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: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: A , Heart weight-to-body weight ratio (HW/BW) in 1-, 4- and 7-month-old CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice. B , Ejection fraction (EF) in 7-month-old R120G and NTG mice. C , Representative Western blots of phosphorylated (P-)STAT3, STAT3 and indicated control in 1-, 4- and 7-month-old R120G and NTG mice. D , P-STAT3/STAT3 quantification of Western blots from 1-, 4- and 7-month-old R120G and NTG mice. E , Stat3 mRNA level determined by RT-qPCR from 7-month-old R120G and NTG mice. Western blot quantification was performed with Image Lab software. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Western Blot, Control, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice treated for 3 weeks with 75 mg/kg ruxolitinib (ruxo) or vehicle (veh) twice-daily oral gavage. Transthoracic echocardiography was performed at the start (before, 21-week-old) and the end of the treatment (veh or ruxo, 24-week-old). A , Scheme of experimental outline. B , WB and quantification of phosphorylated (P-) STAT3, STAT3 and indicated controls at the end of treatment. WB quantification was performed with Image Lab software. C, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW) before and after (veh/ruxo) treatment. D , Heart weight-to-body weight ratio (HW/BW) and body weight (BW) at the end of the treatment. E , Representative images and quantification of R120G and NTG mouse heart sections after vehicle or ruxolitinib treatment. CRYAB is depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 10 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one- way ( B ) or two-way ANOVA and Tukey’s multiple comparisons post-hoc analysis ( C, D ) or unpaired Student’s t-test ( E ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

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: The Journal of pharmacy and pharmacology

Article Title: PPARα-independent action against metabolic syndrome development by fibrates is mediated by inhibition of STAT3 signaling

doi: 10.1111/jphp.13014

Figure Lengend Snippet: differential expressions of STAT3 target genes between two mouse lines. (a, b) Socs3 mRNA levels in the liver and adipose tissues respectively. (c, d) Fga mRNA levels in the wild-type and Ppara-null mice respectively. (e) Fgb mRNA in the liver tissues of wild-type and Ppara-null mice respectively. (f) Fgb mRNA in the adipose tissues of the wild-type and Ppara-null mice respectively. The mRNA levels were measured and normalized by 18S rRNA. The mRNA levels in the vehicle-treated control mice were set as 1 and the results expressed as mean ± SD (n=5, *: compared with the WT-Con or KO-Con groups respectively; ^: compared with the WT-MFD or KO-MFD groups respectively).

Article Snippet: Two mouse Stat3 siRNAs (siStat3–1: 5’-CCAACGACCUGCAGCAAUATT-3’; siStat3–2: 5’-CUCCAACAUCUGUCAGAUGTT-3’) (GenePharma, Shanghai) were transfected into the cells using lipofectamine RNAiMAX before induction of differentiation.

Techniques: Control

Journal: The Journal of pharmacy and pharmacology

Article Title: PPARα-independent action against metabolic syndrome development by fibrates is mediated by inhibition of STAT3 signaling

doi: 10.1111/jphp.13014

Figure Lengend Snippet: Western-blot analysis of STAT3 signaling in the liver and adipose tissue in two mouse lines. Data were from adipose samples collected 5 days after experience and 3/5 of the adipose tissues were randoly selected for western-blot analysis. GAPDH was used as a loading control.

Article Snippet: Two mouse Stat3 siRNAs (siStat3–1: 5’-CCAACGACCUGCAGCAAUATT-3’; siStat3–2: 5’-CUCCAACAUCUGUCAGAUGTT-3’) (GenePharma, Shanghai) were transfected into the cells using lipofectamine RNAiMAX before induction of differentiation.

Techniques: Western Blot, Control

Journal: The Journal of pharmacy and pharmacology

Article Title: PPARα-independent action against metabolic syndrome development by fibrates is mediated by inhibition of STAT3 signaling

doi: 10.1111/jphp.13014

Figure Lengend Snippet: STAT3 knockdown attenuated adipogenesis in 3T3-L1 adipogenic stem cells. (a, b, c) Oil red O staining of siRNA-transfected 3T3-L1 cells after 8 days of differentiation. (d) Reduction of STAT3 was confirmed by western-blot analysis 72 h after siRNA introduction.

Article Snippet: Two mouse Stat3 siRNAs (siStat3–1: 5’-CCAACGACCUGCAGCAAUATT-3’; siStat3–2: 5’-CUCCAACAUCUGUCAGAUGTT-3’) (GenePharma, Shanghai) were transfected into the cells using lipofectamine RNAiMAX before induction of differentiation.

Techniques: Knockdown, Staining, Transfection, Western Blot

Journal: Frontiers in Immunology

Article Title: Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death

doi: 10.3389/fimmu.2021.699128

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Mouse STAT3 , Mm01219775_m1 , 10 ng.

Techniques: Cytometry

Journal: Frontiers in Immunology

Article Title: Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death

doi: 10.3389/fimmu.2021.699128

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: Mouse STAT3 , Mm01219775_m1 , 10 ng.

Techniques: TaqMan Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death

doi: 10.3389/fimmu.2021.699128

Figure Lengend Snippet: Exposing murine DCs to pharmacological inhibitors of Flt3 and STAT3 phenocopies the effect of BEN exposure. (A-K) BALB/c BMDCs were generated following brief exposure to Flt3 inhibitor (ACC20) or STAT3 inhibitor (JSI-124) and characterized by flow cytometry. Data is pooled from 3 independent experiments (n=6-7). (A) Mean percent Flt3 expression among CD11c + BMDCs is shown with SEM, and representative histogram (right). (B-I) Mean percent with SEM of murine DC lineages including (B) total CD11c + , (C) plasmacytoid DCs, (D) conventional DCs, (E) pre-cDC1s, (F) CD8α + cDC1s, (G) CD103 + cDC1s, (H) pre-cDC2s, and (I) SIRPα + cDC2s. (J) Mean percent PD-L1 expression (left) and MFI (middle) among CD11c + FL-BMDCs shown with SEM, and representative histograms (right). (K) Mean percent ICOS-L expression (left) and MFI (middle) among CD11c + FL-BMDCs shown with SEM, and representative histograms (right). One-way ANOVA and Dunnett’s multiple comparisons test were used to determine significance among groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Mouse STAT3 , Mm01219775_m1 , 10 ng.

Techniques: Generated, Flow Cytometry, Expressing

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)-22 receptor 1 is over-expressed in primary Sjogren’s syndrome and Sjögren-associated non-Hodgkin lymphomas and is regulated by IL-18

doi: 10.1111/cei.12643

Figure Lengend Snippet: List of primers for reverse transcription–polymerase chain reaction (RT–PCR) and primary and secondary antibodies used

Article Snippet: Final values were expressed as fold of induction (FOI). table ft1 table-wrap mode="anchored" t5 caption a7 GAPDH Hs99999905_m1 IL-18 Hs01038788_m1 IL-22 Hs01574154_m1 IL-22R1 Hs00222035_m1 IL-22BP Hs00364814_m1 IL-17 Hs00174383_m1 IL-23p19 Hs00372324_m1 RORc Hs01076122_m1 STAT-3 Hs00234174_m1 Rabbit anti-human IL-22 (1 : 100 dilution) Novus Biological, Littleton, CO, USA Rabbit anti-human IL-22R1 (1 : 100 dilution) Novus Biological, Littleton, CO, USA Rabbit anti-human IL-18 (1 : 100 dilution) Novus Biological, Littleton, CO, USA Rabit anti-human IL-22BP (1 : 100 dilution) Novus Biological, Littleton, CO, USA Mouse anti-human CD3 (1 : 100 dilution) Dako, Glostrup, Denmark Mouse anti-human CD68 (1 : 100 dilution) Dako, Glostrup, Denmark Mouse anti-human CD19 (1 : 100 dilution) Dako, Glostrup, Denmark Alexa Fluor® 488 Goat Anti-Rabbit IgG Life Technologies, Europe, BV, Stockholm, Sweden Alexa Fluor® 555 Goat Anti-Mouse IgG Life Technologies, Europe, BV, Stockholm, Sweden Anti-human-CD3-APC-conjugated BD Biosciences, San Jose, CA, USA Anti-human-CD19-FITC-conjugated BD Biosciences, San Jose, CA, USA Anti-human-CD14-FITC-conjugated BD Biosciences, San Jose, CA, USA Anti-human-CD68-FITC-conjugated BD Biosciences, San Jose, CA, USA Anti-human-IL-22R1-PE-conjugated R&D Systems, Minneapolis, MN, USA Anti-human-CD4-APC-conjugated BD Biosciences, San Jose, CA, USA Anti-human-IL-17-FITC-conjugated R&D Systems, Minneapolis, MN, USA Open in a separate window APC = allophycocyanin; FITC = fluorescein isothiocyanate; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; Ig = immunoglobulin; PE = phycoerythrin; STAT-3 = signal transducer and activator of transcription-3.

Techniques: Reverse Transcription Polymerase Chain Reaction