Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 1. Flow cytometric analysis of each lymphocyte subset and expression of phosphorylated proteins. Representative data are shown. Lymphocyte fractions are classified according to surface antibodies including CD3, CD8, and CD56. Natural killer (NK) cells were defined as the CD3-CD56+ immunophenotype and cytotoxic T lymphocytes (CTLs) were CD3+ CD8+ (A). Cells were costained with phospho-specific antibodies, including antibodies targeting pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT, and expression levels of the isotype control and phosphorylated proteins in NK cells are presented (B). The values for the isotype control and each phosphorylated protein are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Expressing, Control, Fluorescence

Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 3. Constitutive levels of phosphorylated proteins including pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT in natural killer (NK) cells (A) and cytotoxic T lymphocytes (CTLs) (B) grouped according to treatment (dasatinib [n = 18] or other TKI [n = 12]) are shown. The values for phosphorylated proteins in each fraction are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Fluorescence

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a qRT-PCR analysis of miR-4293 levels in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. b qRT-PCR analysis of miR-4293 levels in A549, H1299, H1975, and HBE cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. c qRT-PCR analysis of miR-4293 level in A549 cells with transfection of miR-4293 mimic, ASO-4293, mu-4293 (mutant), or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. d MTT assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293 or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01, * p < 0.05; ANOVA test. e FACS analysis of A549 cell apoptosis at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. f Western blot detection of STAT3 and apoptosis-related factors (Bcl-2, Bax, BIRC3, and Caspase 3,8,9) in miR-4293-, ASO-4293-, or scramble-treated A549 cells. g , h Transwell migration assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. i , j Colony formation assays of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Quantitative RT-PCR, MANN-WHITNEY, Transfection, Mutagenesis, MTT Assay, Western Blot, Transwell Migration Assay

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a TargetScan analysis predicted that the DCP2 mRNA-3′-UTR is targeted by miR-4293. We synthesized a mutant miR-4293 (Mu-4293) and cloned DCP2 mRNA-3’-UTR and mu-DCP2–3’-UTR sequences. b Analysis of miR-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. c Analysis of ASO-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. d Western blot detection of DCP2 in miR-4293-, ASO-4293-, or scramble-treated A549 cells. e Western blot detection of DCP2 in siRNA-DCP2–1-, siRNA-DCP2–2-, or scramble-treated A549 cells and H129 cells. f qRT-PCR analysis of WFDC21P level in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. g Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. h qRT-PCR analysis of DCP2 level in pcDNA or pcDNA-DCP2 -treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. i qRT-PCR analysis of WFDC21P level in 0 h, 4 h, 8 h, 12 h, 24 h, 48 h after transfection of pcDNA-DCP2. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. j RIP assay of the interaction between WFDC21P and DCP2. Upper panel, qRT-PCR analysis of WFDC21P pull-down by IgG or DCP2; lower panel, electrophoresis for WFDC21P pull-down by IgG or DCP2 after PCR amplification. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. k qRT-PCR analysis of WFDC21P levels in miR-4293-, ASO-4293, Mu-4293 or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. l qRT-PCR analysis of WFDC21P levels in miR-4293+pcDNA-WFDC21P-, or scramble+ pcDNA-WFDC21P-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. m Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, miR-4293-, miR-4293+pcDNA-WFDC21P-, or scramble-treated A549 cells. n Western blot detection of STAT3 phosphorylation in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. o qRT-PCR analysis of miR-4293 level in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01, * P < 0.05; ANOVA test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Synthesized, Mutagenesis, Clone Assay, Luciferase, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Electrophoresis, Amplification

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a qRT-PCR analysis for WFDC21P level in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. b qRT-PCR analysis for WFDC21P level in A549, H1299, H1975, and HBE cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. c Western blot detection of STAT3 in A549, H1299, H1975, and HBE cells. d qRT-PCR analysis of STAT3 expression in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. e – g Pearson′s correlation analysis of miR-4293 expression and WFDC21P expression ( n = 16, ** P = 0.015) ( e ), miR-4293 expression and STAT3 expression (n = 16, ** P < 0.01) ( f ), WFDC21P expression and STAT3 expression ( n = 16, ** P < 0.01) ( g ).

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a MTT assays of A549 at 24 h post-transfection of pcDNA-WFDC21P and pc-DNA. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. b qRT-PCR analysis of WFDC21P level in si-WFDC21P- and scramble-treated cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. c MTT assays of A549 at 24 h post-transfection of si-WFDC21P and scramble. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. d FACS analysis of A549 cell apoptosis at 24 h post-transfection of pc-DNA, pcDNA-WFDC21P, si-WFDC21P, and scrambled control. e Western blot detection of STAT3 and apoptosis-related factors (Bcl-2, Bax, BIRC3, and Caspase 3,8,9) pcDNA vs pcDNA-WFDC21P; scramble vs si-WFDC. f – i Transwell assays of A549 migrat i on. f , g pcDNA vs pcDNA-WFDC21P; ( h ) and ( i ) scramble vs. si-WFDC. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. j – m Colony formation assays of A549. j , k pcDNA vs pcDNA-WFDC21P; ( l ) and ( m ) scramble vs. si-WFDC. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: The above-mentioned data showed that miR-4293 transfection elevated miR-4293 levels, which could be attenuated by ASO. pcDNA-WFDC21P transfection increased WFDC21P. Si-WFDC treatment reduced WFDC21P level. a MTT assay of A549 at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. b FACS analysis of A549 cell apoptosis at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. c Western blot detection p-STAT3, Bcl-2, BAX, cleaved-caspase 3, 8, and 9 in si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble-treated cells. d , e RTCA station analysis of migration for A549 with transcription of si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble. f MTT of A549 at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. Data are expressed as mean ± SD for triplicate experiments. (* P < 0.05). g FACS analysis of A549 cell apoptosis at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. h Western blot detection of STAT3 phosphorylation and apoptosis-related factors (Bcl-2, Bax and Caspase 3) in pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble-treated A549 cells. i – l RTCA station analysis of migration for A549. i , j pcDNA-WFDC21P vs pc-DNA; ( k ) and ( l ), pcDNA-WFDC21P + scramble vs. pcDNA-WFDC21P + ASO-4293. Data are expressed as mean ± SD for triplicate experiments. * p < 0.05, *** p < 0.001; Student’s t -test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Transfection, MTT Assay, Western Blot, Migration

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: miR-4293 promotes tumor cell proliferation and metastasis but suppresses apoptosis. DCP2 can directly or indirectly bind to and downregulate WFDC21P. By targeting DCP2 mRNA, miR-4293 suppresses the expression of DCP2, and further enhances the expression of WFDC21P and promotes STAT3 phosphorylation. As a result of elevated STAT3 activity, apoptosis is suppressed and proliferation is enhanced through Bcl2 elevation, Bax2 attenuation and reduced caspase cleavage. In addition, WFDC21P elevates miR-4293 expression by promoting STAT3 activation.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Expressing, Activity Assay, Activation Assay

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 1: Comparison on the down-regulation of Moutan Cortex from different regions on protein expression levels of ICAM-1,TGF-β1 and FN. HBZY-1 mesangial cells were treated with 200 μg/mL AGEs in the presence or absence of Moutan Cortex (MC) extract of 200 μg/mL. Aminoguanidine of 10 μM was used as the positive control while BSA (200 μg/mL) as blank control. (A) Western blotting was performed to compare the protein expression levels. (B–D) The grayscale scan results of ICAM-1, TGF-β1 and FN. a, Control; b, 200 μg/mL AGEs; c, Positive control aminoguanidine group; “d-m” represent MC from Anhui, Guizhou, Zhejiang, Henan, Hunan, Hebei, Sichuan, Chongqing, Shandong, Gansu. Data are expressed as means ± SD, n = 3. ###P < 0.001 vs. BSA group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. AGEs group; $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. MC from Anhui group.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Comparison, Expressing, Positive Control, Control, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats. After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues. “a” represents normal control; “b” represents model group (DN rats); “c” represents Positive control 0.1 g/kg AG; “d-m” represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Control, Positive Control

Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: Escherichia coli can induce liver injury in mice. WT mice were injected with PBS or 3 × 10 8 cfu/ml E. coli intraperitoneally and sacrificed 3 hours later. Liver pathological injuries were observed with hematoxylin and eosin staining (a–d). The yellow arrows indicate edema, the blue arrows indicate inflammatory cell infiltration, the black arrows indicate punctate necrosis, and the green arrows indicate binucleate hepatocytes. Bacteria from the liver of PBS- or E. coli -stimulated WT mice were cultured in MH medium (e, f) overnight and identified by Gram staining (g). The serum AST (h) and ALT (i) concentrations were determined by using the detection kits. The mRNA of liver inflammatory cytokines TLR4 (j), NLRP3 (k), IL-1 β (l), and IL-18 (m) of PBS- or 3 × 10 8 cfu/ml E. coli -stimulated mice were measured by RT-qPCR. Data are presented as means ± standard deviation. Statistical significance was determined by the paired t -test ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Injection, Staining, Bacteria, Cell Culture, Quantitative RT-PCR, Standard Deviation

Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: The expression levels of pyroptosis-related markers were detected by Western blot. The expressions of liver pyroptosis proteins in WT, CD38 −/− , and CD38 −/− TLR4 mut mice were detected at 3 hours after E. coli stimulation by Western blot. The expressions of NLRP3, ASC, procaspase-1, cleaved caspase-1, IL-1 β , IL-18, procaspase-3, and cleaved caspase-3 were measured (a). And relative levels of NLRP3 to GAPDH (b), ASC to GAPDH (c), cleaved to procaspase-1 (d), IL-1 β to GAPDH (e), IL-18 to GAPDH (f), and cleaved to procaspase-3 (g) were analyzed by ImageJ software. Data are presented as means ± standard deviation. Statistical significance was determined by one-way ANOVA ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Upregulation of IL-9 and inflammatory cytokines as well as the activation of Notch1 signaling during EAE process. a The level of IL-9 in spinal cords of EAE mice with different clinical scores was measured by real-time PCR and Western blot assay, respectively. b The expressions of GFAP, NICD and p-STAT3 in spinal cords along with EAE process were detected by Western blot assay. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in spinal cords were determined using real-time PCR. d The secretion levels of IL-6, TNF-α, IP-10 and MCP-1 in the sera were detected by Cytometric Bead Array (CBA) during the EAE process. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus PBS group ( n = 6/group, one-way ANOVA). These results were repeated four times. Data are represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: IL-9 activates the Notch1 pathway and promotes inflammatory cytokines production in astrocytes. Primary mouse astrocytes were incubated in a serum-free medium overnight followed by treating with IL-9 at the indicated time point. a The expression changes of GFAP, NICD, and p-STAT3 were analyzed by Western blot assay. b Immunofluorescent staining for GFAP (green), Notch1 (red), and nuclear staining of DAPI (blue) in cultured astrocytes with IL-9 treatment for 6 h. Scale bars, 50 μm. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in astrocytes were detected using real-time PCR assay. d The secretion levels of IL-6, TNF-α, IP-10, and MCP-1 in the supernatant of astrocytes were measured by CBA assay. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus DMEM group (one-way ANOVA). The data are from three independent experiments and represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Incubation, Expressing, Western Blot, Staining, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Inhibition of Gm13568 downregulates Notch1 signaling activation as well as inflammatory cytokine production in astrocytes by IL-9. a Primary mouse astrocytes were infected with recombinant lentivirus, LV-Inhibit-Gm13568 and LV-ctrl, for 72 h, respectively. Then, the astrocytes were incubated in a serum-free medium overnight followed by IL-9 stimulation for 6 h. Western blot assay was used for measuring the protein expressions of GFAP, Notch1/NICD, and p-STAT3. b The mRNA levels of IL-6, TNF-α and IP-10 in astrocytes were measured by real-time PCR assay. c The secretion of IL-6, TNF-α, and IP-10 in the supernatant of astrocytes were analyzed by CBA assay. The data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus DMEM group; ## p < 0.01 and ### p < 0.001 versus LV-ctrl+IL-9 group (one-way ANOVA, the two-tailed Student’s t test)

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Inhibition, Activation Assay, Infection, Recombinant, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Knockdown of Notch1 in astrocytes suppresses inflammation and alleviates EAE in mice. Mice were subjected to recombinant lentiviruses, LV-ctrl or LV-Notch1-shRNA, for 7 days, followed by MOG 35-55 immunization for 23 days ( n = 10 mice per group). a The clinical scores of EAE mice with LV-ctrl and LV-Notch1-shRNA . b The expressions of IL-9, GFAP, NICD, and p-STAT3 in the spinal cords were detected using western blot assay. c , d The changes of IL-6, TNF-α, and IP-10 in the spinal cords and peripheral blood of the LV-ctrl and LV-Notch1-shRNA mice were evaluated by real-time PCR and CBA assay, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PBS group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LV-ctrl group (one-way ANOVA, the two-tailed Student’s t test). Results are represented as mean ± SEM. e The infiltration of inflammatory cells in spinal cords was investigated using hematoxylin and eosin (H&E) staining (scale bars, 50 μm). f The medullary sheath damages from spinal cords were observed via luxol fast blue (LFB) staining (scale bars, 50 μm). Boxed areas in the upper rows are presented enlarged underneath

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Knockdown, Recombinant, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining