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: 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: 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: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Clonogenic survival assay for mGS and mGSRR subjected to irradiation (IR) or control without IR. Left: representative images of colonies formed by surviving cells 13 days after a single dose (4 Gy) of irradiation are shown. Right: fraction of surviving cells after radiation doses of 1, 3, or 5 Gy. Scale bar: 10 mm. (B) Survival curves for mice implanted with 1000 tumor cells (mGS or mGSRR) and subjected to whole-brain irradiation consisting of a daily dose of 2 Gy from days 3 to 7 after cell implantation (10 Gy total). Log-rank test. (C) Cell proliferation analysis for mGS and mGSRR after 72 hours. (D) Self-renewal ability of mGS and mGSRR as evaluated by sphere formation assay in soft agar. Scale bar: 10 mm. (E) Western blot showing expression of stem cell marker (Olig2) progressively increases and neural differentiation maker (Tuj1) is decreased following repeated cycles of irradiation in mGS cells. All blots show representative images (n = 3). (F) Representative images of mGS and mGSRR cells stably expressing a fluorescence marker (mCherry) and grown as spheres in neural stem cell medium are shown. Scale bars: 200 μm. (G) Single-cell suspension of mGS and mGSRR cells were cultured for 4 hours, and then the number of nonattached cells determined by differential centrifugation. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student’s t tests unless otherwise indicated.

Article Snippet: Western blots were performed as described ( 73 ) using antibodies targeting the following proteins: Olig2 (1:1000, Millipore, AB9610), Tuj1 (1:1000, Biolegend, 801202), β-actin (1:2000, Santa Cruz, sc-69879), CD109 (1:100, Santa Cruz, sc-271085), mouse N-cadherin (1:500, Becton Dickinson, 610920), human N-cadherin (1:500, Santa Cruz, sc-7939), E-cadherin (1:1000, Cell Signaling, #3195), VE-cadherin (1:500, Santa Cruz, sc-9989), VCAM1 (1:250, Santa Cruz, sc-8304), Talin1 (1:500, Cell Signaling, #4021), Tensin2 (1:500, Cell Signaling, #11990), Vinculin (1:500, Cell Signaling, #4650), FAK (1:500, Cell Signaling, #3285), β-catenin (1:500, Becton Dickinson, #610193), pan β-catenin (1:500, Cell Signaling, #8480), non-phospho β-catenin (1:500, Cell Signaling, #8814), phospho- (S33/S37/T41) β-catenin (1:500, Cell Signaling, #9561), p120-catenin (1:500, Becton Dickinson, 610133), c-Myc (1:1000, Cell Signaling, #5605), cleaved PARP (1:500, Cell Signaling, #9544), mouse Clusterin (1:500, R&D, AF2747), human Clusterin (1:500, Santa Cruz, sc-5289), Snail1 (1:500, Cell Signaling, #3879), Slug (1:500, Cell Signaling, #9585), Zeb1 (1:1000, Cell Signaling, #3396), and IGF1R (1:500, Cell Signaling, #3018).

Techniques: Clonogenic Cell Survival Assay, Irradiation, Tube Formation Assay, Western Blot, Expressing, Marker, Stable Transfection, Fluorescence, Suspension, Cell Culture, Centrifugation

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Western blot showing expression of cell-cell adhesion molecules following 6 to 12 cycles of 5 Gy irradiation in mGS cells. (B) Fluorescence microscopy shows that N-cad expression is increased on the cell surface of mGSRR cells (green). mGS and mGSRR cells are stably expressing mCherry (red). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 25 μm. (C) Western blot showing expression of N-cad following 3 cycles of irradiation (1–2 Gy) in human GSCs, MGG4. (D) Western blot showing expression of N-cad, Tuj1, and Olig2 in human GSCs (MGG4) transfected with either control (Ctrl) or human N-cad expression vectors (OE). (E) Cell proliferation analysis for MGG4-Ctrl and MGG4 N-cad OE cells. (F) Clonogenic survival assay showing the surviving fraction of MGG4+/– N-cad cells after radiation doses of 1, 3, or 5 Gy. (G) Schematic showing experimental design to establish radioresistant PDX models. Subcutaneous tumors were exposed to repeated irradiation (2 Gy × 6 = 12 Gy total over 2 weeks). (H) Western blot showing expression of N-cad, Tuj1, and Olig2 in primary (P) or adapted to radiation therapy (RT) tumors of 2 PDX models. (I) Kaplan-Meier curve shows that increased N-cad mRNA expression is correlated with reduced survival in the TCGA-GBM data set. Log-rank test. High and low are defined as the top and bottom 15%. All blots show representative images (n = 3 or more). *P < 0.05, **P < 0.01, 2-tailed Student’s t tests unless otherwise indicated. The intensity of the immunoreactive bands was quantified in 3 independent experiments and the average is indicated below the blot.

Article Snippet: Western blots were performed as described ( 73 ) using antibodies targeting the following proteins: Olig2 (1:1000, Millipore, AB9610), Tuj1 (1:1000, Biolegend, 801202), β-actin (1:2000, Santa Cruz, sc-69879), CD109 (1:100, Santa Cruz, sc-271085), mouse N-cadherin (1:500, Becton Dickinson, 610920), human N-cadherin (1:500, Santa Cruz, sc-7939), E-cadherin (1:1000, Cell Signaling, #3195), VE-cadherin (1:500, Santa Cruz, sc-9989), VCAM1 (1:250, Santa Cruz, sc-8304), Talin1 (1:500, Cell Signaling, #4021), Tensin2 (1:500, Cell Signaling, #11990), Vinculin (1:500, Cell Signaling, #4650), FAK (1:500, Cell Signaling, #3285), β-catenin (1:500, Becton Dickinson, #610193), pan β-catenin (1:500, Cell Signaling, #8480), non-phospho β-catenin (1:500, Cell Signaling, #8814), phospho- (S33/S37/T41) β-catenin (1:500, Cell Signaling, #9561), p120-catenin (1:500, Becton Dickinson, 610133), c-Myc (1:1000, Cell Signaling, #5605), cleaved PARP (1:500, Cell Signaling, #9544), mouse Clusterin (1:500, R&D, AF2747), human Clusterin (1:500, Santa Cruz, sc-5289), Snail1 (1:500, Cell Signaling, #3879), Slug (1:500, Cell Signaling, #9585), Zeb1 (1:1000, Cell Signaling, #3396), and IGF1R (1:500, Cell Signaling, #3018).

Techniques: Western Blot, Expressing, Irradiation, Fluorescence, Microscopy, Stable Transfection, Transfection, Clonogenic Cell Survival Assay

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Western blot showing expression changes of several N-cad binding catenins following 6–12 cycles of irradiation (5 Gy) in mGS cells. (B) Fluorescence microscopy shows that β-catenin (green) selectively coaccumulates with N-cad (red) on the cell surface of mGSRR but not mGS cells. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 25 μm. (C) Wnt/β-catenin regulated transcriptional activity in mGS and mGSRR cells measured through transient transfection with a luciferase reporter driven by a WT (TOP) or mutant (FOP) TCF binding site. ***P < 0.001, 2-tailed Student’s t test. (D) TOP/FOP ratio showing Wnt/β-catenin activity in parental N-cad–overexpressing and N-cad–KO mGS cells. **P < 0.01, ***P < 0.001, Tukey’s HSD test. (E) Microarray analysis showing that mRNA expression of multiple Wnt target genes is suppressed in mGSRR compared with mGS cells. Each group contains 2 independent replicates (n = 2). (F) qRT/PCR showing that NeuroD1, Ngn1, and Brn3a mRNAs are reduced in mGSRR cells. Two-tailed Student’s t test. (G) Western blot showing expression change of β-catenin (pan and non-phospho), c-Myc, and Tuj1 by N-cad–overexpressing and N-cad–KO mGS cells. All blots show representative images (n = 3 or more).

Article Snippet: Western blots were performed as described ( 73 ) using antibodies targeting the following proteins: Olig2 (1:1000, Millipore, AB9610), Tuj1 (1:1000, Biolegend, 801202), β-actin (1:2000, Santa Cruz, sc-69879), CD109 (1:100, Santa Cruz, sc-271085), mouse N-cadherin (1:500, Becton Dickinson, 610920), human N-cadherin (1:500, Santa Cruz, sc-7939), E-cadherin (1:1000, Cell Signaling, #3195), VE-cadherin (1:500, Santa Cruz, sc-9989), VCAM1 (1:250, Santa Cruz, sc-8304), Talin1 (1:500, Cell Signaling, #4021), Tensin2 (1:500, Cell Signaling, #11990), Vinculin (1:500, Cell Signaling, #4650), FAK (1:500, Cell Signaling, #3285), β-catenin (1:500, Becton Dickinson, #610193), pan β-catenin (1:500, Cell Signaling, #8480), non-phospho β-catenin (1:500, Cell Signaling, #8814), phospho- (S33/S37/T41) β-catenin (1:500, Cell Signaling, #9561), p120-catenin (1:500, Becton Dickinson, 610133), c-Myc (1:1000, Cell Signaling, #5605), cleaved PARP (1:500, Cell Signaling, #9544), mouse Clusterin (1:500, R&D, AF2747), human Clusterin (1:500, Santa Cruz, sc-5289), Snail1 (1:500, Cell Signaling, #3879), Slug (1:500, Cell Signaling, #9585), Zeb1 (1:1000, Cell Signaling, #3396), and IGF1R (1:500, Cell Signaling, #3018).

Techniques: Western Blot, Expressing, Binding Assay, Irradiation, Fluorescence, Microscopy, Activity Assay, Transfection, Luciferase, Mutagenesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) qRT/PCR showing that mGSRR cells display increased Cdh2 and decreased Cdh1 mRNA expression compared with mGS. Two-tailed Student’s t test. (B) Western blot showing expression of Slug, Snail1, and Zeb1 are gradually increased upon repeated irradiation in mGS cells. (C) Western blot showing that Snail overexpression induces elevation of N-cad, Olig2, and Zeb1, and suppression of Tuj1 in mGS cells. (D) Wnt/β-catenin transcriptional activity is suppressed in mGSRR and mGS with Snail1 overexpression (OE) compared with mGS cells. ***P < 0.001, Tukey’s HSD test. (E) Clonogenic survival assay shows mGS Snail1 OE cells have a higher survival rate than mGS cells. Two-tailed Student’s t test. *P < 0.05, **P < 0.01. (F) Western blot showing increased N-cad, β-catenin, Slug, and Zeb1 expression 2 days after mouse recombinant IGF1 (100 ng/mL), but not TGF-β1 (10 ng/mL) treatment in mGS cells. (G) Western blot showing IGF1 overexpression increases N-cad, β-catenin, Zeb1, and IGF1R expression in mGS cells. (H) Survival curves for mice implanted with 1000 cells (GS with IGF1 expression vector) and subjected to whole-brain irradiation (2 Gy/day, days 3 to 7, 10 Gy total). (I) Left: schematic showing experimental design for clonogenic survival assay with repeated irradiation. Single mGS cells seeded in agarose medium were exposed to repeated irradiation (5 doses of 4 Gy, every 3 days) with or without drug rescue. IGF1R (AEW541 0.5 μM; PPP 0.2 μM) and TGF-β1 (LY2157299 10 μM, SB431542 10 μM) inhibitors were used. Right: quantification of percentage of surviving colonies shows that IGF1R inhibitors selectively decreased survival rate. Drugs alone had no effect on colony formation (data not shown). ***P < 0.001, Dunnett’s test. (J) Mice implanted orthotopically with mGSRR cells had a survival benefit after whole-brain irradiation (2 Gy × 5 days) with adjuvant PPP (15 mg/kg, i.p. twice a day from day 3–7) in contrast to vehicle control, only IR or PPP alone (8 mice/group; log-rank test). All blots show representative images (n = 3 or more).

Article Snippet: Western blots were performed as described ( 73 ) using antibodies targeting the following proteins: Olig2 (1:1000, Millipore, AB9610), Tuj1 (1:1000, Biolegend, 801202), β-actin (1:2000, Santa Cruz, sc-69879), CD109 (1:100, Santa Cruz, sc-271085), mouse N-cadherin (1:500, Becton Dickinson, 610920), human N-cadherin (1:500, Santa Cruz, sc-7939), E-cadherin (1:1000, Cell Signaling, #3195), VE-cadherin (1:500, Santa Cruz, sc-9989), VCAM1 (1:250, Santa Cruz, sc-8304), Talin1 (1:500, Cell Signaling, #4021), Tensin2 (1:500, Cell Signaling, #11990), Vinculin (1:500, Cell Signaling, #4650), FAK (1:500, Cell Signaling, #3285), β-catenin (1:500, Becton Dickinson, #610193), pan β-catenin (1:500, Cell Signaling, #8480), non-phospho β-catenin (1:500, Cell Signaling, #8814), phospho- (S33/S37/T41) β-catenin (1:500, Cell Signaling, #9561), p120-catenin (1:500, Becton Dickinson, 610133), c-Myc (1:1000, Cell Signaling, #5605), cleaved PARP (1:500, Cell Signaling, #9544), mouse Clusterin (1:500, R&D, AF2747), human Clusterin (1:500, Santa Cruz, sc-5289), Snail1 (1:500, Cell Signaling, #3879), Slug (1:500, Cell Signaling, #9585), Zeb1 (1:1000, Cell Signaling, #3396), and IGF1R (1:500, Cell Signaling, #3018).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Irradiation, Over Expression, Activity Assay, Clonogenic Cell Survival Assay, Recombinant, Plasmid Preparation, Adjuvant

Journal: Molecular Therapy. Nucleic Acids

Article Title: SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

doi: 10.1016/j.omtn.2021.12.035

Figure Lengend Snippet: Overexpression of FTO inhibited growth, migration, and invasion of GBM cells in vitro and in vivo (A) Expression levels of FTO in U87MG, A172, and U118MG cells after transduction with control lentivirus (ovNC) and FTO-overexpressing lentivirus (ovFTO). GAPDH was used as control. (B) The proliferation capacity of U87MG and A172 cells transduced with lentivirus was assessed using CCK-8 assay. (C) Transwell assays of U87MG and A172 cells. Representative images are shown. Scale bar, 50 μm. Histograms representing the number of migrating or invading cells. Data are presented as mean ± SD; n = 3. (D) Three-dimensional tumor spheroid invasion assay of U87MG cells transduced with lentivirus. Images at 0, 24, 48, and 72 h are shown; scale bar, 200 μm. (E) Protein levels of ZEB1, CD44, N-cadherin, PCNA, mmp2, and vimentin in U87MG, A172, and U118MG cells transduced with ovNC or ovFTO lentivirus. GAPDH was used as control. (F) Representative images of tumor sphere formation of GSC267 cells transduced with lentivirus and quantified by the diameter of spheres. Scale bar, 100 μm. (G) Extreme limiting dilution assay of GSC267 cells transduced with lentivirus. Data represent mean ± SD from three independent experiments. (H) Protein levels of FTO, CD44, and YKL40 in GSC267 cells transduced with lentivirus. β-Actin was used as control for normalization. (I) In vivo bioluminescent imaging analysis of tumor growth in xenograft nude mice at day 12 after implantation. (J) H&E staining of xenograft sections from FTO-overexpressing or negative control U87MG cell tissues on the day of sacrifice; scale bars, 1,000 μm (left) and 100 μm (right). (K) Survival analysis of nude mice orthotopically implanted with U87MG cells transduced with lentivirus overexpressing the control sequence or FTO (n = 5/group); log rank tests were used to identify the survival significance of the differences. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with serum of bovine or rabbit serum albumin for 30 min, the samples were incubated with primary FTO antibodies (abca dies [9449; Cell Signaling Technology; 1:500] and CD44 [15675-1-AP; Proteintech; 1:1,000]) for 2 h at room temperature or overnight at 4°C.

Techniques: Over Expression, Migration, In Vitro, In Vivo, Expressing, Transduction, Control, CCK-8 Assay, Invasion Assay, Limiting Dilution Assay, Imaging, Staining, Negative Control, Sequencing, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

doi: 10.1016/j.omtn.2021.12.035

Figure Lengend Snippet: Knockdown of FTO promoted growth, migration, and invasion of GBM cells in vitro and in vivo (A) Expression levels of FTO in U251, LN229, and P3 cells transduced with control lentivirus (shNC) or FTO-knockdown lentivirus carrying two short hairpin RNA sequences (shFTO-1 and shFTO-2). GAPDH was used as control. (B) The proliferation capacity of LN229 and U251 cells transduced with lentivirus was assessed using a CCK-8 assay. (C) Transwell assays of LN229 and U251 cells. Representative images are shown. Scale bar, 50 μm. Histograms represent the number of migrating or invading cells. (D) Three-dimensional tumor spheroid invasion assay of LN229 cells transduced with lentivirus. Images at 0, 24, 48, and 72 h are shown; scale bar, 200 μm. (E) Protein levels of ZEB1, CD44, N-cadherin, mmp2, PCNA, and vimentin in P3, LN229, and U251 cells transduced with shNC, shFTO-1, or shFTO-2 lentivirus. GAPDH was used as control. (F) Neural sphere formation assay of GSCs 8–11 transduced with shNC, shFTO-1, or shFTO-2 lentivirus. (G) Extreme limiting dilution assay of GSCs 8–11 transduced with shNC, shFTO-1, or shFTO-2 lentivirus. (H) Protein levels of FTO and YKL40 in GSCs 8–11 transduced with shNC or shFTO lentivirus. β-Actin was used as control for normalization. (I) In vivo bioluminescent imaging analysis of tumor growth in xenograft nude mice at day 6. (J) H&E staining of xenograft sections from FTO-knockdown or negative control LN229 cell tissues on the day of sacrifice; scale bars, 1,000 μm (left) and 100 μm (right). (K) Survival analysis of nude mice orthotopically implanted with LN229 cells transduced with shNC or shFTO lentivirus (n = 5/group); log rank tests were used to identify the survival significance of the differences. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with serum of bovine or rabbit serum albumin for 30 min, the samples were incubated with primary FTO antibodies (abca dies [9449; Cell Signaling Technology; 1:500] and CD44 [15675-1-AP; Proteintech; 1:1,000]) for 2 h at room temperature or overnight at 4°C.

Techniques: Knockdown, Migration, In Vitro, In Vivo, Expressing, Transduction, Control, shRNA, CCK-8 Assay, Invasion Assay, Tube Formation Assay, Limiting Dilution Assay, Imaging, Staining, Negative Control, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

doi: 10.1016/j.omtn.2021.12.035

Figure Lengend Snippet: The inhibitory effects of FTO on GBM progression are reversed by miR-10a (A) Analyses of the correlation between the expression of FTO and MTMR3, a miR-10a target, in five datasets. Statistical analysis was determined using Pearson correlation coefficient analyses. (B) MTMR3 mRNA expression in GBM cells transduced with FTO overexpression, knockdown, and the corresponding control lentiviruses. (C) Protein expression of MTMR3, CD44, PCNA, FTO, and GAPDH in U87MG cells co-transduced with ovNC or ovFTO lentivirus and mimics NC or miR-10a-5p mimics as indicated. Quantification histogram represents relative protein expression. (D) LN229 cells were co-transduced with shNC, shFTO-1, or shFTO-2 lentivirus and inhibitor NC or miR-10a-5p inhibitor as indicated. Quantification histogram represents relative protein expression. RNA expression of (E) miR-10a-5p and (F) MTMR3 in U87MG cells co-transduced with ovNC or ovFTO lentivirus and mimics NC or miR-10a-5p mimics as indicated. LN229 cells were co-transduced with shNC, shFTO-1, or shFTO-2 lentivirus and inhibitor NC or miR-10a-5p inhibitor as indicated. (G) CCK-8 assay of U87MG cells transfected with (left) mimics NC or miR-10a-5p mimics in the presence or absence of FTO overexpression of LN229 cells transfected with inhibitor NC or (right) miR-10a-5p inhibitor in the presence or absence of FTO knockdown. (H) Transwell assay of U87MG cells transfected with mimics NC or miR-10a-5p mimics in the presence or absence of FTO overexpression as indicated. Scale bar, 50 μm. (I) Transwell assay of LN229 cells transfected with inhibitor NC or miR-10a-5p inhibitor in the presence or absence of FTO knockdown. Scale bar, 50 μm. (J) IHC of MTMR3 expression in U87MG cells in xenograft sections of nude mice. Scale bar, 50 μm. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with serum of bovine or rabbit serum albumin for 30 min, the samples were incubated with primary FTO antibodies (abca dies [9449; Cell Signaling Technology; 1:500] and CD44 [15675-1-AP; Proteintech; 1:1,000]) for 2 h at room temperature or overnight at 4°C.

Techniques: Expressing, Transduction, Over Expression, Knockdown, Control, RNA Expression, CCK-8 Assay, Transfection, Transwell Assay, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

doi: 10.1016/j.omtn.2021.12.035

Figure Lengend Snippet: SPI1 inhibited the transcriptional activity of FTO (A) Predicted SPI1 DNA-binding sequences in the FTO promoter region. (B) The correlation between FTO and SPI1 and the correlation between SPI1 and MTMR3 in TCGA and CGGA databases. Statistical analysis was determined using Pearson correlation coefficient analyses. (C) The relative mRNA expression of SPI1 (left) and FTO (right) in GBM cells transfected with siNC, siSPI1, or siSPI1-2. (D) The protein expression of SPI1, FTO, MTMR3, N-cad, CD44, PCNA, and vimentin in GBM cells transfected with siNC, siSPI1, or siSPI1-2. (E) ChIP-PCR of the FTO promoter region in GBM cells. (F) Relative FTO promoter luciferase activity in GBM cells transfected with siNC, siSPI1-1, or siSPI1-2. (G) Relative FTO promoter luciferase activity in the indicated GBM cells with or without the mutation of the SPI1 binding motif. (H) Transwell assays of U87MG and U118MG GBM cells transfected with siNC or siSPI1 in the presence or absence of FTO as indicated. Scale bar, 50 μm. (I) CCK-8 assays of U87MG and U118MG GBM cells transfected with siNC or siSPI1 in the presence or absence of FTO as indicated. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with serum of bovine or rabbit serum albumin for 30 min, the samples were incubated with primary FTO antibodies (abca dies [9449; Cell Signaling Technology; 1:500] and CD44 [15675-1-AP; Proteintech; 1:1,000]) for 2 h at room temperature or overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Expressing, Transfection, Luciferase, Mutagenesis, CCK-8 Assay, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

doi: 10.1016/j.omtn.2021.12.035

Figure Lengend Snippet: The SPI1 inhibitor DB2313 interrupted the transcriptional inhibition of FTO (A) The two-dimensional (2D) binding mode of SPI1 and DB2313. (B) The binding model of DB2313 on molecular surface of SPI1. DB2313 is colored in cyan, and the molecular surface of SPI1 is colored in pale yellow (top); 3D binding mode of SPI1 and DB2313. DB2313 is colored in cyan, the surrounding residues in the binding pockets are colored in yellow, and the backbone of the receptor is depicted as white cartoon with transparency (below). (C) Protein expression of FTO, MTMR3, CD44, PCNA, and GAPDH in GBM cells treated with DMSO or DB2313. (D) CCK-8 assay of GBM cells treated with DMSO or DB2313 (10 μg/mL). (E) Transwell assay of GBM cells treated with DMSO or DB2313 as indicated. Scale bar, 50 μm. (F) DB2313 (10 mg/kg/day) or vehicle was intraperitoneally injected into mice (n = 5 per group) after subcutaneous implantation of U87MG cells. (G) Tumor volumes and weights were measured from sacrificed mice and are shown as box-and-whisker plots. (H) Represented images of immunofluorescence staining for FTO, Ki67, and CD44 in in xenograft sections from U87MG GBM cell subcutaneous xenograft mice treated with DB2313 or matched group. Scale bar, 50 μm. (I) Proposed model underlying the roles of FTO-mediated pri-miR-10a processing in GBM tumors. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with serum of bovine or rabbit serum albumin for 30 min, the samples were incubated with primary FTO antibodies (abca dies [9449; Cell Signaling Technology; 1:500] and CD44 [15675-1-AP; Proteintech; 1:1,000]) for 2 h at room temperature or overnight at 4°C.

Techniques: Inhibition, Binding Assay, Expressing, CCK-8 Assay, Transwell Assay, Injection, Whisker Assay, Immunofluorescence, Staining, 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 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: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: p53 negatively regulates CLP36 expression. A , B human osteosarcoma tissues from a tissue microarray (Tbsbio, Xi'an, China) were stained with antibodies for p53 or CLP36 as described in the “Methods” (Scale bar = 100 µm) ( A ). The clinical information of the tissue samples was shown in . The mean intensity of CLP36 in the p53 positive group (n = 5) was compared to that in the p53 negative group (n = 31) ( B ). C , D , E Skeletal muscle, thymus and bone normal tissue protein and RNA were extracted from p53 wild type (p53 +/+ ) or p53 deficient (p53 -/- ) mice. The protein levels of CLP36 were analyzed by Western blotting. Right panel, the mRNA levels of CLP36 were analyzed by RT-PCR and compared to those in the p53 +/+ tissues (normalized to 1; n = 3). F HT1080, U2OS and Saos-2 cells were analyzed by Western blotting for CLP36, p53 or GAPDH. Right panel, the mRNA levels of CLP36 in the U2OS and Saos-2 cells were analyzed by RT-PCR and compared to that in the HT1080 cells (normalized to 1; n = 3). G, H HT1080 ( G ) or U2OS ( H ) cells were infected with control (Sh-NC) or p53 (Sh-p53) shRNA lentivirus for five days. The cells were analyzed by Western blotting as indicated. Right panels, the mRNA levels of CLP36 in the Sh-p53 cells were analyzed by RT-PCR and compared to those in the Sh-NC cells (normalized to 1; n = 3). I Saos-2 cells were infected with 3xflag-tagged p53 (3fl-p53) or 3xflag vector(3fl) lentivirus for three days. The cells were analyzed by Western blotting as indicated. Right panel, the mRNA level of CLP36 in the 3fl-p53 cells was analyzed by RT-PCR and compared to that in the 3fl cells (normalized to 1; n = 3). Data in B are shown as mean ± SEM. Data in C , D , E , F , G , H , and I are presented as mean ± S.D. Statistical significance was calculated using a two-tailed unpaired Student's t -test, * p < 0.05; ** p < 0.01.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Expressing, Microarray, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Control, shRNA, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: KO of CLP36 inhibits p53 deficiency-induced tumor growth in mouse. A The conditional KO allele of the CLP36 gene was generated as described in the “Methods”. A restriction map of the relevant genomic region of CLP36 (top), the targeting vector (middle), and the mutant locus after homologous recombination (bottom) are shown. The light blue rectangles indicate the targeted exon 2. Red triangles indicate LoxP sites. Dark blue rectangles indicate FRT sites. Yellow line indicates the southern blot probe region. DTA , Diphtheria Toxic A chain gene; Neo , Neomycin resistance gene. Arrows indicate the position for genotyping primers. B DNA from electroporated embryonic stem (ES) cells were digested with Hin dIII and analyzed by Southern blotting as described in the “Methods”. The 8.4 kb and 3.8 kb bands represent wild type (WT) and mutated (MT) alleles. C Skeletal muscle tissue protein samples were extracted from the mice (as indicated) and analyzed by Western blotting with antibodies for CLP36 and GAPDH. D The mRNA level of CLP36 in the CLP36 -/+ p53 +/+ muscle tissues was analyzed by RT-PCR and compared to that in the CLP36 +/+ p53 +/+ muscle tissues (normalized to 1; n = 3). E The numbers of the CLP36 +/+ p53 -/- , CLP36 +/- p53 -/- , CLP36 -/- p53 -/- and CLP36 +/+ p53 +/+ mice with tumors (as indicated) were counted, and the tumors were dissected at the age of six months (n = 21 mice for each group). Red, sarcoma; blue, thymic lymphoma; green, other tumors including head and neck tumor, spine tumor or lung tumor; blank, tumor-free. F Survival of the CLP36 +/+ p53 -/- (n = 26), CLP36 +/- p53 -/- (n = 13), CLP36 -/- p53 -/- (n = 14), and CLP36 +/+ p53 +/+ (n = 10) mice was analyzed by Kaplan-Meier analysis for up to 335 days. Data in F were determined by log-rank test, p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Generated, Plasmid Preparation, Mutagenesis, Homologous Recombination, Southern Blot, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: CLP36 regulates p53 null sarcoma cell proliferation and migration. CLP36 KO Saos-2 cells were infected with lentiviral vectors encoding 3xflag-tagged CLP36 (3fl-CLP36) or 3xflag vector (3fl) for three days. A The cells (as indicated) were analyzed by Western blotting with antibodies for CLP36 and GAPDH. B Cell proliferation was analyzed by CCK-8 assay as described in the “Methods”. The mean absorbance of the cells (as indicated) was compared to that of the Saos-2 cells (normalized to 1; n = 3). C Focus formation assay was performed as described in the “Methods” (scale bar = 5 mm). The mean percentage of focus formation of the cells (as indicated) was compared to that of the Saos-2 cells (right panel). D Anchorage-independent growth was analyzed by soft agar assay as described in the “Methods” (scale bar = 500 pixels). The mean percentage of colony formation of the cells (as indicated) was compared to that of the Saos-2 cells (right panel). E Cell migration was analyzed using transwell motility chambers as described in the “Methods” (scale bar = 200 pixels). Right panel, the mean number of the cells (as indicated) migrated through the membrane was compared to that of the Saos-2 cells (normalized to 1; n = 3). Data in B , C , D , and E are presented as mean ± S.D. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Migration, Infection, Plasmid Preparation, Western Blot, CCK-8 Assay, Tube Formation Assay, Soft Agar Assay, Membrane

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: Depletion of CLP36 reduces cell proliferation and migration in vitro and tumor growth in vivo. Primary cells (LS1) isolated from sarcoma of the CLP36 +/+ p53 -/- mice were infected with mouse control shRNA lentivirus (m-Sh-NC) or mouse CLP36 shRNA lentivirus (m-Sh-CLP36) for three days. A The cells (as indicated) were analyzed by Western blotting with antibodies for CLP36 and GAPDH. B Cell proliferation was analyzed by CCK-8 assay as described in the “Methods”. The mean absorbance of the cells (as indicated) was compared to that of the LS1 cells (normalized to 1; n = 3). C Focus formation assay was performed as described in the “Methods” (scale bar = 5 mm). The mean percentage of focus formation of the cells (as indicated) was compared to that of the LS1 cells (right panel). D Cell migration was analyzed using transwell motility chambers as described in the “Methods” (scale bar = 500 pixels). Right panel, the mean number of the cells (as indicated) migrated through the membrane was compared to that of the LS1 cells (normalized to 1; n = 3). E Tumors derived from subcutaneously inoculated m-Sh-NC or m-Sh-CLP36 infected cells (n = 6, scale bar = 1 cm) were shown. Right panel, the mean weight of the tumors derived from the m-Sh-CLP36 infected cells was compared to that of the tumors derived from the m-Sh-NC infected cells (n = 18 tumors for each group from three independent experiments). Data in B , C and D are presented as mean ± S.D. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis, ** p < 0.01; **** p < 0.0001. Data in the right panel of E were presented as mean ± SEM. Statistical significance was calculated using two-tailed unpaired Student's t -test, ** p < 0.01.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Migration, In Vitro, In Vivo, Isolation, Infection, Control, shRNA, Western Blot, CCK-8 Assay, Tube Formation Assay, Membrane, Derivative Assay, Two Tailed Test

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: Depletion of CLP36 reduces YAP1 expression in vitro and in vivo. A , B Sh-NC or Sh-CLP36 lentivirus infected Saos-2 cells were analyzed by Western blotting for YAP1, CLP36 or GAPDH and the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3) ( A ). The mRNA levels of YAP1 in the cells (as indicated) were analyzed by RT-PCR and compared to that in the Saos-2 cells (normalized to 1; n = 3) ( B ). C , D 3fl-CLP36 or 3fl lentivirus infected CLP36 KO Saos-2 cells were analyzed by Western blotting as indicated and the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3) ( C ). The mRNA level of YAP1 in the cells (as indicated) was analyzed by RT-PCR and compared to that in the Saos-2 cells (normalized to 1; n = 3) ( D ). E Sarcoma tissues from CLP36 +/+ p53 -/- , CLP36 +/- p53 -/- , and CLP36 -/- p53 -/- mice were stained for YAP1 (scale bar = 100 µm). The mean intensity of YAP1 of the samples (as indicated) was compared to that of the CLP36 +/+ p53 -/- (n = 10 fields from two samples in the CLP36 +/+ p53 -/- or CLP36 +/- p53 -/- group; n = 5 fields from one sample in the CLP36 -/- p53 -/- group). F Thymic lymphoma tissues from CLP36 +/+ p53 -/- , CLP36 +/- p53 -/- , and CLP36 -/- p53 -/- mice were stained for YAP1 (scale bar = 50 µm). The mean intensity of YAP1 of the samples (as indicated) was compared to that of the CLP36 +/+ p53 -/- (n = 15 fields from three samples in the CLP36 +/+ p53 -/- or CLP36 +/- p53 -/- group; n = 5 fields from one sample in the CLP36 -/- p53 -/- group). G m-Sh-NC or m-Sh-CLP36 lentivirus infected LS1 cells were analyzed by Western blotting as indicated. H The tumors derived from cells (as indicated) were stained for YAP1 (scale bar = 20 µm). Right panel, the mean intensity of YAP1 in the m-Sh-CLP36 samples was compared to that in the m-Sh-NC samples (n = 15 fields from three samples). Data in A , B , C and D are presented as mean ± S.D. Data in E , F , and H are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis or two-tailed unpaired Student's t-test ( H ), * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Expressing, In Vitro, In Vivo, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Derivative Assay, Two Tailed Test

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: Inhibition of proteasome or depletion of AIP-4 reverses CLP36 deficiency-induced down-regulation of YAP1 expression. A Saos-2 cells were infected with Sh-NC or Sh-CLP36 lentivirus for three days and the Sh-CLP36 infected cells were then treated with MG132 (10 µM) or Leupeptin (10 µM) for 8h. The cells (as indicated) were analyzed by Western blotting for YAP1, CLP36 or GAPDH. Right panel, the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). B CLP36 KO Saos-2 cells were treated with MG132 (10 µM) or Leupeptin (10 µM) for 8 h. The cells (as indicated) were analyzed by Western blotting with antibodies for YAP1, CLP36 or GAPDH. Right panel, the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). C Saos-2 cells were infected with Sh-NC or Sh-CLP36 lentivirus for three days and the Sh-CLP36 infected cells were then transfected with control siRNA (siNC) or AIP-4 targeting siRNA (siAIP4) for three days. The cells (as indicated) were analyzed by Western blotting with antibodies for AIP-4, YAP1, CLP36 or GAPDH. Right panel, the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). D CLP36 KO Saos-2 cells were transfected with control siRNA (siNC) or AIP-4 targeting siRNA (siAIP4) for three days. The cells (as indicated) were analyzed by Western blotting with antibodies for AIP-4, YAP1, CLP36 or GAPDH. Right panel, the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). Data in A , B , C and D are presented as mean ± S.D. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Inhibition, Expressing, Infection, Western Blot, Transfection, Control

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: The α-actinin-binding site is required for CLP36-mediated regulation of YAP1 expression. CLP36 KO Saos-2 cells were infected with 3fl, 3fl-CLP36 or 3xflag-tagged α-actinin-binding defective CLP36 mutant (3fl-ABD) lentivirus for three days. A The cells (as indicated) were analyzed by Western blotting with antibodies for YAP1, CLP36 or GAPDH. Right panel, the YAP1 level in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). B Cell proliferation was analyzed by CCK-8 assay as described in the “Methods”. The mean absorbance in the cells (as indicated) was compared to that in the Saos-2 cells (normalized to 1; n = 3). C Focus formation assay was performed as described in the “Methods” (scale bar = 5 mm). The mean percentage of focus formation in the cells (as indicated) was compared to that in the Saos-2 cells ( F ). D Anchorage-independent growth was analyzed by soft agar assay as described in the “Methods” (scale bar = 100 pixels). The mean percentage of colony formation in the cells (as indicated) was compared to that in the Saos-2 cells ( G ). E Cell migration was analyzed using transwell motility chambers as described in the “Methods” (scale bar = 500 pixels). H The mean number of the cells (as indicated) migrated through the membrane was compared to that of the Saos-2 cells (normalized to 1; n = 3). Data in A , B , F , G and H are presented as mean ± S.D. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis, * p < 0.05; **** p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Binding Assay, Expressing, Infection, Mutagenesis, Western Blot, CCK-8 Assay, Tube Formation Assay, Soft Agar Assay, Migration, Membrane

Journal: Theranostics

Article Title: CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1

doi: 10.7150/thno.72365

Figure Lengend Snippet: Overexpression of YAP1 in CLP36 KO Saos-2 cells restores cell proliferation, focus formation and anchorage-independent growth. CLP36 KO Saos-2 cells were infected with lentiviral vectors encoding 3xflag-tagged YAP1 (3fl-YAP1) or 3xflag vector (3fl) for three days. A The cells (as indicated) were analyzed by Western blotting with antibodies for YAP1, CLP36 or GAPDH. B Cell proliferation was analyzed by CCK-8 assay as described in the “Methods”. The mean absorbance of the cells (as indicated) was compared to that of the Saos-2 cells (normalized to 1; n = 3). C Focus formation assay was performed as described in the “Methods” (scale bar = 5 mm). The mean percentage of focus formation of the cells (as indicated) was compared to that of the Saos-2 cells (right panel). D Anchorage-independent growth was analyzed by soft agar assay as described in the “Methods” (scale bar = 500 pixels). The mean percentage of colony formation of the cells (as indicated) was compared to that of the Saos-2 cells (right panel). E Cell migration was analyzed using transwell motility chambers as described in the “Methods” (scale bar = 200 pixels). Right panel, the mean number of the cells (as indicated) migrated through the membrane was compared to that of the Saos-2 cells (normalized to 1; n = 3). Data in B , C , D , and E are presented as mean ± S.D. Statistical significance was calculated using one-way ANOVA with Tukey-Kramer post-hoc analysis, *** p < 0.001; **** p < 0.0001.

Article Snippet: The pre-cleared cell lysates were incubated with 2 μg YAP1 (Mouse monoclonal, Santa Cruz, sc-101199), CLP36 (Mouse monoclonal, Santa cruz, sc-393084) or normal mouse IgG (Santa Cruz, sc-2025) antibodies and 30 μL of protein A/G-Sepharose beads at 4 °C overnight, followed by washing three times with the PBS and one time with the lysis buffer.

Techniques: Over Expression, Infection, Plasmid Preparation, Western Blot, CCK-8 Assay, Tube Formation Assay, Soft Agar Assay, Migration, Membrane

Journal: Nature cell biology

Article Title: Normal and cancer mammary stem cells evade interferon-induced constraint through the miR-199a-LCOR Axis

doi: 10.1038/ncb3533

Figure Lengend Snippet: ( a ) GSEA demonstrating the enrichment of gene sets related to MaSC , CSC , undifferentiated tumor cells , and Claudin-Low tumors in the ranked gene list of miR-199a-OE vs. control HMLE cells. ( b ) Heat map of HMLE-miR-199a-OE microarray data representing fold change expression of EMT markers, stem cell transcription factors (SC-TFs), and stem cell (SC) markers. Fold change is represented as Log2 ratio. ( c ) Western blot analysis of epithelial (blue) and mesenchymal (red) markers. ( d ) In vitro quantification of mammospheres formed by 2,000 control or miR-199a-OE HMLE cells seeded. ( e ) qRT-PCR of mRNA extracted from 5 day HMLE control or miR-199a-OE mammospheres. ( f-h ) qRT-PCR of miR-199a levels in HMLE-Neu-Twist1-ER-OE tumor initiating cells (TICs) ( f ), CD24 + /Thy1 + TICs isolated from early and late stage spontaneous MMTV-Wnt-1 tumors ( g ), CD24 − /CD44 + TICs isolated from HCI-002 human breast cancer PDX ( h ) as compared to the non-TIC counterparts (n=3 biologically independent samples; data represents mean ± SEM) in d–h . * P <0.05, ** P <0.01, *** P <0.005 by two-tailed Student’s t -test in d–h .

Article Snippet: All cell lines used in the study, including mammary epithelial cell lines (human HMLE, MCF10A and mouse NMuMG cell lines), breast cancer cell lines (human MDA-MB-231, HMLE-Neu, T47D, MCF7, BT474, and mouse 4TO7 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions.

Techniques: Microarray, Expressing, Western Blot, In Vitro, Quantitative RT-PCR, Isolation, Two Tailed Test

Journal: Nature cell biology

Article Title: Normal and cancer mammary stem cells evade interferon-induced constraint through the miR-199a-LCOR Axis

doi: 10.1038/ncb3533

Figure Lengend Snippet: ( a ) Heat map showing the differential expression of predicted miR-199a target genes (TargetScan v7.0) in P4 vs P5 cells. Fold change is represented as Log2 ratio. ( b ) qRT-PCR analysis of top candidate genes in mouse NMuMG and human HMLE cells after transfection with the indicated miRNA mimics. ( c ) Summary of the knockdown effect of the selected candidate miR-199a targets in functional assays using mouse MECs. MS: mammosphere formation assay; CFP: cleared fat pad mammary reconsititution assay. ( d ) qRT-PCR analysis of Lcor expression in different lineages of mouse MECs. ( e ) Immunohistochemistry (IHC) (left panel) analysis of Lcor and immunofluorescence (right panel) of Lcor and K14 localization in the mammary ducts. ( f ) qRT-PCR analysis of LCOR after 48h transfection of miR-199a-3p and miR-199a-5p in HMLE cells. ( g ) Western blot analysis of LCOR in control and miR-199a-OE HMLE cells. ( h ) Schematic diagram of the miR-199a binding sites on the LCOR 3′UTR. ( i ) Normalized activity of the luciferase reporter containing the WT mouse Lcor 3′ UTR or various miR-199a seed sequence mutants, after co-transfection with miR-199a-3p or miR-199a-5p, or control miRNAs in Hela cells. The reporters were divided in two groups, UP and DOWN, containing upper and lower halves of the Lcor 3′-UTR, respectively. Scale bars: 20 μm in e. n=3 biologically independent samples; data represents mean ± SEM in b , d , f and i .* P <0.05, ** P <0.01, *** P <0.005 by two-tailed Student’s t -test in bar graphs.

Article Snippet: All cell lines used in the study, including mammary epithelial cell lines (human HMLE, MCF10A and mouse NMuMG cell lines), breast cancer cell lines (human MDA-MB-231, HMLE-Neu, T47D, MCF7, BT474, and mouse 4TO7 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Functional Assay, Tube Formation Assay, Immunohistochemistry, Immunofluorescence, Western Blot, Binding Assay, Activity Assay, Luciferase, Sequencing, Cotransfection, Two Tailed Test

Journal: Nature cell biology

Article Title: Normal and cancer mammary stem cells evade interferon-induced constraint through the miR-199a-LCOR Axis

doi: 10.1038/ncb3533

Figure Lengend Snippet: ( a ) Quantification of mammosphere formation of 20,000 P4 cells in the indicated conditions (n=3 biologically independent samples; data represents mean ± SEM). ( b–c ) Limited dilution cleared fat pad reconstitution assay of P4 ( b ) and ( c ) P5 cells after transduction with the indicated constructs. Representative images show the outgrowth. Each pie chart represents a mammary gland with blackened area showing the percentage of mammary gland outgrowth. Tables below represent serial dilution injections with the corresponding take rate. n= number of mammary fat pad injections as indicated in the table. Shown in red are the repopulation frequencies for each condition and P value by Pearson’s Chi-squared test, obtained with the ELDA software. ( d ) qRT-PCR of mRNA extracted from mammospheres formed by HMLE cells after transduction with the indicated constructs (n=3 biologically independent samples; data represents mean ± SEM). ( e ) GSEA demonstrating the enrichment of gene sets related to MaSC and Claudin-low tumors in the ranked gene list of LCOR-KD vs control HMLE cells. ( f–g ) qRT-PCR analysis of Lcor expression in Lgr5 + MaSC-enriched P4 cells ( f ), CD24 + /Thy1 + MMTV-Wnt-1 TICs ( g ) and CD24 − /CD44 + TICs isolated from HCI-002 PDX ( h ) as compared to their non-stem cell counterparts (n=3 biologically independent samples; data represents mean ± SEM). Scale bars: 2 mm in b and c . * P <0.05, ** P <0.01 by two-tailed Student’s t -test in bar graphs.

Article Snippet: All cell lines used in the study, including mammary epithelial cell lines (human HMLE, MCF10A and mouse NMuMG cell lines), breast cancer cell lines (human MDA-MB-231, HMLE-Neu, T47D, MCF7, BT474, and mouse 4TO7 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions.

Techniques: Reconstitution Assay, Transduction, Construct, Serial Dilution, Software, Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test

Journal: Nature cell biology

Article Title: Normal and cancer mammary stem cells evade interferon-induced constraint through the miR-199a-LCOR Axis

doi: 10.1038/ncb3533

Figure Lengend Snippet: ( a ) Schematic representation of LCOR mutants. ( b ) Quantification of mammospheres formed by 5,000 HMLE cells with ectopic expression of the indicated LCOR constructs (n=3 biologically independent samples; data represents mean ± SEM). ( c ) Unsupervised hierarchical clustering of the HMLE expressing various LCOR constructs based on transcriptomic profiles. ( d ) GSEA of the IFN-α response gene-set (M5911) in the ranked gene list of LCOR, LSKAA and ΔHTH vs. control HMLE cells. ( e ) GSEA of the IFN-α response gene set in the ranked gene list of the LCOR-KD or miR-199a-OE vs. control HMLE and MDA-MB-231 cells. ( f ) GSEA of the IFN-α response gene set in the indicated gene list from the current study. ( g ) GSEA of the IFN-α response gene set in the gene list of CD24 − /CD44 + CSC vs. non-CSCs in ER − breast cancer . ( h ) Quantification of mammospheres formed by P4 (20,000) and P5 (10,000) cells treated with different doses of IFN-α, from 10 to 1000 U/ml in 8 days (n=3 biologically independent samples; data represented mean ± SEM). * P <0.05, ** P <0.01 by two-tailed Student’s t -test in b and h.

Article Snippet: All cell lines used in the study, including mammary epithelial cell lines (human HMLE, MCF10A and mouse NMuMG cell lines), breast cancer cell lines (human MDA-MB-231, HMLE-Neu, T47D, MCF7, BT474, and mouse 4TO7 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions.

Techniques: Expressing, Construct, 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 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