Journal: The Journal of Biological Chemistry

Article Title: O -Linked N -Acetylglucosamine ( O -GlcNAc) Expression Levels Epigenetically Regulate Colon Cancer Tumorigenesis by Affecting the Cancer Stem Cell Compartment via Modulating Expression of Transcriptional Factor MYBL1 *

doi: 10.1074/jbc.M116.763201

Figure Lengend Snippet: MYBL1 was epigenetically regulated by O-GlcNAc. A, presence of CpG islands was analyzed around TSS site (−1500 to +350) of MYBL1 using the CpG Island Searcher. B, after the genomic DNA was isolated and bisulfate-treated, methylation-specific PCR for CDH1 (top) and MYBL1 (bottom) genes was performed in different colon tumor cells. The presence of a PCR product band in lanes M or U indicates methylated or unmethylated genes, respectively. M-positive, methylated human DNA standard used as a methylation-positive control. Data are representative of two independent experiments. C, tumor cells were treated with the demethylating reagent 5-aza-2′-deoxycytidine (5-Aza) at two different concentrations for 3 days, and total RNA was isolated and used for detection of transcript levels of MYBL1. For each transcript, the values were normalized to control (GAPDH) and expressed as mean ± S.D. from three independent experiments. D, after treatment with OGA inhibitor (TMG, 10 μm) for 24 h, tumor cells were collected for detection of transcripts of MYBL1 by qRT-PCR. E, after genomic DNA was isolated from tumor cells treated with OGA inhibitor (TMG, 10 μm) for 24 h and bisulfate-treated, methylation-specific PCR for the MYBL1 gene was performed. F, genomic DNA was isolated from both scrambled and OGT-shRNA transfected HT-29 cells, and methylation-specific PCR for the MYBL1 gene was performed. Images were representative from two independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: Methylation-specific PCR Purification and bisulfite treatment of genomic DNA samples were performed using the DNeasy tissue kit and EZ DNA Methylation-Gold TM kit (Zymo Research), respectively, according to the manufacturer's instructions.

Techniques: Isolation, Methylation, Positive Control, Quantitative RT-PCR, shRNA, Transfection

Journal: Cell Death & Disease

Article Title: CDC42 governs normal oviduct multiciliogenesis through activating AKT to ensure timely embryo transport

doi: 10.1038/s41419-022-05184-y

Figure Lengend Snippet: a RT-qPCR analysis of Dll1 , Dll4 , Jagged1 , and Jagged2 mRNA in Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. b In situ hybridization of Jagged1 mRNA in Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. c Western blot analysis of CDC42, NICD1, and NICD2 protein in Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. d Immunostaining of NICD1 in the ampulla and isthmus of Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. e RNA-seq analysis of Hes1, Hey1, and Hey2 transcripts (RPKM) in Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. f Immunostaining of HES1 in the ampulla and isthmus of Cdc42 f/f and Pgr cre Cdc42 f/f mouse oviducts on day 2. g Cdc42 f/f organoids were infected with Ad-GFP and Ad-Cre, then subjected to western blot assays. h Immunostaining of HES1 in the organoids before and after E2 and DBZ induced differentiation. Scale bars, 100 μm. Mean ± SD. * P < 0.05, Student’s t test or ANOVA with Tukey’s multiple comparisons test.

Article Snippet: A total of 10 cDNA libraries from organoids were generated using V8 RNA-seq Library Prep Kit (Vazyme, NR605) according to the manufacturer’s instructions, then sequenced using Illumina Nova seq platform (PE150) by Novogene Bioinformatics Technology Co., Ltd (Beijing, China) to generate raw reads.

Techniques: Quantitative RT-PCR, In Situ Hybridization, Western Blot, Immunostaining, RNA Sequencing Assay, Infection

Journal: Chinese medicine

Article Title: Lycium barbarum polysaccharide inhibits ischemia-induced autophagy by promoting the biogenesis of neural stem cells-derived extracellular vesicles to enhance the delivery of miR-133a-3p.

doi: 10.1186/s13020-023-00831-8

Figure Lengend Snippet: Fig. 4 LBP pretreated NSC-EVs suppressed inflammatory response and reduced neuronal apoptosis after stroke. OGD-exposed neurons were incubated with PBS, NSC-EV, L-NSC-EV, and H-NSC-EV. Cells incubated under standard cell culture conditions were used as negative control. A– C The mRNA expression of TNFα, IL-6, and IL-1β in the sham and ipsilateral side of the infarcted brain was measured by RT-qPCR at post stroke day 3 D Representative images and E quantification of TUNEL+ cells in the infarct border. Data are shown as mean ± SD. Data are statistically different from each other with *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: The cell apoptosis of brain tissue was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions.

Techniques: Incubation, Cell Culture, Negative Control, Expressing, Quantitative RT-PCR, TUNEL Assay

Journal: Virus research

Article Title: The neuropathological mechanism of EV-A71 infection attributes to inflammatory pryoptosis and viral replication via activating the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis.

doi: 10.1016/j.virusres.2023.199195

Figure Lengend Snippet: Fig. 1. EV-A71 infection triggers cell death of SH-SY5Y cells. (A) The percentage of cells undergoing cell death was calculated through Annexin V-FITC/PI double staining. (B) The cell viability was conducted with Cell Counting Kit-8. (C) Levels of LDH released into the cell culture medium from membrane pores were examined using an LDH Cytotoxicity Assay Kit. (D) Caspase1 activity was assessed with a Caspase1 Activity Assay Kit. .

Article Snippet: For the colorimetric method, caspase1 activity was determined using a Caspase1 Activity Assay Kit (Beyotime, China) following the recommended protocols.

Techniques: Infection, Double Staining, Cell Counting, Cell Culture, Membrane, LDH Cytotoxicity Assay, Activity Assay

Journal: Virus research

Article Title: The neuropathological mechanism of EV-A71 infection attributes to inflammatory pryoptosis and viral replication via activating the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis.

doi: 10.1016/j.virusres.2023.199195

Figure Lengend Snippet: Fig. 2. EV-A71 infection induces NLRP3-dependent pyroptosis in SH-SY5Y cells. (A) NLRP3, ASC, Casepase1, Gasdermin D, IL-1β and IL-18 mRNA levels were analyzed by qRT-PCR. (B) The abundance of NLRP3, ASC, pro-Caspase1, cleaved Caspase 1, Gasdermin D, IL-1β and IL-18 was determined by WB analysis. (C) At 48 and 72 h post infection, infected SH-SY5Y cells were subjected to indirect immunofluorescence assays with anti-VP1 (red) and anti-Caspase-1 (green) antibodies. (D) Secretion of IL-1β and IL-18 upon infection with EV-A71 was measured with a commercial ELISA kit. .

Article Snippet: For the colorimetric method, caspase1 activity was determined using a Caspase1 Activity Assay Kit (Beyotime, China) following the recommended protocols.

Techniques: Infection, Quantitative RT-PCR, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Virus research

Article Title: The neuropathological mechanism of EV-A71 infection attributes to inflammatory pryoptosis and viral replication via activating the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis.

doi: 10.1016/j.virusres.2023.199195

Figure Lengend Snippet: Fig. 8. has_circ_0045431/has-miR-584/NLRP3 regulatory axis promotes EV-A71-induced pyroptosis formation. (A) SH-SY5Y cells with different treatments were subjected to WB analysis for NLRP3, ASC, pro-Caspase1, cleaved Caspase 1, Gasdermin D, IL-1β and IL-18 expression. (B) The cell culture supernatant was subjected to the examination of ELISA for IL-1β and IL-18 concentration.

Article Snippet: For the colorimetric method, caspase1 activity was determined using a Caspase1 Activity Assay Kit (Beyotime, China) following the recommended protocols.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Mitochondria-targeted nanozyme system for psoriasis treatment

doi: 10.1186/s12951-026-04068-z

Figure Lengend Snippet: Effects of CMH@lip@Res-TCeO2 liposomes on the ROS/mTOR/HIF-1α axis in neutrophils. ( A ) Workflow of neutrophil isolation and experimental grouping; ( B ) Western blot analysis of p-mTOR, mTOR, p-S6K1, and S6K1 expression levels in neutrophils; ( C ) RT-qPCR and Western blot analysis of HIF-1α mRNA and protein expression; ( D ) DCFH-DA fluorescence probe for detecting total intracellular ROS in neutrophils (scale bar: 25 μm); ( E ) MitoSOX staining for mitochondrial ROS levels (scale bar: 25 μm); ( F ) Quantification of oxidative stress markers MDA, GSH, SOD, and LDH using respective assay kits; ( G ) Apoptosis analysis using Annexin V/PI double staining and flow cytometry. All cell experiments were conducted in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Following treatment, neutrophils (1 × 106 cells/mL) were washed with PBS and stained with the Annexin V-FITC Apoptosis Detection Kit (C1062, Beyotime, China).

Techniques: Liposomes, Isolation, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, Staining, Double Staining, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Novel Breast-Specific Long Non-coding RNA LINC00993 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer

doi: 10.3389/fonc.2019.01325

Figure Lengend Snippet: LINC00993 suppresses the growth of triple-negative breast cancer (TNBC) cells in vitro . (A) Structure of LINC00993 expression plasmid for adenovirus. (B) LINC00993 expression adenovirus infection efficiency showed by fluorescence microscope in MDA-MB-231 cells. Original magnification, ×400. Scale bars, 50 μm. (C) Expression of LINC00993 detected by qRT-PCR. MDA-MB-231 cells were infected by adenovirus for 24 h, and RNA was extracted. (D) Image of clone formation assay. (E) Number of clones were counted 2 weeks after plantation. (F) MDA-MB-231 cells were planted into 24-well plates. Twenty-four hours later, adenovirus was added to each well. Three wells of cells were digested and counted every 24 h. (G) LINC00993 expression caused apoptosis shown by TUNEL assay. Green points reflected apoptosis, and we used DAPI to stain DNA. Positive control cells were treated with DNase I, negative control cells were collected without adding TUNEL reaction buffer. Original magnification, ×100. Scale bars, 50 μm. (H) Effect of LINC00993 on cell cycle detected by flow cytometry. (I) Flow cytometry cell cycle results shown in a bar plot. (J) Invasive ability tested by Transwell assay. Twenty thousand cells were plated in each well, cells were observed after 24 h of incubation. (K) Bar plot for the number of cells that migrated across the membrane. *** P < 0.001, based on Student's t -test. Data were presented as mean ± SEM.

Article Snippet: To analyze cell apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed with One Step TUNEL Apoptosis Assay Kit (FITC, Beyotime, China) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Plasmid Preparation, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Tube Formation Assay, Clone Assay, TUNEL Assay, Staining, Positive Control, Negative Control, Flow Cytometry, Transwell Assay, Incubation, Membrane

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Isolated HPC (CD34 + Lin − ) cells were cultured in IL-15-supplemented media, and the culture media was replaced every 48 h. The expression of CD56 as an NK cell surface marker was analyzed using FACS. (b) The kinetics of the mRNA expression of Id2, NKp30, NKG2D and Granzyme B were analyzed by real-time qPCR. (c) Stage 2 progenitors (CD34 + CD117 + CD94 − ) and stage 3 progenitors (CD34 − CD117 + CD94 − ) were isolated from UCB by flow sorting. The mRNA expression of IL2Rγ, NKp46 and NKG2D were analyzed by real-time qPCR. (d) A dendrogram of hierarchical clustering revealed genes that were altered more than 2-fold in 7d- and 14-d (mNK) cultured NK cell compared with 1 d-cultured cells. (E-F) The bar graphs represent the top seven functional categories of upregulated (e) or downregulated (f) genes according to the gene ontology analysis (as determined by DAVID, as described in the Methods). The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing, Marker, Functional Assay

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Negatively correlated miRNA-mRNA interactions were visualized as a network using Magia (miRNAs and genes integrated analysis web-based tool). This network provides for the first time a theoretical outline of the concerted action of regulating miRNAs (red triangles) and their potential target mRNAs (green circles). (b) Isolated HPC (CD34 + Lin − ) cells were cultured as described in the Materials and Methods. After being cultured in IL-15-supplemented media, the cells were collected at the indicated time intervals. The expression of miR-143, miR-223, miR-150 and miR-583 was analyzed by real-time qPCR. The data are representative of five independent experiments performed using two different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: Isolated CB HPCs (CD34 + Lin − cells) were cultured as described in the Materials and Methods. After being cultured in IL-15-supplemented media, the cells were collected at 48 h intervals. (a) The expression of the IL2Rγ gene was analyzed by real-time quantitative RT-PCR. (b) The expression of IL2Rγ protein was analyzed by FACS. (c) Expression profiling of IL2Rγ, miR-583 and miR-143 after IL-15 treatment. The data are representative of three independent experiments performed using five different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56 + CD3 − cells) were analyzed by FACS 10 days after transfection. *, p <0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34 + CD117 + CD94 − ). *, p <0.1. (c) NK cell populations (CD56 + CD3 − cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×10 5 ). *, p <0.01 **, p <0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Cell Differentiation

Journal: Molecular Biotechnology

Article Title: miR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway

doi: 10.1007/s12033-024-01078-w

Figure Lengend Snippet: MiR-324-3p suppresses the activation of transforming growth factor (TGF)-β1- induced LX-2 cells in vitro. A Hepatic stellate cells (HSCs) were transfected with miR-324-3p mimic, and expression of the miR-324-3p was determined by real-time-quantitative polymerase chain reaction (RT-qPCR). B RT-qPCR to analyze the miR-324-3p expression in HSCs transfected with the miR-324-3p inhibitor. C Cell counting kit-8 (CCK-8) to analyze the proliferation of transforming growth factor (TGF)-β1-induced LX-2 cells with indicated treatment. D Flow cytometry to detect the cycle and apoptosis of TGF-β1-induced LX-2 cells. E , G Western blot (WB) assay and RT-qPCR to evaluate α-smooth muscle actin (α-SMA) and Vimentin expression in transfected HSC cells. F , H The α-SMA, and Vimentin levels in transfected cells were analyzed by WB assay as well as RT-qPCR. * P < 0.05 and ** P < 0.01 vs . Control group; # P < 0.05 and ## P < 0.01 vs . NC + TGF-β1 group

Article Snippet: Then, the apoptosis was detected using the Annexin V-FITC/PI Apoptosis Analysis Kit (Bestbio, China) and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

Techniques: Activation Assay, In Vitro, Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Counting, CCK-8 Assay, Flow Cytometry, Western Blot, Control