Journal: Cell Death and Differentiation

Article Title: Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration

doi: 10.1038/s41418-022-00967-4

Figure Lengend Snippet: Key reagents and resources used in this study.

Article Snippet: TBK1/NAK (D1B4) Rabbit mAb , Cell Signaling Technology , cat#3504.

Techniques: Marker, Recombinant, Transfection, Drug discovery, Gene Expression, Microarray, Software

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 1. Rapid testing microfluidic platform for early thrombosis recapitulates SARS-CoV-2 and Spike-mediated technologies thrombotic effect. a) Schematic of SARS-CoV-2 infection-mediated thrombus formation in the microcirculation. In response to viral infection, inflammatory cells are recruited to activate the extrinsic and intrinsic coagulation pathways, leading to thrombin production. Thrombin cleaves fibrinogen to fibrin, which in turn, pro- motes platelet aggregation and fibrin deposition to form blood clots in the microcirculation. b) Similarly, SARS-CoV-2 vaccination therapies Spike-based technologies have the potential role to increment blood coagulation. c) SARS-CoV-2, Spike protein, and Spike variant for mimicking microcirculation environment were assessed for their thrombotic phenotypes in multiple endothelialized microfluidic channels (2 cm x 400 mm x 100 mm). Antibody anti-IL6 and decoy nanoliposome-hACE2 were also tested together with the aforementioned conditions. SARS-CoV-2, Spike protein, and Spike variant expressed using viral vectors were incubated in the PDMS-based microfluidic channels for 12 h at 37 °C, followed by a thrombosis assay in the presence of human subject-specific whole blood at wall shear stress of 25 dyne cm−2. Thrombus formation was quantified in terms of fibrin and platelet deposition.

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Infection, Coagulation, Variant Assay, Incubation, Shear

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 3. SARS-CoV-2 and Spike-mediated inflammatory cytokines regulates coagulation cascade. a) Tissue factor (TF) binds to coagulation factor VII (FVII) to initiate the thrombosis. SARS-CoV-2 infection also induces endothelial release of cytokines (such as IL-1, IL-6, and TNF-𝛼) that mediate platelet activation and coagulation cascades. SARS-COV-2 treatment for 48 h significantly upregulated HAEC mRNA expression of TNF-𝛼, IL-1, IL-6, and IL-15 as demonstrated by the heatmap (*p < 0.05 CTRL vs SARS-COV-2, n = 4 by qRT-PCR). The heat map was constructed by using Euclidean distance with average linkage. The Z-score centered log2-transformed gene in each sample is presented by a color scale, and gene upregulation is denoted in blue, and downregulation in red. b) Spike mutation D614G inflammatory effect was tested HAECs. A microarray heatmap represents 22 genes and selected control genes in HAECs in response to Spike D614G. Hierarchical clustering heatmap reveal differentially expressed genes in response to Lenti-S mutation (in the presence or absence of Lipo-hACE2), normalized to the lenti-CTRL respectively. The heatmap was constructed as previously described, and the Z-score centered log2-transformed gene in each sample was presented as a color scale. Each condition was performed in triplicate (n = 3). In addition to increased cytokines and chemokines mRNA expression level, higher mRNA expression level of endothelial marker of thrombosis, such as vWF and PAI-1, was also observed. Furthermore, the activation of the Toll-like receptor signaling pathway suggests its crucial role in enhancing downstream inflammation and thrombosis (see Figure S5, Supporting Information) (*p < 0.05: n = 3). c) Immunocytochemical analysis showed Lenti-S D614G increasing protein level of IL-6 (in red). Nuclei were stained with DAPI (scale bar: 50 μm).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Coagulation, Infection, Activation Assay, Expressing, Quantitative RT-PCR, Construct, Transformation Assay, Mutagenesis, Microarray, Control, Marker, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 4. Lipo-hACE2 and anti-IL-6 attenuate SARS-CoV-2-mediated inflammation and thrombosis. a) In the endothelialized microfluidic platform, HAECs were exposed to SARS-CoV-2, Lenti-S D614G, or Lipo-S in the presence or absence of Lipo-hACE2 or anti-IL-6 (scale bar = 100 μm). Lipo-hACE2 and anti-IL-6 attenuated SARS-CoV-2, Lenti-S, or Lipo-S-mediated fibrin deposition. b) Both Lipo-hACE2 and anti-IL-6 reduced SARS-CoV-2, Lipo-S, and Lenti-S mediated increases in surface coverage of fibrin and platelet deposition (*p < 0.05 vs Lipo-S or Lenti-S mutation; ***p < 0.001 vs SARS-CoV-2, n = 4).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Mutagenesis

Journal: Cancer Cell International

Article Title: LINC00221 silencing prevents the progression of hepatocellular carcinoma through let-7a-5p-targeted inhibition of MMP11

doi: 10.1186/s12935-021-01819-w

Figure Lengend Snippet: LINC00221 upregulates the MMP11 expression by competitively binding to let-7a-5p. a LncATLAS website for lncRNA subcellular localization revealed that LINC00221 was mainly located in the cytoplasm. b The subcellular localization of LINC00221 verified by FISH assay (× 400; the left) and the co-localization profile intensity (the right). c Venn map ( http://bioinformatics.psb.ugent.be/webtools/Venn/ ) showing downstream regulatory miRNAs of LINC00221 predicted by RNA22 database ( https://cm.jefferson.edu/rna22/Precomputed/ ) (blue circle) and downregulated miRNAs in HCC samples from GSE12717 microarray (red circle). d The binding site between LINC00221 and let-7a-5p sequences predicted on the RNA22 website. e Venn map showing downstream target genes of let-7a-5p predicted by Starbase ( http://starbase.sysu.edu.cn/index.php ) (blue circle), mirDIP ( http://ophid.utoronto.ca/mirDIP/ ) (red circle), and miRDB ( http://www.mirdb.org/ ) (green circle), and upregulated genes in HCC samples from GSE89377 microarray (yellow circle) and from GSE117361 microarray (orange circle). f The binding site between let-7a-5p and MMP11 predicted on the RNA22 website. g The relationships among LINC00221, let-7a-5p and MMP11 identified by dual-luciferase reporter gene assay. h The let-7a-5p expression in HCC and adjacent non-tumor tissues (n = 45) examined by RT-qPCR. i The MMP11 protein expression in HCC and adjacent non-tumor tissues (n = 45) measured by Western blot analysis. j The relationships among LINC00221, let-7a-5p and Ago2 determined by RIP assay. k The binding of LINC00221 to let-7a-5p identified by RNA pull-down. l The regulatory effect of LINC00221 on let-7a-5p expression examined by RT-qPCR. m MMP11 protein expression after upregulation of let-7a-5p measured by western blot analysis. Cell experiments were independently repeated 3 times. * p < 0.05 vs. the mimic NC group, si-NC group or adjacent non-tumor tissues (n = 45); & p < 0.05 vs. the IgG group; # p < 0.05 vs. the Bio-LINC00221-MUT group. Comparison between HCC and adjacent non-tumor tissues was made using paired t- test while comparison between other two groups was performed by unpaired t -test

Article Snippet: The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, transferred onto nitrocellulose membranes, and then blocked with 5% skimmed milk (PBS) for 2 h. The membranes were incubated for 30 min at ambient temperature with rabbit polyclonal antibodies MMP11 (1:1000, ab119284), Bcl-2 (1:1000, ab182858), Cyclin D1 (1:1000, ab40754), CDK4 (1:1000, ab199728), Bax (1:1000, ab182733), and GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK).

Techniques: Expressing, Binding Assay, Microarray, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cancer Cell International

Article Title: LINC00221 silencing prevents the progression of hepatocellular carcinoma through let-7a-5p-targeted inhibition of MMP11

doi: 10.1186/s12935-021-01819-w

Figure Lengend Snippet: Silencing of LINC00221 inhibits the proliferation and invasion but accelerates apoptosis of HCC (Huh7 and MHCC97-H) cells by upregulating let-7a-5p and suppression of MMP11. The Huh7 and MHCC97-H cells were transfected with let-7a-5p mimic, si-LINC00221 or co-transfected with si-LINC00221 and si-MMP11 or si-LINC00221 and oe-MMP11. a The MMP11 protein expression in Huh7 and MHCC97-H cells examined by Western blot analysis. b The proliferation of Huh7 and MHCC97-H cells assessed by MTT assay. c The apoptosis of Huh7 and MHCC97-H cells examined by flow cytometry. d Number of invaded Huh7 and MHCC97-H cells tested by Transwell assay. e Bcl-2, Bax, cyclin D1, and CDK4 protein expression in Huh7 and MHCC97-H cells measured by Western blot analysis. Cell experiment was independently repeated 3 times. * p < 0.05 vs. the miR-NC group; # p < 0.05 vs. the si-LINC00221 + si-NC group; & p < 0.05 vs. the si-LINC00221 + oe-NC group. Comparison among multiple groups was made using one-way ANOVA with Tukey’s post hoc test while data at different time points were compared using two-way ANOVA with Bonferroni post hoc test

Article Snippet: The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, transferred onto nitrocellulose membranes, and then blocked with 5% skimmed milk (PBS) for 2 h. The membranes were incubated for 30 min at ambient temperature with rabbit polyclonal antibodies MMP11 (1:1000, ab119284), Bcl-2 (1:1000, ab182858), Cyclin D1 (1:1000, ab40754), CDK4 (1:1000, ab199728), Bax (1:1000, ab182733), and GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK).

Techniques: Transfection, Expressing, Western Blot, MTT Assay, Flow Cytometry, Transwell Assay, Comparison

Journal: Cancer Cell International

Article Title: LINC00221 silencing prevents the progression of hepatocellular carcinoma through let-7a-5p-targeted inhibition of MMP11

doi: 10.1186/s12935-021-01819-w

Figure Lengend Snippet: The mechanism involved LINC00221 in regulating the progression of HCC. LINC00221 was upregulated while let-7a-5p was downregulated in HCC cells, and highly expressed LINC00221 upregulated MMP11 expression by competitively binding to let-7a-5p, thereby promoting HCC cell viability, migration, and restraining cell apoptosis

Article Snippet: The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, transferred onto nitrocellulose membranes, and then blocked with 5% skimmed milk (PBS) for 2 h. The membranes were incubated for 30 min at ambient temperature with rabbit polyclonal antibodies MMP11 (1:1000, ab119284), Bcl-2 (1:1000, ab182858), Cyclin D1 (1:1000, ab40754), CDK4 (1:1000, ab199728), Bax (1:1000, ab182733), and GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK).

Techniques: Expressing, Binding Assay, Migration

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Table 2 The sequences of sh-NC vector and sh-TERF2

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Plasmid Preparation

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Table 3 The sequences of the primers used in this study

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: TERF2 is a downstream target of miR-29b-1-5p, and miR-29b-1-5p can target and inhibit TERF2 (A) Schematic diagram of miR-29b-1-5p target gene screening. (B) Metascape analysis of miR-29b-1-5p target gene function. (C) GSE185754 microarray analysis of miR-29b-1-5p target gene expression. (D) The ability of the predicted miR-29b-1-5p to target TERF2 was verified by dual-luciferase reporter gene assay. (E) qRT-PCR and (F) western blot analysis showing TERF2 mRNA and protein expression levels in response to LPS and miR-29b-1-5p antagomir transfection in HL-1 cells. (G) Correlation analysis between miR-29b-1-5p and TERF2 in the myocardial tissues of the LPS-treated mice and CLP-treated mice. *P<0.05, **P<0.01, ***P<0.001 vs the control group; ##P<0.01 vs the LPS+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Microarray, Expressing, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Transfection

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Silencing of TERF2 partially reverses the ameliorative effect of miR-29b-1-5p inhibition on LPS-induced cardiomyocyte apoptosis and inflammation (A) HL-1 cells were transfected with sh-TERF2 or sh-NC vector. Forty-eight hours later, HL-1 cells were collected to detect the protein expression of TERF2. (B‒I) HL-1 cells were transfected with sh-TERF2 or the miR-29b-1-5p antagomir for 24 h, followed by stimulation with LPS. MTT assay was performed to examine HL-1 cell viability (B). Flow cytometry was performed to evaluate HL-1 cell apoptosis (C). ELISA was also conducted to evaluate TNF-α, IL-1β, and IL-6 levels in HL-1 cells (D‒F). Western blot analysis was conducted to determine the protein levels of TERF2, cleaved PARP, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II in HL-1 cells (G–I). **P<0.01 vs LPS+NC antagomir+sh-NC; #P<0.05, ##P<0.01 vs LPS+miR-29b-1-5p antagomir+sh-TERF2.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Quality Assurance of RNA Expression Profiling in Clinical Laboratories

doi: 10.1016/j.jmoldx.2011.09.003

Figure Lengend Snippet: Raw data are interpreted after evaluation of virtually all approximately 22,000 human protein-coding genes using Agilent microarrays. A: A heat map shows gene expression profiles of 96 specimens tested for 50 listed genes, and unsupervised hierarchical clustering reveals distinct patterns of expression. B: To classify intrinsic subtypes of breast cancer, a single-sample predictor algorithm compares each patient's expression pattern with the consensus pattern for each of the five intrinsic subtypes, and Spearman's correlation coefficients help estimate the certainty of the classification. In the example shown, the patient's profile matches most closely with luminal (Lum) A, although Lum B subtype cannot be excluded, whereas three other subtypes (normal, basal, and HER2) are excluded based on low correlation coefficients.

Article Snippet: The Clinical and Laboratory Standards Institute produces documents thoroughly describing principles and best practices, such as Diagnostic Nucleic Acid Microarrays, 21 Verification and Validation of Multiplex Nucleic Acid Assays, 22 and Use of External RNA Controls in Gene Expression Assays.

Techniques: Gene Expression, Expressing

Journal: The Journal of Clinical Investigation

Article Title: The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

doi: 10.1172/JCI65703

Figure Lengend Snippet: (A) Morphological changes that take place as neck cells secreting mucus (small white granules) differentiate into ZCs secreting zymogen (large black granules). The cells are oriented with the gastric lumen at the top and the basement membrane at the bottom. Arrows indicate vectors of expansion or contraction during the maturation process. (B) Representative section of the base zone of the gastric unit (thick dashed outline) stained for anti–α-tubulin (green) antibodies and Hoechst (blue, nuclei; thin dashed or solid outlines). A single ZC is highlighted (solid outline). (C) Fluorescent microscopy of the neck, transition (TZ), and base zones of the gastric unit from wild-type mice stained with antibodies against GSII (red, neck cells) and CI-M6PR (green; left) and cathepsin L (green; right). Neck cells and ZCs (solid outline) are shown enlarged in the insets. Expression of CI-M6PR and cathepsin L in transitional cells and parietal cells is indicated by arrowheads and arrows, respectively. (D) Fluorescent microscopy of gastric unit from wild-type mice stained with GSII (magenta, neck cells), anti–α-tubulin (green), and anti-MAP1B (red). Enlarged views of individual neck cell, parietal cell, and ZC stained with α-tubulin and MAP1B, as well as merged images, are shown in the insets. Scale bars: 5 μm (B; C and D, insets), 10 μm (C and D).

Article Snippet: The following primary antibodies were used for immunostaining: goat anti-human GIF (1:2,000; gift of D. Alpers, Washington University School of Medicine); sheep anti-PGC (1:10,000; Abcam); mouse anti–α-tubulin (1:500; Sigma-Aldrich); phalloidin; myosin; integrin; mouse anti–cathepsin L (1:100; Novus Biologicals); rabbit anti–CI-M6PR (1:500; gift of S. Kornfeld, Washington University School of Medicine); mouse anti-MAP1B (1:100; Sigma-Aldrich); rabbit anti–phosphorylated MAP1B (1:200; Novus Biologicals); rabbit anti-DAPK1 (1:200; Sigma-Aldrich); rabbit anti-MIB1 (1:500; Sigma-Aldrich); mouse anti–galectin 8 (1:100; Sigma-Aldrich); rabbit anti-MIST1 (1:200; ref. 15 ).

Techniques: Membrane, Staining, Microscopy, Expressing

Journal: The Journal of Clinical Investigation

Article Title: The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

doi: 10.1172/JCI65703

Figure Lengend Snippet: (A) Fluorescent microscopy of an individual gastric unit (thick dashed outline) stained with GSII (red) and an antibody that specifically recognizes MAP1B phosphorylated at Thr1265 (green). Insets show pan-MAP1B expression (red) and the merged image of the base zone of the gastric unit (far right). (B and C) Gastric units were stained with GSII (red), DAPK1 (B; green) or MIB1 (C; green), and Hoechst (blue, nuclei; thin dashed outline). Enlarged views (×1.5-fold) of representative neck cell and ZC (solid outline) are shown in insets. DAPK1 (B) or MIB1 (C) immunohistochemical staining (brown) is shown below. DAPK1 and MIB1 expression in the transition zone is indicated by arrowheads; lack of DAPK1 or MIB1 expression in parietal cells is indicated by arrows. (D) Western blot of ubiquitin immunoprecipitates for DAPK1. (E) Heatmap representing expression values of DAPK1 and MIB1 probesets in neck cells and ZCs. Scale bars: 10 μm.

Article Snippet: The following primary antibodies were used for immunostaining: goat anti-human GIF (1:2,000; gift of D. Alpers, Washington University School of Medicine); sheep anti-PGC (1:10,000; Abcam); mouse anti–α-tubulin (1:500; Sigma-Aldrich); phalloidin; myosin; integrin; mouse anti–cathepsin L (1:100; Novus Biologicals); rabbit anti–CI-M6PR (1:500; gift of S. Kornfeld, Washington University School of Medicine); mouse anti-MAP1B (1:100; Sigma-Aldrich); rabbit anti–phosphorylated MAP1B (1:200; Novus Biologicals); rabbit anti-DAPK1 (1:200; Sigma-Aldrich); rabbit anti-MIB1 (1:500; Sigma-Aldrich); mouse anti–galectin 8 (1:100; Sigma-Aldrich); rabbit anti-MIST1 (1:200; ref. 15 ).

Techniques: Microscopy, Staining, Expressing, Immunohistochemical staining, Western Blot, Ubiquitin Proteomics

Journal: The Journal of Clinical Investigation

Article Title: The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

doi: 10.1172/JCI65703

Figure Lengend Snippet: (A) Immunohistochemistry using anti-MIB1 antibodies was performed on stomach sections from Mib1Δ/Δ and control (Ctrl) mice 2 weeks after initiation of tamoxifen treatment. Neck, transition, and base zones are indicated. PCR analysis of MIB1 transcript levels and Western blot analysis of MIB1 protein levels in Mib1Δ/Δ and control ZCs are also shown. (B) Fluorescent microscopy of individual gastric units (thick dashed outline) from Mib1Δ/Δ and control mice, 2 weeks after tamoxifen treatment, stained for MIB1 (green; top) and with GSII and DAPK1 (red and green, respectively; bottom). Enlarged views (×1.5-fold) of representative ZCs (solid outline) are shown in the insets. Thin dashed outlines denote nuclei (Hoechst, blue). Note the basal subnuclear distribution of DAPK1 in Mib1Δ/Δ ZCs compared with the apical localization in control mice. (C) Gastric units stained for GSII (red) and phosphorylated MAP1B (green). (D and E) Gastric units stained for CI-M6PR (D; green) or for cathepsin L (E; green). Enlarged views (×1.5-fold) of representative ZCs are shown in the insets. Western blot for pro–cathepsin L and activated cathepsin L is also shown in E. (F) Gastric units stained for galectin 8 (green; top) and with galectin 8 and GIF (green and red, respectively; bottom). Enlarged views (×1.5-fold) of representative ZCs are shown in the insets. Note the accumulation of galectin 8 in the apical compartment of Mib1Δ/Δ ZCs. Scale bars: 10 μm (C–F); 20 μm (B); 40 μm (A).

Article Snippet: The following primary antibodies were used for immunostaining: goat anti-human GIF (1:2,000; gift of D. Alpers, Washington University School of Medicine); sheep anti-PGC (1:10,000; Abcam); mouse anti–α-tubulin (1:500; Sigma-Aldrich); phalloidin; myosin; integrin; mouse anti–cathepsin L (1:100; Novus Biologicals); rabbit anti–CI-M6PR (1:500; gift of S. Kornfeld, Washington University School of Medicine); mouse anti-MAP1B (1:100; Sigma-Aldrich); rabbit anti–phosphorylated MAP1B (1:200; Novus Biologicals); rabbit anti-DAPK1 (1:200; Sigma-Aldrich); rabbit anti-MIB1 (1:500; Sigma-Aldrich); mouse anti–galectin 8 (1:100; Sigma-Aldrich); rabbit anti-MIST1 (1:200; ref. 15 ).

Techniques: Immunohistochemistry, Control, Western Blot, Microscopy, Staining

Journal: The Journal of Clinical Investigation

Article Title: The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

doi: 10.1172/JCI65703

Figure Lengend Snippet: (A) Microarray analysis of Mist1 and Mib1 gene expression from isolated populations of neck cells and ZCs from wild-type and Mist1–/– mice. (B) Mist1–/– gastric unit (thick dashed outline) stained for GSII (red) and MIB1 (green). MIB1 was expressed in the transition zone (white arrow). Western blot of MIB1 protein levels in wild-type and Mist1–/– whole stomach corpus is also shown. (C) The human MIB1 gene, represented to scale, with numbered exons as vertical lines. Note the dramatic conservation of the CATATG sequence, located within the first 1 kb of the end of the first exon, across multiple mammalian species. Also shown are gel electrophoresis images of PCR amplicons from ChIP experiments using anti-MIST1 antibody in HGC-27 cells followed by primers as indicated (see Methods). (D) Mist1–/– gastric unit stained for GSII (red) and DAPK1 (green). Thin dashed outlines denote nuclei (Hoechst, blue). Enlarged view (×1.5-fold) of a representative ZC (solid outline) is shown in the inset. Western blotting of MIB1 immunoprecipitates from wild-type and Mist1–/– mice for DAPK1 is also shown. (E) Mist1–/– gastric unit stained for GSII (red) and phosphorylated MAP1B (green). (F) Mist1–/– gastric unit stained for GSII (red) and for CI-M6PR (green; top) or cathepsin L (green; bottom). Enlarged views (×1.5-fold) of representative ZCs are shown in the insets. Scale bars: 10 μm (E and F); 20 μm (B and D).

Article Snippet: The following primary antibodies were used for immunostaining: goat anti-human GIF (1:2,000; gift of D. Alpers, Washington University School of Medicine); sheep anti-PGC (1:10,000; Abcam); mouse anti–α-tubulin (1:500; Sigma-Aldrich); phalloidin; myosin; integrin; mouse anti–cathepsin L (1:100; Novus Biologicals); rabbit anti–CI-M6PR (1:500; gift of S. Kornfeld, Washington University School of Medicine); mouse anti-MAP1B (1:100; Sigma-Aldrich); rabbit anti–phosphorylated MAP1B (1:200; Novus Biologicals); rabbit anti-DAPK1 (1:200; Sigma-Aldrich); rabbit anti-MIB1 (1:500; Sigma-Aldrich); mouse anti–galectin 8 (1:100; Sigma-Aldrich); rabbit anti-MIST1 (1:200; ref. 15 ).

Techniques: Microarray, Gene Expression, Isolation, Staining, Western Blot, Sequencing, Nucleic Acid Electrophoresis

Journal: Cancers

Article Title: Enhanced Cytotoxicity on Cancer Cells by Combinational Treatment of PARP Inhibitor and 5-Azadeoxycytidine Accompanying Distinct Transcriptional Profiles.

doi: 10.3390/cancers14174171

Figure Lengend Snippet: Figure 2. Effects of PJ-34, 5-aza-dC, and TSA in colon cancer HCT116 and RKO cells. (A,B) Effects of 3 µM PJ-34, 1 µM 5-aza-dC, and 300 nM TSA on the cell growth in colon cancer HCT116 (A) and RKO (B) for 3 days. n = 3. Statistical differences are shown in Table S1. (C) HCT116 cells were treated with 5-aza-dC, PARP inhibitors PJ-34 for 2 days and analyzed with flow cytometry analysis. The cells were stained with PI. The left panel shows the representative cell cycle distribution. The right panel shows the cell population % of subG1, G1, S, G2, and superG2 fractions. N = 3. Mean ± S.E. Statis-tical differences in SubG1 % is indicated. * p < 0.05, ** p < 0.01.

Article Snippet: The human colon cancer cell lines RKO (American Type Culture Collection) and HCT116 (kindly provided by Dr. Bert Vogelstein (The John Hopkins University) were used.

Techniques: Cytometry, Staining

Journal: Cancers

Article Title: Enhanced Cytotoxicity on Cancer Cells by Combinational Treatment of PARP Inhibitor and 5-Azadeoxycytidine Accompanying Distinct Transcriptional Profiles.

doi: 10.3390/cancers14174171

Figure Lengend Snippet: Figure 3. Effect of PJ-34 and 5-aza-dC on SSBs and alkali-labile DNA damage and apoptotic DNA frag-mentation and DNA methylation in colon cancer HCT116 cells. (A) The cells were treated with 5-aza-dC, PARP inhibitors PJ-34 for 3 days and genome DNA was analyzed by pulse-field gel electrophoresis in alkaline treatment to observe SSBs and alkali-labile DNA damage and detected with total genome probe by Southern blot analysis. The band intensity was quantified and plotted in the graph shown below. (B) Induction of apoptotic DNA ladder formation on day 6 with 5-aza-dC treatment but not with PARP inhibitor. No enhancement of DNA ladder formation was observed with

Article Snippet: The human colon cancer cell lines RKO (American Type Culture Collection) and HCT116 (kindly provided by Dr. Bert Vogelstein (The John Hopkins University) were used.

Techniques: DNA Methylation Assay, Nucleic Acid Electrophoresis, Southern Blot

Journal: Cancers

Article Title: Enhanced Cytotoxicity on Cancer Cells by Combinational Treatment of PARP Inhibitor and 5-Azadeoxycytidine Accompanying Distinct Transcriptional Profiles.

doi: 10.3390/cancers14174171

Figure Lengend Snippet: Figure 4. Effect of PJ-34 and 5-aza-dC on the gene expression profiles in HCT116 and RKO cells. HCT116 and RKO cells were treated with 5-aza-dC (5-aza) at 0.5 µM, PJ-34 (PJ) at 3 µM, TSA at 150 nM as a single treatment or in combinations for 3 days and the gene expression profiles were analyzed by microarray analysis. (A) Cluster analysis of gene expression profiles in HCT116 and RKO cells. (B) Cluster analysis of the gene expression profiles in HCT116 using 12,765 genes chosen. (C) Venn diagrams for genes showing 5-fold changes or more compared with the untreated condition in HCT116 and RKO cells. (D) Number of genes showing up-regulation by the single treatments with PJ-34, 5-aza-dC, and TSA in HCT116 and RKO cells (A) 16-fold or more; (B) 32-fold or more). (E) Left panel: Cluster analysis of reactivated genes that were induced by PJ3-4, 5-aza-dC, TSA, and their combinations in HCT116 cells (7742 genes chosen). Right panel: Cluster analysis of reactivated genes by PJ-34, 5-aza-dC, TSA, and their combinations in RKO cells (7720 genes chosen). Genes which showed a control level of raw data lower than 50 and showing reactivation that was 5-fold or more to reach at least to raw data of 50 in at least one single treatment condition were used. (F) Venn

Article Snippet: The human colon cancer cell lines RKO (American Type Culture Collection) and HCT116 (kindly provided by Dr. Bert Vogelstein (The John Hopkins University) were used.

Techniques: Gene Expression, Microarray, Control