). Scale bar: 10 μm. F Average MNs/cell in BMDMos. MN graphs show mean ± SEM ( n = 3 independent experiments) representing eight different microscopic fields with over 200 cells. G Cell death in WT and cGAS −/− BMDMos that were first synchronized at G2/M, then γ‐irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ± SD, x biological triplicates ( n = 3) per treatment group are shown. Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two‐tailed Student's t ‐test. *** P ≤ 0.001 and **** P ≤ 0.0001. Statistical significance in (G) was assessed using two‐way ANOVA test, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Irradiation, Transfection, Plasmid Preparation, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Pulsed‐field gel electrophoresis analysis of γ‐irradiated (10 Gy) WT and cGAS −/− BMDMos. B, C Comet assay in GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells γ‐irradiated (IR: 10 Gy) for 15 min (B). RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293T cells stimulated with transfected DNA for 6 h (C). D, E Comet assay of HEK293 cells stimulated with 10 μg/ml cGAMP for indicate periods, then γ‐irradiated and incubated at 37°C for indicated duration (D). (E) Immunoblots of IRF3 phosphorylation in HEK293 cells treated as in (D). F–H Images (F) and quantifications (G) of comet tails 15 min after irradiation of GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells. RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells stimulated with transfected 23 DNA for 6 h (H). I, J Images (I) and quantifications (J) of micronuclei in GFP‐NLS‐ and GFP‐hcGASΔcGAMP‐expressing HEK293 cells 24 h after γ‐irradiation (IR; 10 Gy). DAPI (DNA). Scale bar: 10 μm. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. K Quantifications of comet tails 15 min after irradiation (10 Gy) of GFP‐NLS‐, GFP‐hcGAS‐, or GFP‐mcGAS‐expressing HEK293 cells. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. Data information: Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS P > 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001. Mean ± SEM of n = 3 independent experiments. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Pulsed-Field Gel, Electrophoresis, Irradiation, Single Cell Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Reporter assays showing the effect of NLS and NES on cGAS‐mediated inhibition of DNA repair. B Both full‐length hcGAS and hcGAS cat (161–522aa) inhibit HR repair. C–E cGAS does not impede ATM activation. ATM phosphorylation in γ‐irradiated (10 Gy) GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells (C), GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells (D), or γ‐irradiated (2.5 Gy) WT, cGAS −/− , and Sting −/− BMDMos (E). Data information: Data are means ± SD, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. *** P < 0.001 **** P < 0.0001, NS: P > 0.05. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A cGAS is not recruited to DSB sites: Confocal microscopic images of GFP‐NLS‐ or GFP‐hcGAS‐expressing U2OS‐DSB reporter cells incubated (or not) with Shield‐1 and 4‐OHT to induce the expression and translocation of mCherry‐LacI‐FokI (red) to specific DSB sites. Scale bar: 10 μm. The arrowheads indicate DSB sites. B cGAS does not co‐localize with γ‐H2AX at DSB sites: GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells exposed (or not) to γ‐irradiation (IR: 10 Gy), then stained for γ‐H2AX. Scale bar: 10 μm. C, D Nuclear cGAS is mainly chromatin‐bound and remains unaltered upon γ‐irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ‐irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear, and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins. E–G cGAS co‐isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co‐isolation of cGAS and DNA repair proteins: Lysates of control (−IR) and γ‐irradiated (+IR, 10 Gy, 30 min) GFP‐hcGAS‐expressing HEK293 cells were treated (or not) with benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co‐isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ‐irradiated HEK293 cells expressing GFP‐hcGAS or GFP‐hcGASΔDNA probed for indicated proteins. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Incubation, Translocation Assay, Irradiation, Staining, Isolation, Control, Immunoprecipitation, Agarose Gel Electrophoresis, Binding Assay

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Confocal images of γ‐irradiated GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293 cells stained for RAD51 (red) with or without γ‐irradiation. Scale bar: 10 μm. Schematics of the D‐loop formation assay, including pre‐incubation of template dsDNA with cGAS cat (i) or with cGAS cat being added after RAD51 was bound to dsDNA (ii). Pre‐incubation of dsDNA with mcGAS cat prevents D‐loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D‐loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D‐loop assay. Pre‐incubation of template dsDNA with hcGAS cat blocks subsequent D‐loop formation. The percentage of D‐loop formation (below) was graphed as the average of triplicates ± SD. Data information: Unpaired two‐tailed Student's t ‐test was used for statistical analyses. NS P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Irradiation, Expressing, Staining, Tube Formation Assay, Incubation, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Negative‐stain electron micrographs of cGAS‐dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation when pre‐incubated with dsDNA. Percentage of D‐loop formed in each reaction (left) graphed as the average of triplicates ± SD. Overview of a single 1:1 hcGAS‐DNA complex depicting the location of the Y215 within the cGAS‐dsDNA interface. DR‐GFP assay showing that hcGASΔDNA‐Y215E is impaired in HR inhibition. hcGASΔDNA‐Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24. hcGASΔDNA‐Y215E and hcGASΔcGAMP are defective in synthase activity. Negative‐stain electron micrographs showing that hcGAScat‐ΔDNA‐Y215E is defective in inducing cGAS‐dsDNA complexes. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation. Data information: Data are means ± SD, n = 3. Unpaired Student's t ‐test was used for statistical analyses: NS P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Staining, Incubation, Inhibition, Activity Assay

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) Level of NFAT isoforms (NFATc1, NFATc2, NFATc3, and NFATc4) was determined in two strains of normal human oral keratinocyte (NHOK-1 and -2), 2 precancerous, non-tumorigenic immortalized oral epithelial cell lines (HOK-16B and NOKSI) and 10 OSCC cell lines (BapT, SCC1, SCC4, SCC9/TNF, SCC15, UM1, UM2, UM6, UM17B, and FaDu) by qPCR. Levels of NFAT isoforms were normalized to GAPDH. ( B ) Level of NFATc3 protein was assessed in normal (NHOK), precancerous (HOK-16B and NOKSI) and OSCC cells (BapT and SCC4) by Western blot analysis. GAPDH was used as loading control. ( C ) Expression of NFAT isoforms was assessed in tumor spheres (Sph.) and their corresponding adherent monolayer cells (Mono.) derived from multiple OSCC cell lines by qPCR. * P < 0.01 compared to Sph. by two-tailed Student’s t test. ( D ) Level of NFATc3 protein was assessed in tumor spheres and their corresponding adherent monolayer cells derived from multiple OSCC cell lines by Western blot analysis.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: Western Blot, Control, Expressing, Derivative Assay, Two Tailed Test

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: NFATc3 expression was forced in non-tumorigenic immortalized oral epithelial cells, NOKSI, by transfecting with vector expressing NFATc3 or empty vector (EV) as a control. ( A ) Ectopic expression of NFATc3 was confirmed by qPCR and Western blot analysis. ( B ) Effect of NFATc3 on cell proliferation was determined by cell counting. Data are means ± SD of triplicate experiments. * P < 0.05 and ** P < 0.01 by two-tailed Student’s t test. ( C ) Effect of NFATc3 on anchorage independent growth ability was determined by soft agar assay. Ten thousand cells were plated in semi-solid agar, and colonies were counted for three weeks. The assay was performed in triplicate with 60-mm dishes. The photographs were taken at a magnification of 40X. ( D ) Effect of NFATc3 on in vivo tumorigenicity was determined by xenograft tumor assay. NOKSI/EV and NOKSI/NFATc3 were injected subcutaneously into 5 nude mice. Tumor sizes were measured for 6 weeks. ** P < 0.01.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Cell Counting, Two Tailed Test, Soft Agar Assay, In Vivo, Injection

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) Effect of NFATc3 on self-renewal capacity of NOKSI was determined by tumor sphere formation assay. Representative image of tumor spheres formed by NOKSI/EV and NOKSI/NFATc3 are shown on the right. Bar indicates 100 μm. ( B ) Effect of NFATc3 on ALDH1 activity of NOKSI was determined by Aldefluor assay. Cells were labeled with Aldefluor combined with or without the ALDH1 inhibitor DEAB and analyzed by flow cytometry. The gate for ALDH1 HIGH cells is determined in relation to the DEAB control (+DEAB) and shows the brightly fluorescent ALDH1 population versus the side scatter, a population that is absent/decreased in the presence of DEAB. The number shown in each panel reflects the percentage of ALDH1 HIGH cells in each cell type. ( C ) Effect of NFATc3 on chemoresistance of NOKSI was determined by MTT assay. Cells were treated with 40 µM of cisplatin for 2, 4, and 6 days, and their viability was determined. Data are expressed as the mean ± SD of triplicate. * P < 0.01 (D ) Effect of NFATc3 on migration ability of NOKSI was determined by transwell migration assay. * P < 0.01. Representative images of transwell migration assay are shown on the right. ( E ) Endogenous NFATc3 was knocked down in multiple OSCC cell lines using siRNA against NFATc3 (NFATc3i). The cells transfected with control siRNA (CTLi) were included for comparison. Knockdown of NFATc3 was confirmed by qPCR. ( F ) The effect of NFATc3 knockdown on self-renewal capacity was determined by tumor sphere formation assay. ( G ) The effect of NFATc3 knockdown on migration ability was determined by transwell migration assay.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: Tube Formation Assay, Activity Assay, Labeling, Flow Cytometry, Control, MTT Assay, Migration, Transwell Migration Assay, Transfection, Comparison, Knockdown

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) Effect of NFATc3 on pluripotent transcription factors (NANOG, OCT4, KLF4, LIN28, and SOX2) expression was determined by qPCR. Their levels in NOKSI/NFATc3 were plotted as fold change against those in NOKSI/EV. * P < 0.001. ( B ) Effect of NFATc3 on OCT4 promoter activity was determined by luciferase promoter assay. Cells were transfected with pGL3-Basic (promoter-less) or pGL3 vectors containing the 1.5-kb upstream (-1545∼ -24) of Oct4. * P < 0.001. ( C ) Sequence analysis reveals a consensus NFAT binding site (5′-GGAAA-3′) at -1088 ∼ -1084 indicated by star (upper diagram). OSCC cells were lysed and performed a ChIP assay. The fragment (-1191∼ -1061) containing the NFAT binding site was enriched with NFATc3, and the fragment (-2930∼ -2783) was amplified as a control. * P < 0.01. ( D ) Correlation analysis of NFATc3 and OCT4 mRNA was determined based on their expression levels in 18 human SCC cell lines by qPCR.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: Expressing, Activity Assay, Luciferase, Promoter Assay, Transfection, Sequencing, Binding Assay, Amplification, Control

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) The effect of OCT4 knockdown on self-renewal capacity of NOKSI/NFATc3 was determined by tumor sphere formation assay. OCT4 was knocked down in NOKSI/NFATc3 using siRNA against OCT4 (Oct4i). The cells transfected with control siRNA (CTLi) were included for comparison. * P < 0.05. ( B ) The effect of OCT4 knockdown on migration ability in NOKSI/NFATc3 was determined by transwell migration assay. ** P < 0.01. ( C ) The effect of OCT4 knockdown on self-renewal capacity of SCC4 was determined by tumor sphere formation assay. ( D ) The effect of OCT4 knockdown on self-renewal capacity of SCC4 was determined by transwell migration assay. ( E ) OCT4 expression was forced in non-tumorigenic immortalized oral epithelial cells, NOKSI, by vector expressing recombinant Myc-DDK-tagged OCT4, and its ectopic expression was confirmed by Western blot analysis using anti-DDK antibody. ( F ) Effect of ectopic OCT4 expression on self-renewal capacity of NOKSI was determined by tumor sphere formation assay. Representative images of tumor spheres formed by NOKSI/EV and NOKSI/Oct4 are shown on the right. ( G ) Effect of ectopic OCT4 expression on migration ability of NOKSI was determined by transwell migration assay. ( H ) Effect of ectopic OCT4 expression on the expression of NFAT isoforms (NFATc1-c4) in NOKSI was determined by qPCR. Their levels in NOKSI/Oct4 were plotted as fold induction against those in NOKSI/EV.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: Knockdown, Tube Formation Assay, Transfection, Control, Comparison, Migration, Transwell Migration Assay, Expressing, Plasmid Preparation, Recombinant, Western Blot

Journal: Oncotarget

Article Title: NFATc3 plays an oncogenic role in oral/oropharyngeal squamous cell carcinomas by promoting cancer stemness via expression of OCT4

doi: 10.18632/oncotarget.26774

Figure Lengend Snippet: ( A ) In vivo NFATc3 expression was determined in normal human oral epithelia (NHOE), oral dysplasia and OSCC tissues by immunohistochemical (IHC) staining. * P < 0.01 and ** P < 0.05. ( B ) Representative examples of NFATc3 IHC staining in NHOE and OSCC tissues in vivo . Bar indicates 100 μm. ( C ) Morphological change of tongues from mice treated without or with 4-NQO (30 µg/ml) for 4 months to induce oral cancer formation. Bar indicates 50 μm. ( D ) Level of NFAT isoforms (NFATc1-c4) was determined in tongues from mice exposed to DMSO or 4-NQO by qPCR. The levels of 4 NFAT isoforms were normalized to GAPDH. qPCR was performed with total RNAs isolated from tongue tissues.

Article Snippet: Immune complexes were obtained using 5 μg of NFATc3 antibody (sc-1152; Santa Cruz Biotech).

Techniques: In Vivo, Expressing, Immunohistochemical staining, Immunohistochemistry, Isolation

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Diagram showing the strategy employed to shortlist AICAR-binding proteins. The three steps include in silico screening using FINDSITE comb2.0 , protein expression assay, and thermal stability assay. b Time-dependent western blotting and relative quantification of protein expression for AICAR-binding proteins. The treatment responses on H1975 cells treated with 1 mM AICAR for 1 and 2 h were grouped by strong inactivation (type 1) and weak response (type 2). GAPDH was used as a loading control. N = 2–3 replicates. c Dose-dependent western blotting and relative quantification of protein expression for MUC1-CT and TMEM70. H1975 cells were treated with increasing doses of AICAR (0, 0.4, 1.3, and 4.4 mM) for 22 h, followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. d Thermal stability assay for MUC1-CT and TMEM70. H1975 cells were treated with 1 mM AICAR for 15 min. The cell pellets were heated for 3 min at their respective temperature (37–55 °C), followed by a western blot assay. N = 2 replicates. e Immunofluorescence staining for MUC1-CT in H441 cells. The cells were treated with 0.3 mM AICAR for 4 h and then incubated with rabbit anti-MUC1-CT primary antibody followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. The nucleus was counterstained with DAPI. The images were taken using a Keyence fluorescent microscope. Scale bar, 50 μm. f qRT-PCR analysis for MUC1-CT targeting genes. Expression levels for CTGF , PGM2 , and ENO1 were analysed by qRT-PCR in H1975 cells treated with 0.3 mM AICAR for 4 h. GAPDH was used as an endogenous control. N = 3 replicates. g qRT-PCR analysis for MUC1 expression in H1975 cells with MUC1 overexpression (OE). The cells were transfected with a lentiviral vector containing MUC1 or scrambled control, followed by 0.5 µg/ml puromycin selection. The relative MUC1 expression level in scrambled control cells was calibrated as 1. GAPDH was used as an endogenous control. N = 3 replicates. h Organoid formation assay for AICAR treatment response in H1975 cells overexpressing MUC1 . The cells with MUC1 overexpression or a scrambled control vector were plated at 2000 cells per well and treated with vehicle or 0.3 mM AICAR continuously for nine days. Images were taken with an EVOS microscope, and the treatment responses were quantified using a 3D Celltiter-Glo assay. N = 4–5 replicates. Scale bar, 300 µm. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , f , h ); Welch’s t -test ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Binding Assay, In Silico, Expressing, Stability Assay, Western Blot, Quantitative Proteomics, Control, Immunofluorescence, Staining, Incubation, Microscopy, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Selection, Tube Formation Assay, Glo Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Top KEGG signalling pathways differentially expressed in H1975 cells treated with AICAR. The cells were treated with 1 mM AICAR for 4 h, followed by whole transcriptomic analysis. N = 3 replicates. b Enrichment plot by gene set enrichment analysis for the JAK-STAT signalling pathway in H1975 cells treated with AICAR. Profile of the running enrichment score (ES) (top) and positions of gene set members on the rank-ordered list (bottom) were shown. N = 3 replicates. c A heat map showing top enriched genes of the JAK-STAT signalling pathway in H1975 cells treated with AICAR compared with vehicle-treated cells. d Longitudinal analysis of p-JAK1, p-TYK2, and MUC1-CT expression. H1975 cells were treated with 1 mM AICAR for 0, 1, and 2 h followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. e Confocal images for co-localisation of MUC1-CT and p-JAK1 in lung cancer cells. H1975 cells were co-incubated with Armenian hamster anti-MUC1-CT and rabbit anti-p-JAK1 primary antibodies for 2 h. Then the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 555. The nucleus was counterstained with DAPI. The images (top: lower magnification; bottom: higher magnification) were taken using a Zeiss confocal fluorescent microscope. Scale Bar, 50 µm (top) and 20 µm (bottom). f Duolink ligation assay and confocal imaging for physical MUC1-JAK1 interactions. H441 cells were treated with vehicle or 1 mM AICAR for 1 h. After treatment, the cells were incubated with mouse anti-ZO-1 primary antibody overnight, followed by anti-mouse IgG conjugated with Alexa Fluor 488. Then the cells were co-incubated with anti-MUC1 and anti-JAK1 primary antibodies, followed by incubation with proximity ligation assay probes conjugated with Cy3, ligation, and amplification steps. The nucleus was counterstained with DAPI. The images were taken using a Zeiss confocal fluorescent microscope, and the Duolink dots were quantified using Image J. Scale Bar, 20 µm. g Cell viability assay of H1975 cells treated with AICAR and VX-509. Cells were plated in a 96-well plate and treated with AICAR (1 mM) with or without VX-509 (10 μM). The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated control group. N = 3–4 replicates. Data are mean ± s.e.m. and were analysed with Welch’s t -test ( a , b , f ); Brown-Forsythe and Welch one-way ANOVA ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Expressing, Western Blot, Control, Incubation, Microscopy, Ligation, Imaging, Proximity Ligation Assay, Amplification, Viability Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL ( T790M; L858R )-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox-impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL -induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL -induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( c , d , e , g ); one-way ANOVA ( a , b ); Brown-Forsythe and Welch ANOVA ( f , h ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, Control, Expressing, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues from transgenic mice. The far normal and tumour tissues from transgenic mice 14 weeks after Dox induction were extracted for RNA analysis. Gapdh was used as an endogenous control. N = 3 replicates. b Differential MUC1 gene expression in lung adenocarcinoma (LUAD) compared with tumour-adjacent tissues by analysing the TCGA_LUAD dataset. N = 59 (normal), and N = 517 (tumour). c Patients’ overall survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 135. d Patients’ disease-free survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 105. e Xenograft tumour growth in mice treated with AICAR. The xenograft tumour was pre-established by implanting 1 million H1975 cells subcutaneously. When the tumour reached 45 mm 3 , the mice were treated with 300 mg/kg/day AICAR in PBS or a vehicle subcutaneously for ten days. The tumour was measured with a digital caliper, and the tumour size was calculated. N = 7 replicates. f Xenograft tumour images and relative weight quantification from mice treated with AICAR or PBS. The average tumour weight from the PBS-treated group is normalised as 1. N = 7 replicates. g Mouse body weight after treatment with AICAR or PBS for ten days. N = 7 replicates. h , i H&E staining of subcutaneous tumours ( h ) and liver tissues ( I ) from H1975 cell line-derived xenograft (CDX) treated with PBS or AICAR. Scale bar, 125 μm. j – l Immunofluorescence staining for Ki-67 ( j ), γ-H 2 AX ( k ), and p21 Cip1 ( l ) in subcutaneous tumours from H1975 CDX treated with PBS or AICAR. Scale bar, 100 μm. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b ); log-rank test ( c , d ); Welch’s t -test ( e – g ). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Quantitative RT-PCR, Gene Expression, Transgenic Assay, Control, Expressing, Staining, Derivative Assay, Immunofluorescence, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1-CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c , d Growth of PDX ( c ) and transgenic mouse EGFR TL -induced lung tumour ( d )-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , d ). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Transgenic Assay, Derivative Assay

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: Circ_0089153 targets miR-198 to regulate miR-198 expression. (a) Sequence of miR-198, the putative miR-198 binding sequence within circ_0089153, and the mutation in the target sites. (b) qRT-PCR analysis showing the overexpression of miR-198 in HCT116 and SW480 cells transfected by miR-198 mimic or miRNA NC control. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. (c and d) Dual-luciferase reporter assays in HCT116 and SW480 cells co-transfected with circ_0089153 reporter construct (WT-circ_0089153) or the mutant reporter (MUT-circ_0089153) and miR-198 mimic or miRNA NC control. Normalized firefly to Renilla ratios were determined in the presence or absence of miR-198 inhibition. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. (e and f) RIP experiments in HCT116 and SW480 cells performed by incubating cell lysates with antibody against Ago2 or IgG control. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. The expression levels of miR-198 by qRT-PCR analysis in 50 pairs of colorectal primary tumors and the adjacent noncancerous colorectal tissues (g), normal colonic FHC cells, HCT116, and SW480 CRC cells (h), HCT116 and SW480 cells transfected by si-NC or si-circ_0089153 (i). P -values based on an unpaired Student’s t -test (two-tailed) or a two-way ANOVA with Sidak’s multiple comparison test. * P < 0.05

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Quantitative RT-PCR, Over Expression, Transfection, Control, Comparison, Luciferase, Construct, Inhibition, Two Tailed Test

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: Circ_0089153 is overexpressed in CRC tissues and cell lines. (a) Representative images depicting the immunohistochemistry assay for Ki67 staining in colorectal primary tumors paired with the adjacent normal tissues from the same patients. (b) qRT-PCR analysis of circ_0089153 in 50 pairs of colorectal primary tumors and the adjacent noncancerous colorectal tissues. P -values based on an unpaired Student’s t -test (two-tailed). (c) The expression of circ_0089153 in normal colonic FHC cells, HCT116 and SW480 CRC cells by qRT-PCR analysis. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. (d and e) RNase R assay showing the RNase R resistance of circ_0089153 in HCT116 and SW480 cells. P -values based on an unpaired Student’s t -test (two-tailed). * P < 0.05

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Two Tailed Test, Expressing, Comparison

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: Circ_0089153 affects cell proliferation, apoptosis, sphere formation, and tube formation in vitro . HCT116 and SW480 cells were transfected with si-circ_0089153 or si-NC. (a) The relative expression of circ_0089153 in transfected cells detected by qRT-PCR analysis. (b) Representative images showing a cell proliferation assay and cell proliferation by EdU assay. (c) Representative images depicting a sphere formation assay and cell sphere formation by sphere formation assay. (d) Representative images presenting the tube formation ability of HUVECs performed by incubating HUVECs with the medium supernatant of transfected cells. (e) Representative images depicting a cell apoptosis assay and flow cytometry for cell apoptosis. (f and g) Western blot showing the expression levels of Bax and Bcl-2 in transfected HCT116 and SW480 cells. * P < 0.05 based on a two-way ANOVA with Sidak’s multiple comparison test

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Transfection, Expressing, Quantitative RT-PCR, Proliferation Assay, EdU Assay, Tube Formation Assay, Apoptosis Assay, Flow Cytometry, Western Blot, Comparison

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: Circ_0089153 silencing affects cell proliferation, apoptosis, sphere formation, and tube formation in vitro by increasing miR-198. (a) qRT-PCR analysis showing the down-regulation of miR-198 expression in HCT116 and SW480 cells transfected by inhibitor NC or miR-198 inhibitor. HCT116 and SW480 cells were transfected with si-NC, si-circ_0089153, si-circ_0089153+ inhibitor NC, or si-circ_0089153+ miR-198 inhibitor and checked for cell proliferation by EdU assay (b) and sphere formation ability by sphere formation assay (c). (d) Tube formation assay for tube formation ability of HUVECs incubated with the medium supernatant of HCT116 and SW480 cells transfected by si-NC, si-circ_0089153, si-circ_0089153+ inhibitor NC, or si-circ_0089153+ miR-198 inhibitor. HCT116 and SW480 cells were transfected with si-NC, si-circ_0089153, si-circ_0089153+ inhibitor NC, or si-circ_0089153+ miR-198 inhibitor, followed by the determination of cell apoptosis by flow cytometry (e), Bax and Bcl-2 levels by western blot (f and g). P -values based on a two-way ANOVA with Sidak’s multiple comparison test. * P < 0.05

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Transfection, EdU Assay, Tube Formation Assay, Incubation, Flow Cytometry, Western Blot, Comparison

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: Circ_0089153 operates as a regulator of SENP1 expression by competing for binding to miR-198. (a) Sequence of miR-198, the putative binding sequence for miR-198 in the 3ʹUTR of SENP1 and the mutation in the seed region. (b and c) Dual-luciferase reporter assays in HCT116 and SW480 cells co-transfected with SENP1 3ʹUTR reporter construct (WT-SENP1-3ʹUTR) or the mutant reporter (MUT-SENP1-3ʹUTR) and miR-198 mimic or miRNA NC control. Normalized firefly to Renilla ratios were determined in the presence or absence of miR-198 inhibition. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. (d and e) RIP experiments in HCT116 and SW480 cells performed by incubating cell lysates with antibody against Ago2 or IgG control. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. qRT-PCR analysis of SENP1 mRNA and western blot of SENP1 protein level in colorectal primary tumors and the adjacent noncancerous colorectal tissues (f and g), normal colonic FHC cells, HCT116, and SW480 CRC cells (h), HCT116 and SW480 cells transfected by miR-198 mimic or miRNA NC control (i), HCT116 and SW480 cells transfected with si-NC, si-circ_0089153, si-circ_0089153+ inhibitor NC, or si-circ_0089153+ miR-198 inhibitor (j). P -values based on an unpaired Student’s t -test (two-tailed) or a two-way ANOVA with Sidak’s multiple comparison test. * P < 0.05

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Binding Assay, Sequencing, Mutagenesis, Luciferase, Transfection, Construct, Control, Inhibition, Comparison, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Bioengineered

Article Title: Circular RNA circ_0089153 acts as a competing endogenous RNA to regulate colorectal cancer development by the miR-198/SUMO-specific peptidase 1 (SENP1) axis

doi: 10.1080/21655979.2021.1967076

Figure Lengend Snippet: MiR-198 overexpression impacts cell proliferation, apoptosis, sphere formation, and tube formation in vitro by suppressing SENP1. (a) Western blot showing the up-regulation of SENP1 protein in HCT116 and SW480 cells transfected by SENP1 overexpression plasmid (pc-SENP1) or pc-NC control plasmid. P -values based on a two-way ANOVA with Sidak’s multiple comparison test. HCT116 and SW480 cells were transfected with miRNA NC control, miR-198 mimic, miR-198 mimic+pc-NC, or miR-198 mimic+pc-SENP1, followed by the assessment of cell proliferation by EdU assay (b), and sphere formation ability by sphere formation assay (c). P -values based on a two-way ANOVA with Tukey’s post hoc test. (d) Tube formation assay for tube formation ability of HUVECs incubated with the medium supernatant of HCT116 and SW480 cells transfected with miRNA NC control, miR-198 mimic, miR-198 mimic+pc-NC, or miR-198 mimic+pc-SENP1. P -values based on a two-way ANOVA with Tukey’s post hoc test. HCT116 and SW480 cells were transfected with miRNA NC control, miR-198 mimic, miR-198 mimic+pc-NC, or miR-198 mimic+pc-SENP1 and checked for cell apoptosis by flow cytometry (e), Bax and Bcl-2 levels by western blot (f and g). P -values based on a two-way ANOVA with Tukey’s post hoc test. * P < 0.05

Article Snippet: We obtained normal colonic epithelial FHC cells, HCT116, and SW480 CRC cells, and human umbilical vein endothelial cells (HUVECs) from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, In Vitro, Western Blot, Transfection, Plasmid Preparation, Control, Comparison, EdU Assay, Tube Formation Assay, Incubation, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A CHRM4 and AR protein levels of LNCaP, 22Rv1, C4-2, C4-2-MDVR, PC3, and LASCPC01 cells, measured by a western blot analysis. B , C CHRM4 mRNA abundances in LNCaP and C4-2 cells during 1, 2, 3, 4, and 5 months of 20 μM MDV3100 treatment, measured by an RT-qPCR analysis. * vs. parental LNCaP or C4-2 cells, by a one-way ANOVA. D Relative CHRM4 mRNA levels of C4-2 cells cultured in charcoal-stripped serum (CSS)-containing medium for 5 and 10 days, followed by treatment with 10 nM dihydrotestosterone (DHT) for 24 h. Quantification of relative mRNA levels is presented as the mean ± SEM of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.0001. E CHRM4, androgen-responsive markers (KLK3 and NKX3-1), and AR protein levels in C4-2 cells cultured in CSS-containing medium for 5 and 10 days, followed by treatment with 10 nM DHT for 24 h. F CHRM4, KLK3, NKX3-1, and AR protein levels of LNCaP and C4-2 cells cultured in 20 μM MDV3100 for 1 week. G Relative mean expressions of the AR, KLK3, NKX3-1, and CHRM4 in LNCaP cells from 3 weeks to 11 months of androgen withdrawal (ADT) in the GDS3358 database. * vs. the control, by a one-way ANOVA. H GSEAs of TCGA prostate dataset revealing negative associations between high CHRM4 expression in prostate tissues with gene signatures representing androgen-responsive signaling (GO, Nelson, Wang, PID, and Hallmark). NES normalized enrichment score, FDR false discovery rate.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Western Blot, Quantitative RT-PCR, Cell Culture, Control, Expressing

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A ChIP-sequencing analysis of the detected DNA-binding sites for the AR of the CHRM4 gene in cells in response to 0.5 or 4 h of AR-ligand R1881 treatment labeled as black boxes in the tracks. ChIP-sequencing data were downloaded from the Gene Expression Omnibus (GEO) (GSE84432) and analyzed by Genome Brower (Genomics Institute). B Schematic of the predicted AR resonse elements (AREs) and an introduced single- or double-binding site mutant in regulatory sequence reporter constructs of human CHRM4 (GRCh38:11). ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence following treatment of C4-2 cells with 10 nM dihydrotestosterone (DHT) ( C ) or 20 μM MDV3100 ( D ) for 24 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to the AR and acetyl-H3, and predictive primers ( B , black arrows) were used to quantify the precipitated DNA by a qPCR. Enrichment of each protein to each site is given as a percentage of the total input and then normalized to IgG. * vs. the vehicle (Veh) ( C ) or DMSO ( D ), by a one-way ANOVA. E , F ChIP assay showing binding of the AR and acetyl-H3 to predicted AREs of the CHRM4 gene regulatory sequence in PC3 cells following stable transfection with an empty vector (EV) or AR cDNA vector ( E ) or in C4-2 cells with a non-targeting control (NC) or AR siRNA transfection ( F ). * vs. the EV ( E ) or NC ( F ), by a one-way ANOVA. G Relative CHRM4 and AR mRNA levels of C4-2 cells transfected with the NC or AR siRNA, measured by an RT-qPCR analysis, * vs. the NC. H , I Relative mean florescence intensity (MFI) of the GFP reporter gene containing a wild-type (WT)- or mutant (M)-ARE from the CHRM4 regulatory sequence in C4-2 cells following treatment with 10 nM DHT ( H ) or 20 μM MDV3100 ( I ) for 48 h. * vs. WT; # vs. the vehicle (Veh) ( H ) or DMSO ( I ), by a two-way ANOVA. J , K Relative MFI of the GFP reporter gene containing a WT- or M-ARE from the CHRM4 regulatory sequence in PC3 cells following stable transfection with the EV or AR cDNA vector ( J ) or in C4-2 cells following NC or AR siRNA transfection ( K ). * vs. the EV ( J ) or NC ( K ), by a two-way ANOVA. Quantification of the ChIP assay, relative MFI values, and mRNA levels are presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: ChIP-sequencing, Binding Assay, Labeling, Gene Expression, Mutagenesis, Sequencing, Construct, Stable Transfection, Plasmid Preparation, Control, Transfection, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A , B CHRM4 and NE marker (CHGA, SYP, and ENO2) mRNA levels in C4-2 cells stably transfected with an empty vector (EV) or a CHRM4-expressing vector ( A ) or in PC3 cells stably transfected with the non-targeting control (NC) or CHRM4 shRNA vector (B) , measured by an RT-qPCR analysis, * vs. the EV ( A ) or NC ( B ), by a one-way ANOVA. C Western blot showing CHRM4 and NE marker protein levels in CHRM4-modified C4-2 and PC3 cells. D GSEA analysis of TCGA prostate cancer dataset revealed positive correlations between higher CHRM4 expression in prostate tissues and gene profiles reflecting NEPC-responsiveness. NES, normalized enrichment score; FDR, false discovery rate. E – H Relative cell migration and invasion through Matrigel (E , F) and proliferation (G , H) of CHRM4-overexpressing C4-2 ( E , G ) or CHRM4-knockdown (KD) PC3 cells ( F , H ). n = 5 per group. * vs. the EV ( E , G ) or NC ( F , H ), by a one-way ANOVA. I – K Tumor growth analysis ( I , J ) and tumor weights ( K ) of CHRM4-KD PC3 cells subcutaneously inoculated into male nude mice for 8 weeks. Tumor weights were measured on the day tumors were collected. Tumor sizes were measured once a week and analyzed by a one-way ANOVA. IHC staining ( L ) and representative intensities ( M ) of CHRM4, ENO2, CHGA, and KI67 in subcutaneous tumors from J . * vs. NC-bearing tumors, by a two-tailed Student’s t -test. Quantification of relative mRNA levels, and migration, invasion, and proliferation are presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Marker, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Modification, Migration, Knockdown, Immunohistochemistry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: A GSEAs of TCCA prostate cancer dataset showing that high abundance of CHRM4 mRNA in prostate cancer samples was positively linked to a wide range of cytokine-responsive gene signatures (GO, KEGG, and BIOCARTA). NES, normalized enrichment score; FDR, false discovery rate. B Venn diagram showing the number of overlapping genes identified in the top four cytokine-responsive gene sets. C The list of five overlapping gene candidates includes INHBC, IFNA17, IFNG, IL1RN , and TNFSF8 from ( B ). Pearson correlations among the five candidate genes and CHRM4 were analyzed in TCGA prostate cancer dataset by XY correlation analyses in GraphPad Prism. Relative mRNA levels of INHBC, IFNA17, IFNG , and IL1RN in C4-2 cells stably transfected with an empty vector (EV) or a CHRM4-expressing vector ( D ) or in PC3 cells stably transfected with a non-targeting control (NC) or CHRM4 shRNA vector, examined by an RT-qPCR. * vs. the EV ( D ) or NC ( E ), by a one-way ANOVA. F Kaplan–Meier analyses of IFNA17 alterations in the GSE21032 dataset. A log-rank (Mantel–Cox) test was used for the survival curve analysis. Hazard ratio = 0.3806, p = 0.0378. G Mean expression levels of IFNA17 in normal prostate ( n = 28), primary prostate cancer ( n = 111), and metastatic prostate cancer ( n = 13) samples in the GSE21032 dataset. * vs. normal prostate; # vs. Primary, *p < 0.05, ***p < 0.001, by a two-way ANOVA. H Mean expression levels of IFNA17 in prostate cancer patient samples in the GSE21032 dataset by pathologic Gleason scores (GSs). * vs. GS6. * p < 0.05, by a one-way ANOVA. I IFNA17 protein levels in LNCaP, VCaP, C4-2, PC3, LASCPC01, and C4-2-MDVR cells, measured by a Western blot analysis. IFNA17 cytokine concentrations in supernatants of cultured medium derived from LNCaP, VCaP, C4-2, PC3, LASCPC01, and C4-2-MDVR cells ( J ) or C4-2 and PC3 cells expressing the EV and CHRM4 cDNA or the NC and CHRM4 shRNA vectors ( K ), measured with an ELISA kit. * vs. LNCaP cells ( J ) or the EV ( K ); # vs. the NC ( K ), by a two-way ANOVA. Quantification of relative mRNA levels and IFNA17 contents is presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. L GSEAs of TCGA prostate dataset revealing negative associations between high IFNA17 expression in prostate tissues with gene signatures representing androgen-responsive signaling (GO, Nelson, Wang, PID, and Hallmark). NES normalized enrichment score, FDR false discovery rate. M IFNA17 cytokine concentrations in patient sera derived from benign prostatic hyperplasia (BHP; n = 23), primary prostate cancer ( n = 16), and castration-resistant prostate cancer (CRPC) samples ( n = 8). * vs. BPH; # vs. primary prostate cancer, by a two-way ANOVA. N Representative images of IHC staining of CHRM4 and CHGA in selected tissue sections from patients diagnosed with BHP, primary prostate cancer, and CRPC from ( M ).

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer

doi: 10.1038/s41419-023-05836-7

Figure Lengend Snippet: Various prostate cancer cells were treated with 0, 1, 5, 10, 25, 50, and 100 μM of the small-molecule drugs, ceritinib ( A ), PD102807 ( B ), and LY2033298 ( C ) for 24 h, and cell viability was assessed by an MTT colorimetric assay. * vs. the control (0 μM). n = 8 per group. D , E Sphere-formation assay of PC3 and LASCPC01 cells treated with DMSO or 5 μM ceritinib during 1 week. * vs. DMSO. n = 5 per group by a t -test. F , G Relative CHRM4, MYCN, IFNA17, NE marker (CHGA, ENO2, and SYP), and immune checkpoint (PDL1 and CTLA4) mRNA levels in PC3 and LASCPC01 cells treated with DMSO or ceritinib at 10 and 25 μM for 24 h, as measured by an RT-qPCR analysis. * vs. DMSO, by a one-way ANOVA. Quantification of relative mRNA expressions is presented as the mean ± SEM from three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. H , I Tumor growth monitoring of LASCPC01 cells subcutaneously injected into male nude mice. One month after injection, DMSO or ceritinib (25 mg/kg) was intraperitoneally inoculated into mice once a week for 4 weeks. The tumor volume was measured every week, and tumor tissues were collected on the last day of the experiment. DMSO-injected mice ( n = 13); ceritinib-injected mice ( n = 14). * vs. DMSO, *** p < 0.001, by a t -test. IHC staining ( J ) and representa t ive intensities ( K ) of CHRM4, IFNA17, MYCN, KI67, ENO2, and PDL1 in subcutaneous tumors from I . * vs. DMSO. ** p < 0.01, *** p < 0.001. Significance was examined by a two-tailed Student’s t -test. L A schematic summary of this study. Our study focused on androgen-deprivation therapy (ADT)-induced NE prostate cancer (NEPC) to determine the mechanism by which androgen receptor (AR) loss of function might promote CHRM4-driven AKT/MYCN signaling leading to increased IFNA17 and PDL1 expressions. Increased abundances of IFNA17 and PDL1 may be regulated by the MYCN transcription factor through a positive feedback mechanism. Serum IFNA17 levels can be considered a prognostic biomarker in NEPC-like prostate cancer, and targeting CHRM4 may have the potential to inhibit NEPC progression.

Article Snippet: AR-positive prostate cancer cell lines (LNCaP, C4-2, and 22Rv1) and an AR-negative prostate cancer cell line (PC3) were obtained from ATCC and cultured in RPMI-1640 medium (Thermo Fisher Scientific, 11875-085) supplemented with 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR) and 1% penicillin.

Techniques: Colorimetric Assay, Control, Tube Formation Assay, Marker, Quantitative RT-PCR, Injection, Immunohistochemistry, Two Tailed Test, Biomarker Discovery

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured in low or high density. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without serum. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without aphidicolin. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. cGAS in nuclear/cytosolic fractions of indicated cell types. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Western Blot, Cell Culture

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Fluorescence images of GFP‐hcGAS, GFP‐hcGASΔcGAMP, GFP‐hcGASΔDNA, and GFP‐hcGASΔOligo in HEK293 cells cultured with or without aphidicolin. Scale bar: 20 μm. B Corresponding quantification of (A). The nuclear cGAS/total cGAS was calculated from 6 different fields with n > 50 cells. C, D A nuclear export signaling (NES) is not sufficient to dislodge chromatin‐bound cGAS from the nucleus. (C) Fluorescence images of GFP‐hcGAS, GFP‐hcGAS‐NLS, and GFP‐hcGAS‐NES in HEK293 cells. Scale bar: 10 μm. (D) Immunoblots of subcellular fractions of GFP‐hCGAS‐, GFP‐hCGAS‐NLS‐, and GFP‐hCGAS‐NES‐expressing HEK293 cells. Data information: Data are presented as means ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05 and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Fluorescence, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Micronuclei (indicated by arrowhead) in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells before (0 h) or 24 h after γ‐irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show mean ± SEM ( n = 3 independent experiments) representing six different microscopic fields with over 200 cells. C IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n = 3 independent experiments. D Experimental outline for micronucleus generation and cell death after γ‐irradiation. E Micronucleus (indicated by arrowhead) and cGAS staining in WT and cGAS −/− BMDMos exposed to γ‐irradiation (10 Gy). Scale bar: 10 μm. F Average MNs/cell in BMDMos. MN graphs show mean ± SEM ( n = 3 independent experiments) representing eight different microscopic fields with over 200 cells. G Cell death in WT and cGAS −/− BMDMos that were first synchronized at G2/M, then γ‐irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ± SD, x biological triplicates ( n = 3) per treatment group are shown. Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two‐tailed Student's t ‐test. *** P ≤ 0.001 and **** P ≤ 0.0001. Statistical significance in (G) was assessed using two‐way ANOVA test, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Expressing, Irradiation, Transfection, Plasmid Preparation, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Pulsed‐field gel electrophoresis analysis of γ‐irradiated (10 Gy) WT and cGAS −/− BMDMos. B, C Comet assay in GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells γ‐irradiated (IR: 10 Gy) for 15 min (B). RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293T cells stimulated with transfected DNA for 6 h (C). D, E Comet assay of HEK293 cells stimulated with 10 μg/ml cGAMP for indicate periods, then γ‐irradiated and incubated at 37°C for indicated duration (D). (E) Immunoblots of IRF3 phosphorylation in HEK293 cells treated as in (D). F–H Images (F) and quantifications (G) of comet tails 15 min after irradiation of GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells. RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells stimulated with transfected 23 DNA for 6 h (H). I, J Images (I) and quantifications (J) of micronuclei in GFP‐NLS‐ and GFP‐hcGASΔcGAMP‐expressing HEK293 cells 24 h after γ‐irradiation (IR; 10 Gy). DAPI (DNA). Scale bar: 10 μm. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. K Quantifications of comet tails 15 min after irradiation (10 Gy) of GFP‐NLS‐, GFP‐hcGAS‐, or GFP‐mcGAS‐expressing HEK293 cells. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. Data information: Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS P > 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001. Mean ± SEM of n = 3 independent experiments. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Pulsed-Field Gel, Electrophoresis, Irradiation, Single Cell Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Schematics of HR and NHEJ reporter assays. C, D Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts depict knockdown efficiency of cGAS and histone H1.2. E, F Results showing the effect of hcGAS, hcGAS▵cGAMP, hcGAS▵DNA, or hcGAS▵Oligo on HR (E) or NHEJ (F) in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression. Data information: Data are means with SEM, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05, * P ≤ 0.05, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Knockdown, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Reporter assays showing the effect of NLS and NES on cGAS‐mediated inhibition of DNA repair. B Both full‐length hcGAS and hcGAS cat (161–522aa) inhibit HR repair. C–E cGAS does not impede ATM activation. ATM phosphorylation in γ‐irradiated (10 Gy) GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells (C), GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells (D), or γ‐irradiated (2.5 Gy) WT, cGAS −/− , and Sting −/− BMDMos (E). Data information: Data are means ± SD, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. *** P < 0.001 **** P < 0.0001, NS: P > 0.05. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A cGAS is not recruited to DSB sites: Confocal microscopic images of GFP‐NLS‐ or GFP‐hcGAS‐expressing U2OS‐DSB reporter cells incubated (or not) with Shield‐1 and 4‐OHT to induce the expression and translocation of mCherry‐LacI‐FokI (red) to specific DSB sites. Scale bar: 10 μm. The arrowheads indicate DSB sites. B cGAS does not co‐localize with γ‐H2AX at DSB sites: GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells exposed (or not) to γ‐irradiation (IR: 10 Gy), then stained for γ‐H2AX. Scale bar: 10 μm. C, D Nuclear cGAS is mainly chromatin‐bound and remains unaltered upon γ‐irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ‐irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear, and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins. E–G cGAS co‐isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co‐isolation of cGAS and DNA repair proteins: Lysates of control (−IR) and γ‐irradiated (+IR, 10 Gy, 30 min) GFP‐hcGAS‐expressing HEK293 cells were treated (or not) with benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co‐isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ‐irradiated HEK293 cells expressing GFP‐hcGAS or GFP‐hcGASΔDNA probed for indicated proteins. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Expressing, Incubation, Translocation Assay, Irradiation, Staining, Isolation, Control, Immunoprecipitation, Agarose Gel Electrophoresis, Binding Assay

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Confocal images of γ‐irradiated GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293 cells stained for RAD51 (red) with or without γ‐irradiation. Scale bar: 10 μm. Schematics of the D‐loop formation assay, including pre‐incubation of template dsDNA with cGAS cat (i) or with cGAS cat being added after RAD51 was bound to dsDNA (ii). Pre‐incubation of dsDNA with mcGAS cat prevents D‐loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D‐loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D‐loop assay. Pre‐incubation of template dsDNA with hcGAS cat blocks subsequent D‐loop formation. The percentage of D‐loop formation (below) was graphed as the average of triplicates ± SD. Data information: Unpaired two‐tailed Student's t ‐test was used for statistical analyses. NS P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: HEK293, HEK293T, U2OS, HeLa, Raw 264.7, and THP‐1(ATCC) cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) penicillin (100 IU/ml)+streptomycin (100 μg/ml).

Techniques: Irradiation, Expressing, Staining, Tube Formation Assay, Incubation, Activity Assay, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation in NB organoids. A Western blot analysis of GSPT1 protein levels in NB organoids (LU-NB-1, LU-NB-2, LU-NB-3R) after treatment with CTX-56 for 24 h. Quantification shows the fold change expression compared with DMSO control presented as the mean of 3 biological replicates; B ) Cell viability and cell death in NB organoids following GSPT1 degradation with CTX-56. Organoids were treated for 72 h. 3 biological replicates (x3 technical replicates) were performed for each model; C ) Western blot analysis of GSPT1 protein levels in NB organoids after treatment with CTX-18 for 24 h. Quantification shows the fold change expression compared with DMSO control presented as the mean of 3 biological replicates; D ) Cell viability and cell death in NB organoids following GSPT1 degradation with CTX-18. Organoids were treated for 72 h. 3 biological replicates (x3 technical replicates) were performed for each model; E ) Cell viability of NB organoids LU-NB-1, LU-NB-2, LU-NB-3R, and HEK293 cells after CTX-18 treatment for 72 h. 3 biological replicates were performed; F ) Western blot analysis of GSPT1 levels following co-treatment with CTX-18 and siCRBN in the LU-NB-1 model. Quantification shows the fold-change expression compared with DMSO control presented as the mean of 3 biological replicates; G ) Western blot analysis of GSPT1 levels following co-treatment with CTX-18 and proteasome inhibitor MG132 in LU-NB-1 organoids. Cells were pre-treated with 5 µM MG132 for 1 h, followed by 6-hour co-treatment with CTX-18 at 10 nM and 100 nM. Quantification shows the fold change in expression compared with DMSO control presented as the mean of 3 biological replicates; H ) Analysis of compound selectivity in the AlphaLISA-based ternary complex formation assay. Luminescence observed for the neosubstrate-CRBN system at 10 µM of tested MGs (CC-90009, CTX-56, CTX-18) was normalized to signal observed for the reference compound at the same concentration. The normalized response is expressed as a percentage of the reference value (mean ± SD): 0–25% (green), 26–75% (orange), 76–100% (red). NA – not analyzed; BLD – below limit of detection; I ) Western blot analysis of CRBN-neosubstrate levels in LU-NB-1 organoids treated with CTX-18 for 24 h. Quantification shows the fold change in expression compared with DMSO control presented as the mean of 3 biological replicates. Samples were run on 2 separate gels, and appropriate loading controls were used for each gel (representative loading control is shown); J ) Proteomic analysis showing downregulation of proteins in LU-NB-1 organoids after treatment with 10 nM CTX-18 for 6 h (left) or 24 h (right). ~6770 proteins were detected; downregulated proteins are marked in red. A p -value < 0.01 was considered significant. The data show the results from 4 biological replicates; K ) Proteomic analysis showing downregulation of proteins in LU-NB-1 organoids after treatment with 1 µM CTX-18 for 6 h (left) or 24 h (right). ~6770 proteins were detected; downregulated proteins are marked in red, downregulated NB-related proteins are marked in orange. A p -value < 0.01 was considered significant. The data show the results from 4 biological replicates

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Western Blot, Expressing, Control, Tube Formation Assay, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation induces caspase-mediated apoptosis in NB organoids. A Western blot analysis of PARP/cleaved PARP levels in LU-NB-1 organoids following 24 h treatment with CTX-18. Quantification shows the cleaved PARP/PARP to actin ratio presented as fold change compared with DMSO control (mean of 3 independent experiments); B ) Flow cytometry analysis of Annexin V/PI-positive LU-NB-1 cells 48 h after treatment with CTX-56 at 100 nM and 1 µM concentrations; C ) Morphological changes (brightfield imaging, upper panel) and activity of caspase 3 (brightfield + fluorescence, lower panel) in LU-NB-1 organoids 48 h after treatment with 1 µM CTX-56. 50 μm (upper panel) and 100 μm (lower panel) scale were used; D ) Cell death and cell viability of LU-NB-1 organoids after co-treatment with 100 nM CTX-56 and the pan-caspase inhibitor Z-VAD, compared with CTX-56 treatment alone. 4 biological replicates were performed. t-test with Welch correction was used for statistical analysis

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Western Blot, Control, Flow Cytometry, Imaging, Activity Assay, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation leads to MYCN suppression in chemoresistant MYCN -amplified neuroblastoma. A Gene Set Enrichment Analysis (GSEA) of PDX1 tumors showing pathways upregulated (red) and downregulated (blue) following treatment with CTX-18 (30 mg/kg for 1 or 5 days) relative to control tumors. Pathways with p < 0.05 were considered statistically significant; B ) Changes in mRNA expression of genes associated to cycling persisters, non-cycling cells, and stress response-related genes following treatment with CTX-18 in vivo. Statistical significance was assessed using a two-tailed t -test; C ) Western blot analysis of MYCN protein levels in LU-NB-1 organoids following CTX-18 treatment for 24 h. The quantification is a mean of 3 biological replicates presented as fold-change levels compared with DMSO; D ) Western blot analysis of MYCN protein levels in PDX1 tumors from mice treated with vehicle (Ctrl2, Ctrl3) or CTX-18 (30 mg/kg) for 1 day (1, 3) or 5 days (3, 4); E ) mRNA levels of MYCN and MYCN -associated core regulatory circuitry genes in PDX1 tumors following treatment with CTX-18 in vivo (1 day and 5 days treatment combined). Statistical significance was assessed using a two-tailed t -test

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Amplification, Control, Expressing, In Vivo, Two Tailed Test, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: The conditioned medium from let-7d-overexpressing RCC cells had no effects on the proliferation, migration and tube formation of HUVECs. a, b The cell proliferation assay showed that conditioned medium from let-7d transfected 786O and 769P cells had no significant influence on proliferation of HUVEC cells compared with that from vehicle control transfected cells. The data represent the mean ± SD of three independent experiments with triplicates of each group. c, d Boyden chamber assays were performed, showing that HUVEC cell migration was not significantly different in conditioned medium from let-7d-overexpressing 786O and 769P cells compared with that in conditioned medium from respective control cells. Original magnification:×20. e, f Tube formation assay showed that the number of tubes formed by HUVEC cells in conditioned medium from let-7d-overexpressing 786O and 769P cells was not significantly different from those in conditioned medium from respective control cells. Bar, 500 μm. The quantification results of migrated cells and number of tubes per well were plotted in the right. Results were displayed as the mean ± SD of three independent experiments with five random fields counted for each chamber/well. Different numbers between two groups were analyzed by two-tailed Student’s t test. *p < 0.05

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: Migration, Proliferation Assay, Transfection, Control, Tube Formation Assay, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: The conditioned medium from THP-1 cells cocultured with let-7d-overexpressing RCC cells inhibited the proliferation, migration, and tube formation of HUVEC cells. a, b The cell proliferation assay showed that the proliferation of HUVEC cells in conditioned medium from coculture system with vector control-transfected 786O and 769P cells was significantly higher than those in conditioned medium from M0 cells cultured alone or coculture system with let-7d-transfected cells. The data represented the mean ± SD of three independent experiments with triplicates of each group. c, d Boyden chamber assay was performed, showing that HUVEC migration in conditioned medium from coculture system with vector control-transfected 786O and 769P cells was significantly higher than those in conditioned medium from M0 cells cultured alone or coculture system with let-7d-transfected cells. Original magnification: ×20. e, f Tube formation assay showed that the number of tubes formed by HUVEC cells in conditioned medium from coculture system with vector control transfected 786O and 769P cells was significantly higher than those in conditioned medium from M0 cells cultured alone or coculture system with let-7d-transfected cells. Bar, 500 μm. The quantification results of migrated cells and number of tubes are plotted in the right. Results were displayed as the mean ± SD of three independent experiments with five random fields counted for each chamber/well. Different numbers between three groups were analyzed by one-way ANOVA followed by the Bonferroni-Holm procedure. *p < 0.05

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: Migration, Proliferation Assay, Plasmid Preparation, Control, Transfection, Cell Culture, Boyden Chamber Assay, Tube Formation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: Let-7d overexpression in RCC cells inhibited the M2 polarization of THP-1 cells cocultured with RCC cells. a, b Flow cytometry analysis showed that the percentage of CD204 (marker for M2 macrophage) positive cells were lower in M0 (THP-1 treated with 200 nM PMA for 48 h) cells cocultured with let-7d-overexpressing RCC cells, as compared with those cocultured with respective control RCC cells. c Real-time RT-PCR results showed that M0 cells cocultured with let-7d-overexpressing 786O cells showed decreased M2 markers, including CD163, CD206, TGF-β and CCL22, as well as increased M1 marker, including CD80, CD86, TNF-α, HLA-DR, IL-6 and IL-1β. The expression levels of CD86 and HLA-DR remain unchanged in M0 cells cocultured with let-7d-overexpressing 769P cells. The mRNA expression was normalized to GAPDH mRNA. The data represented the mean ± SD of three independent RT–PCRs (RNAs were from the same set). d The protein levels of pro-inflammatory cytokine TNF-α and IL-6 as well as anti-inflammatory cytokine TGF-β in conditioned medium from M0 cells cocultured with let-7d-overexpressing or control RCC cells were analyzed by ELISA assay. e The expression of PIGF was significantly decreased in M0 macrophages cocultured with let-7d-overexpressing RCC cells, compared with those cocultured with control RCC cells, at mRNA level and protein level as detected by real time RT–PCR (left) and ELISA assay (right). The data in d and e represented the mean ± SD of three independent experiments with triplicates of each sample. Student’s t test. *p < 0.05,**p < 0.01

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: Over Expression, Flow Cytometry, Marker, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: Let-7d overexpression inhibited macrophage M2 polarization and tumor angiogenesis in vivo. a Representative microphotograph of IHC staining shows the microvessels (CD31 positive) in subcutaneous xenografts formed by inoculating let-7d-overexpressing 786O cells or control 786O cells together with M0 macrophages. Bar, 100 μm. Three random sections for each xenograft were subjected to IHC staining and calculation of MVD. Quantitative data of the mean MVD in each group were compared. b Photograph of subcutaneous xenografts formed by inoculating let-7d-overexpressing 786O cells or control 786O cells together with M0 macrophages. Quantitative data of the mean tumor weight were shown in the right. c Real-time RT-PCR quantification of relative CD204 expression to CD68 expression in let-7d-overexpressing groups was significantly lower than that in the control groups. The mRNA expression was normalized to GAPDH mRNA. Data represented the mean ± SD of five mice. p values in a–c were obtained by two-tailed Student’s t-test. d IHC staining of CD68, CD204 and CD31 (MVD). Bar, 100 μm. The correlation between mean CD68 (e), CD204 (f) positive cell counts and MVD in each tumor sample were analyzed with r (Spearman) and p-values indicated (n = 80). (g) A linear regression and correlation among data from qRT-PCR of let-7d and the CD204 positive cell counts in 80 RCC tissues samples were shown with r (Spearman) and p-values indicated. Five independent areas with the highest numbers of discrete microvessels, CD68 or CD204 positive cells were selected and manually counted under a microscope using a 20 × objective lens.The average positive cell counts and vessel counts for each tumor tissue were used for the statistical analysis. Expression status of let-7d was shown in a log scale

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Control, Quantitative RT-PCR, Expressing, Two Tailed Test, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: IL-13 and IL-10 were direct target genes of let-7d in RCC cells. a The expression of endogenous IL-10 and IL-13 was inhibited in the let-7d-overexpressing 786O and 769P cells, compared with the control at mRNA level as detected by real time RT–PCR; The data represented the mean ± SD of three independent RT–PCRs (RNAs were from the same set). b Representative Western blot results showed that the protein level of IL-10 and IL-13 were also downregulated following lenti–pri–let-7d infection. GAPDH served as an internal loading control. The corresponding densitometry of each band was presented in a bar graph in c. d Sequence alignment of human let-7d seed sequence with the 3′-UTRs of IL-10 and IL-13. The mutated sequence in the putative let-7d binding sites for each gene was shown above each gene set (Mut). e Luciferase activity of various reporter plasmids. Statistical significance was obtained using one way ANOVA. Data presented in a, c, e were the mean ± SD of three independent experiments.*p < 0.05

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Infection, Sequencing, Binding Assay, Luciferase, Activity Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: Rescue of IL-10 and IL-13 eliminated the effects of let-7d in vitro. a Flow cytometry analysis showed that the percentage of CD204 positive cells was restored in the presence of purified IL-10 or IL-13 (100 ng/ml) respectively, as compared with control (PBS phosphate buffer solution). b The cell proliferation assay showed that the proliferation of HUVEC cells in conditioned medium from coculture system with let-7d-overexpressing RCC cells in the presence of 100 ng/ml IL-10 or IL-13 was significantly increased compared with that in the presence of control. The data represented the mean ± SD of three independent experiments with triplicates of each sample. *p < 0.05. Statistical significance was obtained using two way ANOVA. c Represented micrographs and quantitative data of a Boyden chamber assay showed that the migration of HUVEC cells was significantly restored in the conditioned medium from M0 macrophages cocultured with let-7d-overexpressing RCC cells in the presence of 100 ng/ml IL-10 or IL-13. Original magnification: ×40. d Photograph showed that the tubes formed by HUVEC cells following the addition of IL-10 or IL-13 in the coculture system. Bar, 500 μm. Histogram displayed the quantitative data of the number tubes formed by HUVEC cells in conditioned medium added with IL-10, IL-13 or control respectively. (c-d) The data presented are the mean ± SD of three independent experiments with five random fields counted for each chamber/well. e Real-time RT-PCR results showed that the expression of CD163, CD206, TGF-β and CCL22 were significantly increased and the expression of CD80, CD86, TNF-α, HLA-DR, IL-6 and IL-1β were significantly decreased in M0 cells cocultured with let-7d-overexpressing 786O after addition of 100 ng/ml IL-10 or IL-13 in the coculture system. The mRNA expression was normalized to GAPDH mRNA. The data represented the mean ± SD of three independent RT–PCRs (RNAs were from the same set). f ELISA assay showed decreased protein levels of TNF-α and IL-6 as well as increased protein level of TGF-β in conditioned medium from M0 cells cocultured with let-7d-overexpressing 786O after addition of 100 ng/ml IL-10 or IL-13 in the coculture system. g, h The expression of PIGF was significantly decreased in conditioned medium from M0 macrophages cocultured with let-7d-overexpressing 786O cells in the presence of control, compared with those in the presence of 100 ng/ml IL-10 or IL-13, at mRNA level (g) and protein level (h) as detected by real time RT–PCR and ELISA assay respectively. The data represented the mean ± SD of three independent experiments with triplicates of each sample. One-way ANOVA followed by the Bonferroni–Holm procedure. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: In Vitro, Flow Cytometry, Purification, Control, Proliferation Assay, Boyden Chamber Assay, Migration, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10

doi: 10.1007/s00262-020-02791-6

Figure Lengend Snippet: Rescue of IL-10 and IL-13 overcame the effects of let-7d in vivo. Let-7d expression inversely correlated with the IL-10 and IL-13 mRNA levels in RCC tissues. a Western blot results demonstrated that the protein levels of IL-10 and IL-13 were increased in the let-7d-overexpressing 7860 cells infected with lentivirus carrying the expression cassette without the 3′-UTRs of IL-10 or IL-13 respectively. The corresponding densitometry of each band was presented in a bar graph. The data represented the mean ± SD of three independent experiments. b Photograph of subcutaneous xenografts from the three groups. Quantitative data of the mean tumor weight were shown in the right. c Representative graphs of IHC staining of MVD (CD31 positive) in xenografts formed by inoculating M0 macrophages together with let-7d-overexpressing 786O cells infected with IL-10, IL-13 expressing or vector control lentiviruses. Bar, 100 μm. Three random sections for each xenograft were subjected to IHC staining and calculation of MVD. Quantitative data of the mean MVD in each group were compared in the right. d Real-time RT-PCR quantification of relative CD204 expression to CD68 expression in xenografts formed by inoculating M0 macrophages together with let-7d-overexpressing 786O cells infected with IL-10, IL-13 expressing or vector control lentiviruses. The mRNA expression was normalized to GAPDH mRNA. Data in b, c and d represented the mean ± SD of five mice. p values were obtained by one-way ANOVA followed by the Bonferroni–Holm procedure. e, f The relationship between let-7d expression and IL-10 (e) and IL-13 (f) mRNA levels in RCC samples (80 cases). Statistically significant inverse correlations were observed between let-7d and IL-10 and between let-7d and IL-13 mRNA levels using the two-tailed Spearman’s test. *p < 0.05, **p < 0.01

Article Snippet: Cell lines and reagents Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA).

Techniques: In Vivo, Expressing, Western Blot, Infection, Immunohistochemistry, Plasmid Preparation, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Protein & Cell

Article Title: JMJD1C forms condensate to facilitate a RUNX1-dependent gene expression program shared by multiple types of AML cells

doi: 10.1093/procel/pwae059

Figure Lengend Snippet: N-terminal region of JMJD1C interacts with RUNX1 and is critical for survival of multiple AML cells. (A) Schematics of different truncations of JMJD1C protein used in . (B) Immunoprecipitation of GFP nanobody-conjugated beads with 293T NEs expressing different mEGFP-f-JMJD1C truncations (described in ) and HA-RUNX1. Bound proteins were detected with antibodies shown on left. Quantified and normalized values of co-IPed HA-RUNX1 are indicated below each lane. The co-IPed HA-RUNX1 value was determined by the HA signals in the IP group normalized by HA-RUNX1 in the input group, then further normalized to the bait mEGFP-f-JMJD1C signals. The value in Lane 7 was set as “1”. (C) Direct interaction assay using purified f-JMJD1C truncations and HA-RUNX1 protein. Purified f-JMJD1C truncated proteins were incubated with HA-RUNX1 immobilized on anti-HA agarose beads. Immunoprecipitants were analyzed by immunoblot with antibodies indicated on left. (D–F) Fold changes in GFP percentages (Day 0/Day 11 or Day 0/Day 14) of MOLM-13 (D), Kasumi-1 (E), and MV4-11 (F) cells. Day 0: the day when sgRNA expressing cells reach maximal levels of GFP expression, and when Cas9 was induced with doxycycline. Each dot represents an individual sgRNA targeting JMJD1C gene. Shaded areas are regions with depleted sgRNAs targeting N-terminal region of JMJD1C, zinc finger domain, and JmjC domain of JMJD1C. (G–I) Bar plots of growth competition assays showing the changes of GFP + cells expressing sgRNAs targeting Rosa , PCNA , 5ʹ-exon, N-terminal region (NTR), Zinc finger domain (ZFD), and JmjC domain of JMJD1C in MOLM-13 (G), Kasumi-1 (H) and MV4-11 (I) cells. sgRNA targeting Rosa serves as a negative control and sgRNA targeting PCNA serves as a positive control. Y-axis represents the GFP percentages of Day 12 divided by Day 0 after sgRNA-expressing cells reach maximal levels of GFP expression, at which point Cas9 was induced with doxycycline. Data are presented as mean ± SD. P -values were determined using unpaired two-tailed Student’s t -test, *** *P < 0.0001.

Article Snippet: Immunoblots were performed using primary antibodies against JMJD1C (Chen et al. 2015) (custom-made, 1:5,000); RUNX1 (Santa Cruz Biotechnology, sc-365644, 1:500); GFP (CST, 2955S, 1:3,000); mCherry (Easybio, BE2026, 1:3,000); Flag-HRP (Sigma, A8592, 1:5,000); HA (Easybio, BE2007, 1:1000; abcam, ab9110, 1:5,000); LDB1 (abcam, ab96799, 1 :2,000); GAPDH (Easybio, BE0024, 1:5,000) and ACTB (Easybio, BE0021, 1:5,000).

Techniques: Immunoprecipitation, Expressing, Purification, Incubation, Western Blot, Negative Control, Positive Control, Two Tailed Test

Journal: Protein & Cell

Article Title: JMJD1C forms condensate to facilitate a RUNX1-dependent gene expression program shared by multiple types of AML cells

doi: 10.1093/procel/pwae059

Figure Lengend Snippet: JMJD1C N-terminus mediates formation of RUNX1-DNA droplets. (A) Representative images for immunofluorescence staining of JMJD1C and RUNX1 in primary cells from three AML patients. The scale bar represents 2 μm. (B) Schematic of the ABA-inducible CRISPR-mediated genomic imaging system to visualize the self-interaction ability of JMJD1C (1–757) and its ability to recruit RUNX1 at target genomic loci (left panel, created in BioRender. C, Q. (2023)). Representative images depicting the interaction dynamics between sfGFP-JMJD1C (1–757), Halo-JMJD1C (1–757), and mCherry-RUNX1 at selected genomic loci on chromosome 3 in living 293T cells (right panel). sfGFP was fused with PYL1 and can be recruited to chromosome 3 through the dCas9-ABI system. sfGFP, Halo, or mCherry proteins serve as negative controls for sfGFP-JMJD1C (1–757), Halo-JMJD1C (1–757), or mCherry-RUNX1. The scale bars represent 2 μm. (C) Representative images of in vitro droplets formation assay to indicate JMJD1C N-terminus formed droplets could incorporate RUNX1 and RUNX1-bound DNA. mEGFP or mCherry proteins serve as negative controls for mEGFP-tagged proteins or mCherry-RUNX1. DNA with mutant RUNX1 motif serves as a control for non-RUNX1 binding sequence. (D) Quantification of droplet numbers formed by indicated proteins, related to . P -values were determined using ANOVA followed by Dunn’s multiple comparison test, **** P < 0.0001, ns represents not significant. (E) Quantification of mCherry signals that are incorporated into GFP droplets. mCherry intensity was divided by GFP signal intensity in each droplet formed by mEGFP-tagged proteins to indicate the incorporation levels of mCherry-RUNX1 into condensates, related to . P -values were determined using ANOVA followed by Dunn’s multiple comparison test, **** P < 0.0001. (F) Quantification of DAPI signals that are incorporated into GFP droplets. DAPI intensity was divided by GFP signal intensity in each mEGFP-tagged protein-formed droplet to show the incorporation levels of DNA into condensates, related to . P -values were determined using ANOVA followed by Dunn’s multiple comparison test, **** P < 0.0001.

Article Snippet: Immunoblots were performed using primary antibodies against JMJD1C (Chen et al. 2015) (custom-made, 1:5,000); RUNX1 (Santa Cruz Biotechnology, sc-365644, 1:500); GFP (CST, 2955S, 1:3,000); mCherry (Easybio, BE2026, 1:3,000); Flag-HRP (Sigma, A8592, 1:5,000); HA (Easybio, BE2007, 1:1000; abcam, ab9110, 1:5,000); LDB1 (abcam, ab96799, 1 :2,000); GAPDH (Easybio, BE0024, 1:5,000) and ACTB (Easybio, BE0021, 1:5,000).

Techniques: Immunofluorescence, Staining, CRISPR, Imaging, In Vitro, Tube Formation Assay, Mutagenesis, Control, Binding Assay, Sequencing, Comparison

Journal: Journal of advanced research

Article Title: P53-response circRNA_0006420 aggravates lung cancer radiotherapy resistance by promoting formation of HUR/PTBP1 complex.

doi: 10.1016/j.jare.2023.07.011

Figure Lengend Snippet: Fig. 3. Promotion effect of IRSense on DDR in vitro. A. WB analysis of the protein levels of ATM, CHK2, p-Cdc25c, and Cdc2 after radiation treatment of A549 and H1299 cells. B. Quantitative analysis of the mean protein levels of ATM, CHK2, p-Cdc25c, and Cdc2 performed using ImageJ software. C. WB analysis of the protein levels of ATM, CHK2, rH2AX, p-ATM, and Rad62 after 4 Gy radiation treatment of IRSense-depleted A549 (p53-wt) and A549 (p53-KO) cells. D. Quantitative analysis of the mean protein levels of ATM, CHK2, rH2AX, p-ATM, and Rad62 performed using ImageJ software. E. WB analysis of the protein levels of ATM, CHK2, rH2AX, p-ATM, and Rad62 after 4 Gy radiation treatment in A549 (p53-wt) and A549 (p53-KO) cells treated with an IRSense overexpression plasmid. F. Quantitative analysis of the mean protein levels of ATM, CHK2, rH2AX, p-ATM, and Rad62 using ImageJ software. G. IHC analysis of CHK2 expression in mice. Scale bar, 50 lm. H. IF analysis of ATM expression and IHC analysis of CHK2 expression in mice. I. rH2AX foci in IRSense-depleted A549 (p53-wt) and A549(p53-KO) cells. rH2AX foci in A549 (p53-wt) and A549 (p53-KO) cells treated with an IRSense overexpression plasmid. GAPDH was used as the loading control. Data are means ± SD. N = 3 independent experiments, two-tailed Student’s t-test.*p < 0.05.

Article Snippet: The antibodies used for western blot analysis targeted the following proteins: GAPDH (glyceraldehyde-3-phosphate dehydrogenase, CST, 5174), Ecadherin (epithelial cadherin, CST, 14472), ATM (serine-protein kinase ATM, Abcam, ab201022), CHK2 (serine/threonine-protein kinase, Abcam, ab109413), Cdc2 (cell division control protein 2 homolog, Abcam, ab32444), p-Cdc25c (M phase inducer phosphatase 3, Abcam, ab47322), Rad52 (DNA repair protein RAD52 homolog, Abcam, ab124971), rH2AX (histone H2AX, Abcam, ab229914), p53 (CST, 2527S), N-cadherin (CST, 31593), vimentin (CST, 14472S), HUR (CST,12582S), EIF4A3 (protein CASC3, Abcam, ab32485), PTBP1 (Thermo Fisher Scientific, PA5-18197), Wee1 (Wee1-like protein kinase, CST, 13084S), CDK1 (cyclin-dependent kinase 1, CST, 4539S), and p21(CST, 2947S).

Techniques: In Vitro, Software, Over Expression, Plasmid Preparation, Expressing, Control, Two Tailed Test

Journal: Journal of advanced research

Article Title: P53-response circRNA_0006420 aggravates lung cancer radiotherapy resistance by promoting formation of HUR/PTBP1 complex.

doi: 10.1016/j.jare.2023.07.011

Figure Lengend Snippet: Fig. 4. Silencing IRSense suppresses the EMT in a p53-dependent manner in vitro and in vivo. A. WB analysis of the protein levels of E-cadherin, N-cadherin, and vimentin after 4 Gy radiation treatment in IRSense-depleted A549 (p53-wt) and A549 (p53-KO) cells. B. WB analysis of the protein levels of E-cadherin, N-cadherin, and vimentin after radiation treatment in IRSense-depleted A549 (p53-wt) and A549 (p53-KO) cells. C. WB analysis of the protein levels of E-cadherin, N-cadherin, and vimentin after 4 Gy radiation treatment in A549 (p53-wt) and A549 (p53-KO) cells treated with an IRSense overexpression plasmid. D. WB analysis of the protein levels of E-cadherin, N-cadherin, and vimentin after radiation treatment in A549 (p53-wt) and A549 (p53-KO) cells treated with an IRSense overexpression plasmid. E. HE staining of lung tissues of IRSense- depleted p53-wt and p53-KO mice. Scale bar, 50 lm. F. HE staining of lung tissues of p53-wt and p53-KO mice injected (or not) with an IRSense-stable overexpression vector. Scale bar, 50 lm. G. Electron micrograph of the lung structure of IRSense-depleted p53-wt and p53-KO mice. Scale bar, 1 lm. H. Electron micrograph of lung structure in p53- wt and p53-KO mice injected (or not) with an IRSense-stable overexpression vector. Scale bar, 1 lm. I. IHC analysis of E-cadherin expression in lung tissues of IRSense- depleted p53-wt and p53-KO mice. Scale bar, 50 lm. J. p53-wt and p53-KO mice injected (or not) with an IRSense-stable overexpression vector. Scale bar, 50 lm. GAPDH was used as the loading control.

Article Snippet: The antibodies used for western blot analysis targeted the following proteins: GAPDH (glyceraldehyde-3-phosphate dehydrogenase, CST, 5174), Ecadherin (epithelial cadherin, CST, 14472), ATM (serine-protein kinase ATM, Abcam, ab201022), CHK2 (serine/threonine-protein kinase, Abcam, ab109413), Cdc2 (cell division control protein 2 homolog, Abcam, ab32444), p-Cdc25c (M phase inducer phosphatase 3, Abcam, ab47322), Rad52 (DNA repair protein RAD52 homolog, Abcam, ab124971), rH2AX (histone H2AX, Abcam, ab229914), p53 (CST, 2527S), N-cadherin (CST, 31593), vimentin (CST, 14472S), HUR (CST,12582S), EIF4A3 (protein CASC3, Abcam, ab32485), PTBP1 (Thermo Fisher Scientific, PA5-18197), Wee1 (Wee1-like protein kinase, CST, 13084S), CDK1 (cyclin-dependent kinase 1, CST, 4539S), and p21(CST, 2947S).

Techniques: In Vitro, In Vivo, Over Expression, Plasmid Preparation, Staining, Injection, Expressing, Control

Journal: Journal of advanced research

Article Title: P53-response circRNA_0006420 aggravates lung cancer radiotherapy resistance by promoting formation of HUR/PTBP1 complex.

doi: 10.1016/j.jare.2023.07.011

Figure Lengend Snippet: Fig. 6. P53 promotes IRSense/HUR/PTBP1 complex formation to promotion of radiation resistance. A. WB analysis of the protein levels of E-cadherin, N-cadherin, and vimentin in A549 (p53-wt) and A549 (p53-KO) cells, treated (or not) with radiation, with or without silencing of PTBP1 expression. B. KEGG analysis of proteins binding HUR regulated by IRSense after 4 Gy radiation treatment. C. Co-localization of HUR (red) using an antibody against PTBP1 (green) in A549 (p53-wt) and A549 (p53-KO) cells after 4 Gy radiation treatment, with or without an IRSense-depleted siRNA; nuclei were stained with DAPI (blue). Scale bar, 10 lm. D. WB analysis of the protein levels of HUR and PTBP1 in A549 (p53-wt) and A549 (p53-KO) cells treated (or not) with radiation after IRSense silencing or overexpression. E. Nuclear and cytoplasmic HUR and PTBP1 levels determined by WB after IRSense overexpression. F. Quantification of PTBP1 in nuclear and cytoplasmic expression after IRSense overexpression. G. Colon formation assay of A549 cells treated or not with radiation after IRSense silencing or overexpression, or co-silencing of IRSense and PTBP1. GAPDH was used as the loading control and H3 as a nuclear biomarker. Data are means ± SD. N = 3 independent experiments, two-tailed Student’s t-test.*p < 0.05.

Article Snippet: The antibodies used for western blot analysis targeted the following proteins: GAPDH (glyceraldehyde-3-phosphate dehydrogenase, CST, 5174), Ecadherin (epithelial cadherin, CST, 14472), ATM (serine-protein kinase ATM, Abcam, ab201022), CHK2 (serine/threonine-protein kinase, Abcam, ab109413), Cdc2 (cell division control protein 2 homolog, Abcam, ab32444), p-Cdc25c (M phase inducer phosphatase 3, Abcam, ab47322), Rad52 (DNA repair protein RAD52 homolog, Abcam, ab124971), rH2AX (histone H2AX, Abcam, ab229914), p53 (CST, 2527S), N-cadherin (CST, 31593), vimentin (CST, 14472S), HUR (CST,12582S), EIF4A3 (protein CASC3, Abcam, ab32485), PTBP1 (Thermo Fisher Scientific, PA5-18197), Wee1 (Wee1-like protein kinase, CST, 13084S), CDK1 (cyclin-dependent kinase 1, CST, 4539S), and p21(CST, 2947S).

Techniques: Expressing, Binding Assay, Staining, Over Expression, Tube Formation Assay, Control, Biomarker Discovery, Two Tailed Test

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a Western blot analysis of NELF-A and NELF-E in non-tumorigenic breast epithelial cell line MCF10A, as well as in different breast cancer cell lines T-47D (ER + , PR + ), SK-BR-3 (HER2 + ), MCF7 (ER + ), BT-474 (HER2 + ), BT-549 (triple-negative), MDA-MB-231 (triple-negative), and SUM159 (triple-negative). β-actin was used as the loading control. b , c Left: MCF7 cells ( n = 3) and BT-549 cells ( n = 4) were transfected with non-targeting siRNA (‘scrambled, siSCR’) and siRNAs targeting NELF-A and NELF-E, respectively, followed by western blot analysis. GAPDH was used as the loading control. Right: Representative images and quantification of soft agar assays. d Left: Western blot analysis of NELF-A, NELF-E, NELF-B, and NELF-C/D in WT and NELF-A/NELF-E KO SUM159 cells. β-actin was used as the loading control. Right: Representative images and quantification of soft agar assays ( n = 4). e 5 × 10 6 WT, NELF-A KO, and NELF-E KO SUM159 cells were injected into the mammary fat pads of NOD/SCID mice, respectively. Tumor volume was measured every 3 days after the tumor was palpable; mean ± SEM, n = 4/group. f Left: Tumors harvested from WT, NELF-A KO, and NELF-E KO groups; Right: Quantification of tumor weights; mean ± SEM, n = 4/group. Blots and images are representative of at least three independent experiments. Data in ( b – d ) are presented as mean ± SD. p -values are determined by a two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Western Blot, Transfection, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a MA plot showing gene expression changes between NELF-E KO and WT SUM159 cells. Red and blue points indicate significantly upregulated ( n = 388) and downregulated ( n = 540) genes, respectively. b GSEA enrichment plot for stemness and epithelial-mesenchymal transition pathways in NELF-E KO SUM159 cells. c Western blot analysis of NELF-E and NELF-A in WT, NELF-E KO, and NELF-E rescue SUM159 cells. β-actin was used as the loading control. d Quantification of wound healing assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells. Scale bar = 100 μm. e Quantification of mammosphere formation assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells ( n = 4). Scale bar = 100 μm. f Quantification of invasion assay in WT, NELF-E KO, and NELF-E rescue SUM159 cells ( n = 3). Scale bar = 100 μm. g Body weight (mean ± SEM) of mice injected with 1.25 × 10 6 WT and NELF-E KO SUM159 cells, respectively ( n = 8, each group). h Lungs inflated and fixed with 10% neutral buffer formalin from mice in WT ( n = 8) and NELF-E KO group ( n = 8). i Representative images of H&E staining in lung tissues at three magnifications (mf: metastatic foci). From top to bottom, Bar = 2 mm, 500 μm, and 100 μm, respectively. j Quantitative analysis of lung metastasis presented as % of occupation by metastasis and the number of metastatic sites per lung ( n = 8/group). k Graph showing quantification of mammospheres in WT, NELF-E KO, and NELF-E rescue MCF7 cells at primary ( n = 3), secondary ( n = 4), and tertiary passages ( n = 4). l MCF7 cells from tertiary spheroids were seeded at a density of 1000 cells per well and incubated for 2 weeks. The colonies were then fixed and stained with crystal violet. m Flow cytometry analysis and quantification of the CD24 low /CD44 high population in MCF7 tertiary mammospheres ( n = 3). Blots and images are representative of at least three independent experiments. Data in ( d , e , f , j , k , m ) are presented as mean ± SD. p -values are determined by a two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Expressing, Western Blot, Wound Healing Assay, Tube Formation Assay, Invasion Assay, Injection, Staining, Incubation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a Barplot showing the top perturbed pathways from Molecular Signatures Database (MSigDB) in Doxycycline (Dox)-treated MCF7ras+SS cells compared to the vehicle control. b Volcano plot depicting the significantly upregulated genes ( n = 1750) and downregulated genes ( n = 2223). Select mesenchymal/stemness-related genes are labeled in red, while epithelial genes are labeled in blue. adj. p -value ≤ 0.05; |fold change| ≥ 1.5. p -value was calculated using DESeq2 package (see details in “Methods”) c Western blot analysis for different markers in Dox-induced MCF7ras+SS cells transduced with scrambled or two independent shRNAs targeting NELF-E. GAPDH was used as the loading control. d GSEA plot of shNELF-E+Dox vs SCR+Dox cells. p -value was calculated from gene set enrichment analysis. (see detail in “Methods”). e Representative images and quantification of the migration and invasion assays. MCF7ras+SS cells were transduced with scrambled or NELF-E shRNA and treated with Dox to induce SOX9 and SLUG expression for 72 h prior to the assays ( n = 3). Scale Bar = 100 μm. f Representative images and quantification of the sphere-formation assay. MCF7ras+SS cells were transduced with scrambled or NELF-E shRNA, followed by 72-h Dox treatment. Cells were seeded at a density of 10K per well, and the number of spheres was counted 5–7 days later ( n = 4). Scale Bar = 100 μm. g Representative images and quantification of the soft agar assays for SCR+Dox and shNELF-E+Dox cells ( n = 3). h Flow cytometry analysis and quantification of the CD24 low /CD44 high population in NELF-E KD+Dox cells compared to SCR+Dox cells ( n = 3). Blots and images are representative of at least three independent experiments. p -values in ( e – h ) are determined by a two-tailed Student’s t -test. Mean ± SD is represented by bar graphs. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Labeling, Western Blot, Transduction, Migration, shRNA, Expressing, Tube Formation Assay, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a Log 2 fold change of UP-genes ( n = 1750), DOWN-genes ( n = 2223), and unchanged genes ( n = 1581) in the two different conditions. Gene categorization was based on the condition of SCR+Dox vs SCR-Con with the cutoffs of adj. p -value ≤ 0.05 and |fold change| ≥ 1.5. Center lines show median values, box limits represent the upper and lower quartiles, and whiskers show 1.5× the interquartile range. Two-tailed Student’s t -tests were used for all comparisons. p -values were not adjusted for multiple tests, and t -statistics are provided in source data. b Genomic distribution of NELF-E binding sites in Dox-treated MCF7ras+SS cells. c Metagene plots of NELF-E ChIP-seq signals in SCR-Con and SCR+Dox cells for UP-genes (top panel) and DOWN-genes (bottom panel). d Metagene plots of RNAPII ChIP-seq signals in SCR-Con, SCR+Dox, and shNELF-E+Dox cells for UP-genes and DOWN-genes. e Genome browser tracks showing NELF-E and RNAPII occupancies at the promoters of mesenchymal gene FN1 (left) and stemness-related gene CD44 (right). Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Two Tailed Test, Binding Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a Volcano plot of NELF-E qPLEX-RIME analysis of WT+Dox vs WT-Con. Proteins that satisfy the significance threshold of |log 2 (fold change)| ≥0.5 and adj. p -value <0.05 are labeled with their gene names and colored red. The p -value was adjusted by Benjamini–Hochberg multiple hypothesis correction. b Interaction network plot of proteins enriched in Dox induction by qPLEX-RIME, superimposed on the STRING interaction network. Interactions detected by qPLEX-RIME are colored in blue, while interactions from the STRING database are colored in gray. Proteins/nodes are colored based on log 2 (fold change) value. c Western blot analysis of SOX9, SLUG, and NELF-E following NELF-E immunoprecipitation (IP), performed on nuclear extracts from Dox-treated MCF7ras+SS cells. Blots are representative of three independent experiments. d Genomic distribution of SLUG binding sites in MCF7ras+SS cells transduced with scrambled shRNA and treated with Dox (SCR+Dox). e Metaplots of SLUG binding across the three different categories in SCR+Dox and shNELF-E+Dox MCF7ras+SS cells. f , g Pie chart and heatmap depiction of SLUG and NELF-E co-bound peaks at TSS and distal regions. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Labeling, Western Blot, Immunoprecipitation, Binding Assay, Transduction, shRNA

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a Dot plot showing KAT2B as one of the top genes ( n = 41) whose SLUG-mediated activation was most significantly attenuated by NELF-E KD. Expressions of corresponding genes in the two comparisons are connected by gray lines. b Genome browser tracks showing the occupancies of NELF-E, SLUG, and RNAPII on the KAT2B locus. c RT-qPCR analysis (left) and western blot analysis (right) of KAT2B in MCF7ras+SS cells treated with Dox for different time points ( n = 3). d Western blot analysis of KAT2B in MCF7ras+SS cells transduced with scrambled shRNA or two independent NELF-E shRNAs and treated with or without Dox. GAPDH was used as the loading control. e Heatmap depiction of SLUG/NELF-E/KAT2B/RNAPII signals at NELF-E-alone, SLUG-alone and NELF-E/SLUG co-bound regions. f Metaplot of KAT2B ChIP-Seq signals over the different cohorts of SLUG-bound regions. g Venn diagram showing the overlap between SLUG-bound UP-genes and NELF-E/KAT2B targets. The percentage overlap of SLUG-bound UP-genes compared to other categories is also indicated. h Genome browser tracks showing the occupancies of NELF-E, SLUG, and KAT2B on LAMC1 and ZEB1 loci. Blots are representative of three independent experiments. p -values are determined by a two-tailed Student’s t -test. Mean ± SD is represented by bar graphs. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Transduction, shRNA, ChIP-sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

doi: 10.1038/s41467-023-38132-1

Figure Lengend Snippet: a GSEA plot showing significantly enriched pathways in siKAT2B+Dox vs siSCR+Dox MCF7ras+SS cells. p -value was calculated from gene set enrichment analysis (see details in “Methods”). b Western blot analysis of ECM1 and CD44 in MCF7ras+SS cells treated with or without GA and AA. GAPDH was used as the loading control. Images and blots are representative of three independent experiments. c qRT-PCR analysis of FN1 and ECM1 in GA- and AA-treated Dox-induced MCF7ras+SS cells ( n = 3). d Flow cytometry analysis and quantification of the CD24 low /CD44 high population in vehicle control, GA-, and AA-treated MCF7ras+SS cells, as a function of Dox induction. e Flow cytometry analysis and quantification of the CD24 low /CD44 high population following KAT2B overexpression in MCF7ras+SS cells. Vector control (GFP only; pCAGIG) and KAT2B-overexpression (KAT2B+GFP; pCAGIG-KAT2B) plasmids were transfected into MCF7ras+SS cells, respectively. The GFP − and GFP + populations were isolated and analyzed for the CD24 low /CD44 high population ( n = 3). f , g Patient-derived breast cancer organoids were transduced with scrambled shRNA or two independent KAT2B or NELF-E shRNAs, followed by western blot and sphere-formation assay ( n = 3). β-actin was used as the loading control. h Patient-derived breast cancer organoids were transduced with vector control or KAT2B overexpressing plasmid, followed by western blot and sphere-formation assay ( n = 4). GAPDH was used as the loading control. Blots and images are representative of at least three independent experiments. p -values in ( c – h ) are determined by a two-tailed Student’s t -test. Mean ± SD is represented by bar graphs. Source data are provided as a Source Data file.

Article Snippet: Then, 200 μl of Dynabeads® Protein A (Thermo Fisher Scientific, 1002D) and 10 μg of NELF-E antibody (Abcam, ab170104) or Rabbit IgG antibody (Invitrogen, TH275005) were used for each sample.

Techniques: Western Blot, Quantitative RT-PCR, Flow Cytometry, Over Expression, Plasmid Preparation, Transfection, Isolation, Derivative Assay, Transduction, shRNA, Tube Formation Assay, Two Tailed Test