Journal: Oncogenesis

Article Title: BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling

doi: 10.1038/s41389-020-0218-z

Figure Lengend Snippet: a Overall chromatin accessibility changes of AGS cells following a 72 h treatment of JQ1 revealed by ATAC-seq analysis. b The location of the differentially accessible peaks were shown. c Motif-enrichment analysis of the differentially accessible sites. d The total number of differentially expressed genes of AGS cells after JQ1 treatment for 72 h revealed by RNA-seq analysis. e GO cluster analysis showed that the differentially expressed mRNAs were subjected to multiple GO terms. f The top 20 upregulated and top 20 downregulated genes after JQ1 treatment for 72 h demonstrated by RNA-seq analysis. g WB and qRT-PCR analysis of the mRNA and protein expression of NID1 in HGC27 and AGS cells treated with JQ1 (0, 200 nM, 500 nM, 1 μM, 2 μM, and 5 μM) for 72 h (mean ± SEM, **** p < 0.0001).

Article Snippet: We utilized the following antibodies in our research: anti-BRD4 (ab128874), anti-NID1 (ab133686), anti-c-MYC (ab32072), anti-E-cadherin (ab231303), and anti-vimentin (ab8978) antibodies from Abcam (Cambridge, MA, USA); anti-BRD4 (13440S), anti-E-cadherin (3195S), anti-Snail (3879S), anti-caspase-3 (14220), and anti-pro-caspase-3 (9661) antibodies from Cell Signaling Technology (Danvers, MA, USA); anti‐RUNX2 (sc-390715) antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti‐vimentin (10366-1-AP) antibody from ProteinTech Group (Rosemont, IL, USA); the anti-NID1 (AF2570) antibody from R&D Systems (Minneapolis, MN, USA); and the anti‐β‐actin (A5441) antibody from Sigma‐Aldrich (St Louis, MO, USA).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: Oncogenesis

Article Title: BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling

doi: 10.1038/s41389-020-0218-z

Figure Lengend Snippet: a Luciferase reporter gene assay showed that the transcriptional activity of RUNX2 promoter in AGS cells was repressed by JQ1 (0, 200 nM, 500 nM, 1 μM, 2 μM, and 5 μM) (mean ± SEM, **** p < 0.0001). b ChIP-qRT-PCR analysis of BRD4 occupancy of the RUNX2 gene in AGS cells after JQ1 (0, 1 μM) treatment for 72 h (mean ± SEM, * p < 0.05). c , d RUNX2 overexpression significantly antagonized the downregulation of the migration and invasion induced by JQ1 in HGC27 and AGS cells. Representative images of the migrated and invaded GC cells in each group were shown on the left. Cells that migrated and invaded through the pores of transwell plates were counted in five random fields and were reported on the right (mmean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). e RUNX2 overexpression significantly attenuated the downregulation of NID1 expression induced by JQ1 in HGC27 and AGS cells detected by WB analysis. f , g NID1 overexpression significantly repressed the downregulation of the migration and invasion induced by JQ1 in HGC27 and AGS cells. Representative images of the migrated and invaded GC cells in each group were shown on the left. Cells that migrated and invaded through the pores of transwell plates were counted in five random fields and were reported on the right (mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: We utilized the following antibodies in our research: anti-BRD4 (ab128874), anti-NID1 (ab133686), anti-c-MYC (ab32072), anti-E-cadherin (ab231303), and anti-vimentin (ab8978) antibodies from Abcam (Cambridge, MA, USA); anti-BRD4 (13440S), anti-E-cadherin (3195S), anti-Snail (3879S), anti-caspase-3 (14220), and anti-pro-caspase-3 (9661) antibodies from Cell Signaling Technology (Danvers, MA, USA); anti‐RUNX2 (sc-390715) antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti‐vimentin (10366-1-AP) antibody from ProteinTech Group (Rosemont, IL, USA); the anti-NID1 (AF2570) antibody from R&D Systems (Minneapolis, MN, USA); and the anti‐β‐actin (A5441) antibody from Sigma‐Aldrich (St Louis, MO, USA).

Techniques: Luciferase, Reporter Gene Assay, Activity Assay, Quantitative RT-PCR, Over Expression, Migration, Expressing

Journal: Oncogenesis

Article Title: BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling

doi: 10.1038/s41389-020-0218-z

Figure Lengend Snippet: a , b Knockdown of NID1 impeded the migration and invasion of HGC27 and AGS cells. c , d Overexpression of NID1 increased the migration and invasion of HGC27 and AGS cells. For Fig. 6a–d, representative images of the migrated and invaded GC cells in each group were shown on the left. Cells that migrated and invaded through the pores of transwell plates were counted in five random fields and were reported on the right (mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). e Knockdown of NID1 inhibited the protein expression of Vimentin in HGC27 and AGS cells shown by WB analysis. f Overexpression of NID1 enhanced the protein expression of Vimentin in HGC27 and AGS cells indicated by WB analysis. g Differential expression of NID1 in patients with AGC, EGC, and PL compared with NG (mean ± SEM, NS > 0.05, * p < 0.05, **** p < 0.0001). h Immunohistochemistry staining showing weak expression and strong expression of NID1 in the tumor tissues from two patients with AGC. i Kaplan–Meier curves comparing overall survival in AGC patients with negative and positive expression of NID1 ( n = 82; P = 0.031, log-rank test). j GEPIA (Gene Expression Profiling Interactive Analysis) data from the GEO databases (dataset 79,973) demonstrates elevated mRNA levels of NID1 in GC, compared with non-tumor tissues (mean ± SEM, **** p < 0.0001). k Kaplan–Meier curves comparing overall survival in GC patients from TCGA dataset ( n = 374) with negative ( n = 92) and positive ( n = 282) expression of NID1 ( n = 82; P = 0.0167, log-rank test).

Article Snippet: We utilized the following antibodies in our research: anti-BRD4 (ab128874), anti-NID1 (ab133686), anti-c-MYC (ab32072), anti-E-cadherin (ab231303), and anti-vimentin (ab8978) antibodies from Abcam (Cambridge, MA, USA); anti-BRD4 (13440S), anti-E-cadherin (3195S), anti-Snail (3879S), anti-caspase-3 (14220), and anti-pro-caspase-3 (9661) antibodies from Cell Signaling Technology (Danvers, MA, USA); anti‐RUNX2 (sc-390715) antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti‐vimentin (10366-1-AP) antibody from ProteinTech Group (Rosemont, IL, USA); the anti-NID1 (AF2570) antibody from R&D Systems (Minneapolis, MN, USA); and the anti‐β‐actin (A5441) antibody from Sigma‐Aldrich (St Louis, MO, USA).

Techniques: Knockdown, Migration, Over Expression, Expressing, Quantitative Proteomics, Immunohistochemistry, Staining, Gene Expression

Journal: Oncogenesis

Article Title: BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling

doi: 10.1038/s41389-020-0218-z

Figure Lengend Snippet: a Twelve mice were divided into two groups and were treated with DMSO and JQ1, respectively. All mice and resected tumors were photographed after killing. b The weights and volumes of resected tumors from each mouse were recorded. c The body weights of each mouse were recorded. d The protein expression of NID1 of the resected tumors revealed by WB analysis. e The mRNA expression of NID1 of the resected tumors revealed by qRT-PCR analysis (mean ± SEM, ** p < 0.01). f Schematic model for how JQ1 regulated the RUNX2/NID1 signaling via altering c hromatin accessibility in GC cells.

Article Snippet: We utilized the following antibodies in our research: anti-BRD4 (ab128874), anti-NID1 (ab133686), anti-c-MYC (ab32072), anti-E-cadherin (ab231303), and anti-vimentin (ab8978) antibodies from Abcam (Cambridge, MA, USA); anti-BRD4 (13440S), anti-E-cadherin (3195S), anti-Snail (3879S), anti-caspase-3 (14220), and anti-pro-caspase-3 (9661) antibodies from Cell Signaling Technology (Danvers, MA, USA); anti‐RUNX2 (sc-390715) antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti‐vimentin (10366-1-AP) antibody from ProteinTech Group (Rosemont, IL, USA); the anti-NID1 (AF2570) antibody from R&D Systems (Minneapolis, MN, USA); and the anti‐β‐actin (A5441) antibody from Sigma‐Aldrich (St Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

Article Title: The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

doi: 10.1007/s00417-021-05156-6

Figure Lengend Snippet: Chain-specific laminin expression in ILM and iERMs. a–c Idiopathic ERM/ILM double labelled with anti-type IV collagen (red) and anti laminin 1+2 (green) antibodies. The iERM expresses both antigens. The ILM (arrowheads) shows an exuberant immunoreactivity for the anti-laminin 1+2 antibody. d–f Idiopathic ERM/ILM triple labelled with anti-α5(IV) (cyan), anti-entactin (red), and anti-laminin β2 (green) antibodies. The α5 chain of collagen IV and the laminin β2 are expressed exclusively in the ILM (arrowheads). Entactin, in contrast, is expressed either in the iERM or in the ILM. g–i Idiopathic ERM/ILM double labelled with anti-entactin (red) and anti-laminin α5 (green) antibodies. Whereas laminin α5 is present almost exclusively in the ILM (arrowheads), entactin is expressed in both membranes. V vitreal side of the iERM, R retinal side of the ILM. j–m Idiopathic ERM/ILM triple labelled with anti-laminin γ1 (cyan), anti-entactin (red), and anti-laminin β1 (green) antibodies. Though with a different degree of intensity, all antigens are expressed either in the ILM (arrowheads) or in the iERM. Entactin and the laminin γ1 are prevalently located in the ILM, whereas the laminin β1 fluorescence is more evident in the iERM. ERM epiretinal membrane. a–f Magnification bars = 10 μm. g – m Magnification bars = 20 μm

Article Snippet: The following primary antibodies were used: mouse anti-vimentin (clone V9) monoclonal antibody (code V6389) was from Sigma-Aldrich (Saint Louis, MO); monoclonal mouse anti-laminin α5 (clone CL3118) (code NBP2-42391) and rabbit anti-collagen I (code NB600-408) antibodies were from Novus Biologicals Europe (Abingdon, UK); goat anti-collagen IV (code 1340-01) was purchased from Southern Biotech (Birmingham, AL); rabbit anti-collagen VI (code ab6588), rabbit anti-collagen III (ab7778), and rabbit anti-laminin 1+2 (code ab7463) antibodies were obtained from Abcam (Cambridge, UK); mouse anti-laminin-2, α2 chain specific, monoclonal antibody (clone 5H2) was from Gibco BRL (Gaithersburg, MD); mouse anti-laminin-5, γ2 chain-specific, monoclonal antibody (clone 4G1, code M7262) was obtained from Dako (Glostrup, Denmark); mouse anti-laminin γ1 (clone 2E8, code MAB1920), mouse anti-laminin α4 (clone 6C3, code sc-130541), and mouse anti-laminin-5, γ2 chain-specific (clone D4B5, code MAB19562), monoclonal antibodies were obtained from Chemicon (Temecula, CA); rat anti-α5(IV) (NC1 domain) collagen monoclonal antibody (clone 5H2, code 7077) was purchased from Chondrex Inc. (Woodinville, WA); rat anti-laminin β1 (clone LT3, code sc-33709), mouse anti-laminin β2 (clone C4, code sc-59980), and mouse anti-laminin β3 (clone A-6, code sc-133178) monoclonal antibodies were from Santa Cruz (Dallas, TX); and goat anti-entactin antibody (code AF2570) and rabbit anti-fibronectin antibody (code GTX112794) were obtained from R&D Systems (Minneapolis, MN) and GeneTex Inc. (Irvine, CA) respectively.

Techniques: Expressing, Fluorescence, Membrane

Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

Article Title: The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

doi: 10.1007/s00417-021-05156-6

Figure Lengend Snippet: Anti-ECM protein (excluding laminin chains) antibodies employed in the study and their reactivity with the ILMs and the ERMs

Article Snippet: The following primary antibodies were used: mouse anti-vimentin (clone V9) monoclonal antibody (code V6389) was from Sigma-Aldrich (Saint Louis, MO); monoclonal mouse anti-laminin α5 (clone CL3118) (code NBP2-42391) and rabbit anti-collagen I (code NB600-408) antibodies were from Novus Biologicals Europe (Abingdon, UK); goat anti-collagen IV (code 1340-01) was purchased from Southern Biotech (Birmingham, AL); rabbit anti-collagen VI (code ab6588), rabbit anti-collagen III (ab7778), and rabbit anti-laminin 1+2 (code ab7463) antibodies were obtained from Abcam (Cambridge, UK); mouse anti-laminin-2, α2 chain specific, monoclonal antibody (clone 5H2) was from Gibco BRL (Gaithersburg, MD); mouse anti-laminin-5, γ2 chain-specific, monoclonal antibody (clone 4G1, code M7262) was obtained from Dako (Glostrup, Denmark); mouse anti-laminin γ1 (clone 2E8, code MAB1920), mouse anti-laminin α4 (clone 6C3, code sc-130541), and mouse anti-laminin-5, γ2 chain-specific (clone D4B5, code MAB19562), monoclonal antibodies were obtained from Chemicon (Temecula, CA); rat anti-α5(IV) (NC1 domain) collagen monoclonal antibody (clone 5H2, code 7077) was purchased from Chondrex Inc. (Woodinville, WA); rat anti-laminin β1 (clone LT3, code sc-33709), mouse anti-laminin β2 (clone C4, code sc-59980), and mouse anti-laminin β3 (clone A-6, code sc-133178) monoclonal antibodies were from Santa Cruz (Dallas, TX); and goat anti-entactin antibody (code AF2570) and rabbit anti-fibronectin antibody (code GTX112794) were obtained from R&D Systems (Minneapolis, MN) and GeneTex Inc. (Irvine, CA) respectively.

Techniques: