Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPINs suppress the LPS-mediated NF-κB and IFN antiviral pathways. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 20 μg/ml LPS for the indicated period with HOIPINs. The cell lysates were immunoblotted with the indicated antibodies. b LPS-induced gene expression is suppressed by HOIPINs. BMDM cells were pre-treated with the indicated concentrations of HOIPINs for 30 min, and stimulated with 100 ng/ml LPS for 1 h. The mRNA levels were assessed by qPCR. c Suppression of IRF3 targets by HOIPIN-1. BMDM cells were stimulated with 100 ng/ml LPS for 8 h with HOIPIN-1, and interferon β ( n = 13) and Cxcl10 ( n = 10) were quantified by ELISA. d Suppression of antiviral signaling by HOIPIN-8. MEFs were stimulated with 10 μg/ml poly(I:C) for the indicated period with HOIPIN-8, and subjected to immunoblotting analysis. e Suppression of IRF3 targets by HOIPINs. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 10 μg/ml poly(I:C) for 2 h with HOIPINs. The mRNA levels were assessed by qPCR. f HOIPIN-1 inhibits ISRE-luciferase activity. MEF cells, transfected with the ISRE-luciferase reporter, were stimulated with 10 μg/ml poly(I:C) with or without HOIPIN-1 for 6 h, and the luciferase activities were analyzed. g LUBAC activity is indispensable for the IFN pathway. WT- and HOIP −/− -MEFs were treated with 10 μg/ml poly(I:C) and HOIPIN-1 for 2 h. Cell lysates were subjected to immunoblotting analysis. h HOIP is critical for the expression of IRF3-target genes. WT- and HOIP −/− -MEFs were treated as in g , and qPCR analyses were performed. i The Sendai virus (SeV)-induced antiviral response is suppressed by HOIPINs. MEFs were infected with SeV at a multiplicity of infection (MOI) of 10 for 8 h, and treated with the indicated concentrations of HOIPINs for 30 min. qPCR analyses were performed. In b , c , e , f , h , i , data are shown as mean ± SEM, n = 3 (sample numbers in c are indicated in the legend). NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Transfection, Expressing, Virus, Infection

Journal: Communications Biology

Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

doi: 10.1038/s42003-020-0882-8

Figure Lengend Snippet: a HOIPIN-1 shows potent toxicity to ABC-DLBCL cell lines. GCB-DLBCL cells (BJAB, SU-DHL-4, and HT) and ABC-DLBCL cells (TK, DLBCL2, and OYB) were cultured in the presence of DMSO or 10 μM HOIPIN-1. Taking the cell viabilities in the presence of DMSO as 100%, the relative cell viabilities of respective cell line in the presence of HOIPIN-1 were assessed by a CellTiter-Glo luminescent cell viability assay. b HOIPIN-8 enhanced the cell death of ABC-DLBCL cells than HOIPIN-1. DLBCL2 cells or BJAB cells were treated with DMSO, 10 μM HOIPIN-1, or 10 μM HOIPIN-8 for the indicated period, and the relative cell viabilities were assessed as in a . c Caspase activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated with or without 10 μM HOIPIN-8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. d Reduced NF-κB activation in ABC-DLBCL cells by HOIPIN-8. Cells were treated and analyzed as in c . e HOIPIN-1 suppresses the expression of NF-κB target genes in ABC-DLBCL cells. Cells were treated with 10 μM HOIPIN-1 for 24 h, and qPCR analyses were performed. f Intracellular linear ubiquitin and IκBα phosphorylation in ABC-DLBCL cells are diminished by HOIPIN-1. HBL1 and BJAB cells were treated with 10 μM HOIPIN-1 for the indicated period, and cell lysates were immunoblotted with the indicated antibodies. g HOIPIN-8 has potent inhibitory effects on NF-κB activation and linear ubiquitination in ABC-DLBCL cells than those in HOIPIN-1. HBL1 cells were treated with the indicated concentrations of HOIPIN-1 or -8 for 24 h, and cell lysates were immunoblotted with the indicated antibodies. In b , e , data are shown as mean ± SEM, NS not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions.

Techniques: Cell Culture, Cell Viability Assay, Activation Assay, Expressing, Ubiquitin Proteomics, Phospho-proteomics

Journal: Molecular Systems Biology

Article Title: A ubiquitous GC content signature underlies multimodal mRNA regulation by DDX3X

doi: 10.1038/s44320-024-00013-0

Figure Lengend Snippet: Reagents and tools.

Article Snippet: For the RNA-seq, RNA was extracted from 25 μl intact lysate (non-digested) using the Direct-zol kit (Zymo Research) and stranded total RNA libraries were prepared using the TruSeq Stranded Total RNA Human/Mouse/Rat kit (Illumina), following manufacturer’s instructions.

Techniques: Sequencing, Software, Flow Cytometry, Staining, Real-time Polymerase Chain Reaction

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: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A GPR4 regulates JAK2 and STAT3 phosphorylation via western blotting. n = 3. Scale bar: 100 µm. B Representative IF images of STAT3-Y705 phosphorylation regulated by GPR4. C Quantitative analysis of STAT3-Y705 phosphorylation in GPR4 - OE SW480 cell line and ( D ) GPR4 - KD HCT116 cell lline. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. E Static treatment reduces both pSTAT3-Y705 and the expression of LOXL2, COL1A1, and TGF-β in GPR4 - OE SW480 cells. F RT-PCR analysis for LOXL2 in SW480 cell lines after receiving Stattic treatment. Two-tailed t test, n = 3 biologically independent samples. G RT-PCR analysis for TGF-β in SW480 cell lines after receiving Stattic treatment. Data are presented as mean ± SD.Two-tailed t test, n = 3 biologically independent samples. H STAT3 transcription factor binding site. I In SW480 cells, STAT3 binds to the LOXL2 promoter region confirmed via ChIP assays. J STAT3 binds to the TGF-β promoter region confirmed via ChIP assays. K RT-PCR assesses the transcriptional activity of LOXL2 promoter regions and TGF-β promoter region L . Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: Subsequently, Transcription Factor Staining Kit (Elabscience, E-CK-A108) was applied for cells fixation and Permeabilization as recommended method.

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay

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

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 1. CXCR4+ DTC cells exhibit enhanced self-renewal activity, tumorigenic potential and IR resistance compared to CXCR4- cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR cl, 22onogenic survival assay (black bar 50 mm), and (f) Western blot (left) and RT-PCR (right) analyses of DTC cells after sorting with apc-conjugated CXCR4 antibody. *p < .05. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 2. Silencing of CXCR4 via shRNA infection suppresses self-renewal activity, tumorigenic potential and IR resistance in DTC cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells infected with shControl (shCTL), shCXCR4 a or D (upper) and quantification of the results (lower). *p < .05, **p < .01, ***p < .001. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: shRNA, Infection, Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 5. CXCR4 overexpressing U-CH1 cells exhibited higher self-renewal activity, tumorigenic potential and IR resistance than control cells. (a) MTT assay, (b) sphere- forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells of transduced with control (pQCXIP) and CXCR4 carrying retroviruses in U-CH1 cells (upper) and quantification of the results (lower). *p < .05, **p < .01. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, Control, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transduction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 6. Suppression of CXCR4 reduced tumorigenic potential and enhanced IR sensitivity of DTC cell in an in vivo xenograft model. (a) A representative image of tumor-bearing mice (left) and measurement of in vivo tumor growth rate (right) in shControl (shCTL) and shCXCR4-infected DTC cells subcutaneously transplanted in mice. (b) Combination therapy of IR and CXCR4 inhibitor (AMD3100, 5 mg/kg) in xenograft mouse model. (c) Immunohistochemistry (white bar 50 μm) of xenograft tissues probed with Sox2 antibody (left) and quantitation of the results (right) *p < .05, ***p < .001.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: In Vivo, Infection, Immunohistochemistry, Quantitation Assay