Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 1. Identification of linc-AAM in Activated Macrophages (A) Quantitative real-time PCR verification of top six upregulated lncRNAs in RAW264.7 cells treated with medium (control [Ctrl]) or AEPS (50 mg/mL) for 1 h (n = 3). (B) Quantitative real-time PCR analysis of linc-AAM, COX-2, IL-1b, IL-6, IL-10, TNF-a, CCL2, CCL5, and CCL22 in RAW264.7 cells treated with AEPS (50 mg/mL) for different times (n = 3). (C) The RACE analysis of linc-AAM using total RNA extracted from RAW264.7 cells treated by AEPS (50 mg/mL) for 1 h. (D) Northern blotting of linc-AAM in RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA served as a loading control. The images shown are representative of three independent experiments. (E and F) Electrophoretogram (E) and quantitative real-time PCR analysis (F) of linc-AAM in cytoplasm and nuclei of RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h (n = 3). (G) linc-AAM (red) was detected in RAW264.7 cells by RNA fluorescence in situ hybridization (FISH). Nuclei (blue) were counterstained with DAPI. Scale bars, 10 mm. (H) Identification of coding capability of linc-AAM using in vitro transcription and translation system. Full-length linc-AAM was cloned into the eukaryotic expression vector pcDNA3.1() with N-terminal start codon ATG and C-terminal FLAG tag in all three coding patterns, and these plasmids were subsequently transfected into HEK293T cells, respectively. Immunoblotting was used to detect the FLAG-tagged protein. STAT3 with FLAG tag severs as a positive control. The images shown were representative of three independent experiments. (I) Quantitative real-time PCR analysis of linc-AAM in different tissues from six C57BL/6 mice per group, male and female in half. (J) Quantitative real-time PCR analysis of linc-AAM in RAW264.7 cells, BMDMs, PMs, and BMDCs treated with medium (Ctrl) or AEPS (50 mg/mL) for 1 h (n = 3). (K) Quantitative real-time PCR analysis of linc-AAM and IL-1b in BMDMs treated with AEPS (50 mg/mL) for different times (n = 3). (L) Quantitative real-time PCR analysis of linc-AAM, IL-1b, and COX-2 in RAW264.7 cells stimulated with AEPS (50 mg/mL), LPS (100 ng/mL), poly(I:C) (50 mg/mL), Alum (200 mg/mL), or Quil A (20 mg/mL) for 1 or 4 h, respectively (n = 3). Data are presented as mean ± SEM. ***p < 0.001. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Real-time Polymerase Chain Reaction, Control, Northern Blot, In Situ Hybridization, In Vitro, Clone Assay, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Western Blot, Positive Control

Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 2. linc-AAM Silencing or Knockout (KO) Inhibits Macrophage Activation and the Expression of IRGs in Vitro and ex Vivo (A) Heatmap of differentially expressed genes in linc-AAM-RNAi RAW264.7 cells (linc-AAM KD) relative to Ctrl-RNAi cells (Scramble) treated with AEPS for 4 h. The most downregulated genes in linc-AAM KD cells compared with Scramble cells are magnified into view. (B) Quantitative real-time PCR analysis of linc-AAM, COX-2, IL-1b, IL-10, TNF-a, CCL2, CCL3, CCL4, CCL5, and CCL22 in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 4 h (n = 3). (C) The production of IL-1b and IL-6 from linc-AAM KD and Scramble RAW264.7 cells treated with medium (Ctrl) or AEPS (50 mg/mL) for 24 h using ELISA (n = 3). nd, not detectable. (D) Western bolt analysis of COX-2 in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 12 and 24 h. (E) Fluorescence-activated cell sorting (FACS) analysis of surface molecules (Ly6G, CD40, CD80, CD86, MHC I, and MHC II) in linc-AAM KD and Scramble RAW264.7 cells treated with AEPS (50 mg/mL) for 24 h (n = 3). (F) Quantitative real-time PCR analysis of linc-AAM potential target genes IL-1b, IL-6, COX-2, TNF-a, CCL2, and CXCL10 in BMDMs from WT and linc-AAM KO mice treated with medium (untreatment) or AEPS (25 mg/mL) for 4 or 8 h (n = 3). (G) The production of IL-6 and TNF-a in BMDMs from WT and linc-AAM KO mice treated with medium (Ctrl) or AEPS (25 mg/mL) for 24 h using ELISA (n = 3). (H) FACS analysis of surface molecules (CD40, CD80, CD86, MHC I, and MHC II) of BMDMs from WT or linc-AAM KO mice treated with medium (Ctrl) or AEPS (25 mg/mL) for 24 h (n = 3). (I) Quantitative real-time PCR analysis of miR155hg in linc-AAM KD and Scramble RAW264.7 cells treated by AEPS (50 mg/mL) for 4 h. (J) Quantitative real-time PCR analysis of miR155hg in BMDMs from WT and linc-AAM KO mice after treated by AEPS (25 mg/mL) for 4 h. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figures S2 and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Knock-Out, Activation Assay, Expressing, In Vitro, Ex Vivo, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, FACS

Journal: Cell reports

Article Title: linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

doi: 10.1016/j.celrep.2020.108584

Figure Lengend Snippet: Figure 6. linc-AAM Facilitates Chromatin Activation to Promote the Transcription of IRGs (A) Cross-linked RIP to detect linc-AAM association with H3 and H3K4me3. The nuclear lysates of RAW264.7 cells were IPed with control IgG, anti-histone H3, or anti-histone H3K4me3 antibody, and then the complexes were analyzed for the presence of linc-AAM by Quantitative real-time PCR (n = 3). Signals were normalized to 10% input samples. (B) Western blot analysis of histone H3 in RNA pull-down assay samples of linc-AAM and its different mutants as in Figure 5G. (C and D) coIP analysis of the interaction between hnRNPL and histone H3 or H3K4me3 in RAW264.7 cells treated by medium or AEPS for 1 h (n = 3). (E) coIP analysis of the interaction between hnRNPL and histone H3 in BMDMs from WT or linc-AAM KO mice treated by medium or AEPS for 2 h (n = 3). (F) Mouse IL-1b promoter-driven Luc activities in HEK293T cells transfected with linc-AAM overexpression plasmids or empty plasmids (Ctrl). (G) ChIRP enrichment analysis for linc-AAM and control IL-1b. LacZ antisense DNA probes are used as negative controls. (H) linc-AAM ChIRP-qPCR in AEPS-treated RAW264.7 cells. The sequences of primers used in qPCR for CCL2, IL-1b, COX-2, CCL5, TNF-a, CXCL10, IL-6, and GAPDH are within their promoter regions. GAPDH served as a negative control. (I) Integrated model depicting linc-AAM functioning to facilitate inducible expression of IRGs in macrophages. TF, transcription factor. Data are presented as mean ± SEM. **p < 0.01 and ***p < 0.001. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pyrrolidinedithiocarbamate ammonium (PDTC) Beyotime Cat# S1808 Red blood cell lysis buffer Beyotime C3702 Enhanced chemiluminescence (ECL) kit Beyotime P0018AS Proteinase K Beyotime Cat# ST533 Radioimmunoprecipitation (RIPA) buffer Beyotime Cat# P0013B TRIzol reagent Ambion Cat# 15596026 Lipofectamine 3000 Invitrogen Cat# L3000015 Lipofectamine LTX reagent with PLUS reagent Invitrogen Cat# 15338100 Rela (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227671 Stat3 (Myc-DDK-tagged)-pCMV6 OriGene Cat# MR227265 pCMV6-Entry OriGene Cat# PS100001 PrimeSTAR max DNA polymerase Takara Cat# R045A pLXSN retroviral Vector Clontech Cat# 631509 pcDNA3.1(-) eukaryotic expression vector Invitrogen Cat# V79520 pGL3-Basic Vector Promega Cat# E1751 Stat3 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29494-V NF-kB p65 shRNA(m) lentiviral particles Santa Cruz Cat# sc-29411-V Control shRNA(m) lentiviral particles Santa Cruz Cat# sc-108080 G418 sulfate BBI Cat# A600958-0001 Puromycin InvivoGen Cat# ANT-PR Blasticidin InvivoGen Cat# ant-bl-05 FastStart Universal SYBR Green Master (Rox) Roche Cat# 4913850001 CSPD Roche Cat#11655884001 Anti-digoxigenin AP-conjungate Roche Cat#11093274910 Ribonucleoside vanadyl complex New England BioLabs Cat# S1402S MyOne T1 streptavidin beads Invitrogen Cat# 65604D Albumin from chicken egg white Sigma-Aldrich Cat# A5503 Concanavalin A Sigma-Aldrich Cat# L6397 Critical Commercial Assays Mouse IL-10 ELISA kit Boster Cat# EK0417 Mouse TNF-a ELISA kit Boster Cat# EK0527 Mouse IL-1b ELISA kit Boster Cat# EK0394 Mouse CCL2 ELISA kit Boster Cat# EK0568 Mouse IL-6 ELISA kit Boster Cat# EK0411 Mouse tail direct PCR Kit Bimake Cat# B40013 BLOCK-iT Pol II miR RNAi Expression Vector Kits with EmGFP Invitrogen Cat# K4936-00 Maxima First Strand cDNA Synthesis Kit Thermo Fisher Cat# K1671 NorthernMax Gly Kit Ambion Cat# AM1946 PrimeScript RT reagent Kit with gDNA Eraser Takara Cat# RR047A SMARTer RACE kit Clontech Cat# 634858 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript T7 Transcription Kit Ambion Cat# AM1333 Pierce RNA 30 End Desthiobiotinylation Kit Thermo Scientific Cat# 20163 PierceMagnetic RNA-Protein Pull-Down Kit Thermo Scientific Cat# 20164 (Continued on next page) Cell Reports 34, 108584, January 5, 2021 e2

Techniques: Activation Assay, Control, Real-time Polymerase Chain Reaction, Western Blot, Pull Down Assay, Transfection, Over Expression, Negative Control, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: ANKRD22 deletion reduces the inflammatory response by inhibiting activation of the macrophages after gastric mucosal injury. ( A and B ) Ankrd22 knockout decreased the number of CD45 + cells in damaged mouse gastric mucosa detected by FCM. Rat IgG2b was used as an isotype control (n = 4). ( C and D ) Effect of Ankrd22 knockout on the expression profiles of inflammatory factors in damaged mouse gastric mucosa detected by Luminex assay (n = 4). ( E ) Ankrd22 knockout reduced IL1α and TNF-α in mouse damaged gastric mucosa detected by ELISA (n = 3). ( A – E ) Ankrd22 +/+ and Ankrd22 -/- mice were examined 24 hours after intragastric administration of EtOH/HCl or saline solution (Ctrl). ( F ) Expression levels of ANKRD22 in the macrophages activated by different concentrations of IFN-γ. THP-1 and RAW264.7 macrophages were stimulated with 0, 50, 80, or 100 ng/mL IFN-γ for 24 hours. Subsequently, the expression of ANKRD22 was determined by qRT-PCR. The data from the 0 ng/mL group were normalized to 1.0. TUBB and Tubb were used as internal references. ( G ) Expression levels of Ankrd22 in the activated mouse macrophages detected by qRT-PCR. Data of the PBS-treated group (Ctrl) were normalized to 1.0. Tubb was used as an internal reference. ( H and I ) Determination of IL1α and TNF-α in supernatant of activated macrophages from Ankrd22 +/+ or Ankrd22 -/- mice by ELISA. ( J and K ) Effects of Ankrd22 knockout on the percentages of CD86 + and CD206 + cells in activated Ankrd22 +/+ and Ankrd22 -/- mouse macrophages detected by FCM. Rat IgG2b was used as an isotype control (n = 3). ( L and M ) Determination of IL12 (p70) and IL10 in the supernatant of activated macrophages from Ankrd22 +/+ or Ankrd22 -/- mice by ELISA. ( E – M ) Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS or PBS (Ctrl) for 24 hours. The data were analyzed by the Student t test and are presented as means ± SD. ∗ P < .05, ∗ P < .01, and ∗∗∗ P < .001. APC-A, Allophycocyanin Area; FITC-A, Fluorescein Isothiocyanate Area; FSC-A, Forward Scatter Area.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Activation Assay, Knock-Out, Control, Expressing, Luminex, Enzyme-linked Immunosorbent Assay, Saline, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: ANKRD22 deletion reduces the expression levels of mitochondrial Ca 2+ and cytoplasmic NFAT in macrophages. ( A ) Mitochondrial colocalization of exogenous-expressing ANKRD22 in THP-1 macrophages detected by confocal microscopy. ( B – E ) Ankrd22 knockout reduced the mitochondrial Ca 2+ level in activated mouse macrophages. Confocal microscopy was used to compare the fluorescence intensity and colocalization with mitochondria. Macrophages treated with 50 μmol/L 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for 24 hours was used as a negative control. The fluorescence intensity was detected by Rhod-2–based FCM. ( F ) Determination of IL1α and TNF-α in supernatant of activated mouse macrophages by ELISA after NFAT inhibition. The activated macrophages were treated with 100 ng/mL NFAT inhibitor for 1 hour. ( B – F ) Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS or PBS (Ctrl) for 24 hours. ( G ) Determination of TNF-α in the supernatant of activated macrophages by ELISA after NFAT inhibition. The LPS-activated macrophages were treated with 100 ng/mL NFAT inhibitor for 1 hour. ( H ) Detection of NFAT in the activated mouse macrophages by Western blot. ( I ) Detection of NFAT in the activated macrophages by Western blot. ( G – I ) THP-1 macrophages were treated with 100 ng/mL phorbol 12-myristate 13-acetate for 24 hours in advance. ( J ) Effects of Ankrd22 knockout on the expression levels of NFAT in activated mouse macrophages detected by Western blot. Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS for 24 hours. Macrophages were stimulated with 0, 50, or 100 ng/mL IFN-γ or 0, 0.1, or 1.0 μg/mL LPS for 24 hours. Data are presented as means ± SD and analyzed using the Student t test. ∗ P < .05 and ∗∗ P < .01.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Expressing, Confocal Microscopy, Knock-Out, Fluorescence, Negative Control, Enzyme-linked Immunosorbent Assay, Inhibition, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: Identification of a lead compound that inhibits the activity of ANKRD22. ( A ) Small molecules interacting with the L122/D132 sites of ANKRD22 predicted by protein ligand interface fingerprint software. ( B ) Expression levels of ANKRD22 in the WT, empty vector–transfected, or ANKRD22/pcDNA3.1(-)–transfected SGC7901 cells detected by Western blot. ( C ) Effect of the E115A/D123A mutant on intracellular Ca 2+ levels in ANKRD22 + gastric cancer SGC7901 cells. The fluorescence intensity was detected using Fluo-4–based FCM. ( D ) Effect of the L122A/D132A mutant on the intracellular Ca 2+ levels in ANKRD22 + gastric cancer SGC7901 cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( E ) Schematic diagram of the chemical structure and acting sites of the ANKRD22 inhibitory lead compound-AV023. ( F ) Effects of AV023 on LGR5 in ANKRD22 + SGC7901 cells detected by qRT-PCR. SGC7901 cells without ANKRD22 overexpression were used as the control groups and the cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. TUBB was used as an internal reference. ( G ) AV023 increased the clone number of primary mouse gastric epithelial cells in organoid culture. Ankrd22 +/+ mouse gastric EPCs were treated with 0 (Ctrl) or 1.0 μmol/L AV023 for 24 hours. ( H ) AV023 reduced the intracellular Ca 2+ levels in Ankrd22 +/+ mouse gastric epithelial cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( I ) AV023 reduced the intracellular Ca 2+ levels in ANKRD22 + SGC7901 cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( J ) AV023 increased the Wnt pathway activity in Ankrd22 +/+ mouse gastric organoids detected by Western blot. ( K ) AV023 increased the Wnt pathway activity in ANKRD22 + SGC7901 cells detected by Western blot. ( L ) AV023 increased the Wnt transcriptional activity of ANKRD22 + SGC7901 cells detected by TOPflash luciferase reporter assay. ( H – L ) Ankrd22 +/+ and Ankrd22 -/- mouse gastric cells were treated with 0, 0.5, and 1.0 μmol/L AV023 for 24 hours. SGC7901 cells without ANKRD22 overexpression were used as the control groups and the cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. ( M and N ) Effect of AV023 on the release of IL1α and TNF-α in the activated mouse macrophages detected by ELISA. Mouse macrophages were stimulated with 100 ng/mL LPS for 24 hours, and the control group was not treated with LPS. Cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. The data were analyzed by the Student t test and are presented as means ± SD. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Activity Assay, Software, Expressing, Plasmid Preparation, Transfection, Western Blot, Mutagenesis, Fluorescence, Quantitative RT-PCR, Over Expression, Control, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay