Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A–D) Relative expression of selected genes, obtained from a publicly available database. Expression of each gene is shown in healthy skin and in lesional psoriatic skin. Boxplots report ±SEM (*** p < 0.001). (A) ZFP36 expression in psoriatic skin samples is significantly reduced, compared to healthy skin samples [Healthy donor: n = 21, median 10.83, range 8.59–12.85; Lesional psoriatic fibroblasts (PSO): n = 33, median 9.23, range 8.79–9.78]. (B) NLRP3 levels show no significant variation among the two groups of healthy and psoriatic skin (Healthy donor: n = 21, median 2.73, range 2.58–3.13; Lesional PSO: n = 33, median 2.80, range 2.56–3.13). (C) CASP1 expression results increased in lesional psoriatic samples compared to normal skin samples (Healthy donor: n = 21, median 6.88, range 5.13–8.52; Lesional PSO: n = 33, median 8.00, range 5.45–8.77). (D) IL1B is significantly more expressed in lesional skin samples from psoriatic patients than skin from healthy donors (Healthy donor: n = 21, median 4.04, range 3.69–6.67; Lesional PSO: n = 33, median 5.05, range 4.06–7.03). (E,F) Relative quantity of mRNA measured by real-time (RT) PCR in healthy and psoriatic fibroblasts samples. (E) Differential expression of TTP mRNA in psoriatic and healthy fibroblasts, measured through qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Results are represented as means of three experiments (±SEM) (* p < 0.05). (F) IL1β mRNA levels in psoriatic and healthy fibroblasts, measured through qRT-PCR. GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM) (** p < 0.01).

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) Tristetraprolin (TTP) protein levels in healthy and psoriatic fibroblasts. Two different healthy samples and two different psoriatic samples were divided into two series. In “Fibroblasts II series,” psoriatic cell samples from the same donor are distinguished in lesional and non-lesional. Vinculin was used as loading control. (B) NLRP3, pro-IL1β, and IL1β (p17) protein levels in healthy and psoriatic fibroblasts. (C) Pro-CASP1 and active CASP1 (p20) protein levels in healthy and psoriatic fibroblasts. Vinculin was used in (B,C) as a loading control.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques:

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) qRT-PCR shows a decrease in NLRP3 mRNA in HaCaT cells transfected with a TTP-expressing vector (pcDNA3.1-ZFP36), compared to control transfection (pcDNA3.1). GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM) (** p < 0.01). (B) Luciferase reporter assay shows a decrease in luciferase activity in HEK293T cell line cotransfected with luciferase reporter vector and the TTP-expressing vector, compared to the control cotransfection with the empty vector. Measured luciferase activity was normalized over β-gal signals. Results are represented as the means of three experiments (±SEM) (* p < 0.05). (C) Human NLRP3 3′UTR displaying TTP binding site (highlighted). (D) NLRP3 mRNA levels measured by qRT-PCR in HaCaT cells transfected at time 0 with a TTP-overexpressing vector or an empty vector. After transfection (48 h), cells were treated with Act.D to block transcription. NLRP3 mRNA levels were recorded at the moment of ActD treatment (Ctr), after 1 h and after 3 h. Results are represented as the means of three experiments (±SEM). Statistical analysis was performed comparing ZFP36-transfected and empty vector-transfected cells at each timepoint (* p < 0.05). GAPDH was used as endogenous control.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Binding Assay, Blocking Assay

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) TTP silencing achieved by lentiviral infection (sample labeled shZFP36) was verified by Western blot, in comparison to endogenous TTP levels measured after infection with a vector expressing an aspecific scrambled short hairpin (labeled EV). Actin was used as loading control. (B) Western blot displaying the expression levels of inflammasome components. Inflammasome interactor levels vary after lentiviral infection with a short hairpin directed against TTP mRNA. Vinculin is included as loading control. (C) NLRP3, IL1β, and TTP mRNA levels have been analyzed by qRT-PCR in both cell samples. GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM).

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Infection, Labeling, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) NLRP3 inflammasome protein levels are displayed by Western blot in three different fibroblast cell populations: healthy fibroblasts (Healthy), psoriatic fibroblasts (PSO), and psoriatic fibroblasts infected with a pRRL-TTP-overexpressing vector (PSO TTP). Tubulin was used as loading control. (B) TTP overexpression is shown in psoriatic fibroblast sample. Actin was used as loading control. (C) Inflammasome components variation pattern already seen in primary fibroblasts is maintained in the HaCaT keratinocyte cell line. Actin was used as loading control. (D) pRRL-empty vector was used to obtain the overexpression vector pRRL-TTP, by substitution of ΔNGFR sequence with TTP cDNA.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Western Blot, Infection, Plasmid Preparation, Over Expression, Sequencing

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: iNSCs transplantation ameliorated pyroptosis in ICH rats (A) The representative western blot images of pyroptosis pathway related proteins in each group. (B–L) Quantitative analysis of target proteins of NF-κB p-P65, NLRP3, ASC, pro-Caspase-1, Caspase-1 p10, Caspase-1 p20, GSDMD, pro-IL-18, IL-18, pro-IL-1β, IL-1β, respectively, and normalized to β-actin. The data are expressed as mean ± SD, (n = 5). Comparison of means among multiple groups was performed using one-way ANOVA followed by Tukey’s post hoc test, and compared with the Sham group ∗p < 0.05; compared with the ICH group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Transplantation Assay, Western Blot

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: ICH induced pyroptosis in astrocytes (A and B) The representative images of GFAP/NLRP3 immunofluorescence co-labeling staining around hematomas and quantitative analysis of the percentage of NLRP3 labeled GFAP positive cells in each group. (C and D) The representative images of GFAP/Caspase-1 immunofluorescence co-labeling staining around hematoma and quantitative analysis of the percentage of Caspase-1 labeled GFAP positive cells in each group. Arrows pointed co-labeled positive cells, scale bar = 50 μm. The data are expressed as median with IQR, (n = 5). The Mann-Whitney test in nonparametric test was used to analyze the differences among the groups, and compared with the Sham group ∗p < 0.05; compared with the ICH group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Immunofluorescence, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: ICH induced pyroptosis in microglia (A and B) The representative images of Iba-1/NLRP3 immunofluorescence co-labeling staining around hematomas and quantitative analysis of the percentage of NLRP3 labeled Iba-1 positive cells in each group. (C and D) The representative images of Iba-1/Caspase-1 immunofluorescence co-labeling staining around hematoma and quantitative analysis of the percentage of Caspase-1 labeled Iba-1 positive cells in each group. Arrows pointed the co-labeled positive cells, scale bar = 50 μm. The data are expressed as median with IQR, (n = 5). The Mann-Whitney test in nonparametric test was used to analyze the differences among the groups, and compared with the Sham group ∗p < 0.05; compared with the ICH group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Immunofluorescence, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: ICH induced pyroptosis in neurons (A and B) The representative images of NeuN/NLRP3 immunofluorescence co-labeling staining around hematomas and quantitative analysis of the percentage of NLRP3 labeled NeuN positive cells in each group. (C and D) The representative images of NeuN/Caspase-1 immunofluorescence co-labeling staining around hematoma and quantitative analysis of the percentage of Caspase-1 labeled NeuN positive cells in each group. Arrows pointed the co-labeled positive cells, scale bar = 50 μm. The data are expressed as median with IQR, (n = 5). The Mann-Whitney test in nonparametric test was used to analyze the differences among the groups, and compared with the Sham group ∗p < 0.05; compared with the ICH group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Immunofluorescence, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: Microglia pyroptosis induced in vitro (A–C) The representative western blot images of Iba-1, CD68, NLRP3 and NF-κB p-P65 expression in microglia under different conditions. (D–G) Quantitative analysis the expression level of Iba-1, CD68, NLRP3 and NF-κB p-P65, respectively. (H) Schematic drawing of the next step of co-culture system. The data are expressed as mean ± SD, (n = 3). Comparison of means among multiple groups was performed using one-way ANOVA followed by Tukey’s post hoc test, and compared with the Normal group, ∗p < 0.05; compared with the LPS (1.0 μg/mL)/ATP group or the MCC950 (2.0 μM) group or the Bay 11–7082 (10.0 μM) group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: In Vitro, Western Blot, Expressing, Co-Culture Assay

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: Immunofluorescence staining of NLRP3 in microglia (A) The representative images of Iba-1/NLRP3 immunofluorescence co-labeling staining in each group. Arrows indicated co-labeled positive cells, scale bar = 20 μm. (B) Quantitative analysis of Iba-1/NLRP3 co-labeled positive cells in each group. The data are expressed as mean ± SD, (n = 5). Comparison of means among multiple groups was performed using one-way ANOVA followed by Tukey’s post hoc test, and compared with the LPS/ATP group ∗p < 0.05; compared with the iNSCs group # p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Immunofluorescence, Staining, Labeling

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet: The effects of iNSCs on pyroptosis in microglia (A)The representative western blot images of pyroptosis pathway related proteins in each group. (B–N) Quantitative analysis the expression level of NF-κB P65, NF-κB p-P65, NLRP3, ASC, pro-Caspase-1, Caspase-1 p10, Caspase-1 p20, GSDMD, GSDMD-N, pro-IL-18, IL-18, pro-IL-1β, and IL-1β, respectively, and normalized to β-actin. The data are expressed as mean ± SD, (n = 3). Comparison of means among multiple groups was performed using one-way ANOVA followed by Tukey’s post hoc test, and compared with the LPS/ATP group ∗p < 0.05; compared with the MCC950 group # p < 0.05; compared with the Bay 11–7082 group & p < 0.05; compared with the iNSCs + MCC950 group @ p < 0.05; compared with the iNSCs + Bay 11–7082 group $ p < 0.05.

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Western Blot, Expressing

Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet:

Article Snippet: Brain tissue or cell samples with a total protein content of 50 μg were loaded onto 10% – 12.5% SDS‒PAGE gels to separate various types of proteins, wet transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab108319, abcam), NGF (1:1000, ab52918, abcam), GDNF (1:1000, ab176564, abcam), GAP-43 (1:1000, ab16053, abcam), NF-κB p-P65 (1:1000, ab76302, abcam), NF-κB P65 (1:10000, 80979-1-RR, Proteintech), NLRP3 (1:1000, ab263899, abcam), ASC (1:1000, 10500-1-AP, Proteintech), Caspase-1 (1:1000, ab179515, abcam), GSDMD (1:1000, ab209845, abcam), GSDMD-N (1:1000, 20770-1-AP, Proteintech), pro-IL-18 (1:1000, 10663-1-AP, Proteintech), IL-18 (1:1000, 60070-1-Ig, Proteintech), pro-IL-1β (1:1000, ab254360, abcam), IL-1β (1:1000, 16806-1-AP, Proteintech), CD68 (1:1000, 28058-1-AP, Proteintech), Iba-1 (1:1000, ab283319, abcam), Arg-1(1:10000, 16001-1-AP, Proteintech), iNOS (1:1000, 18985-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), TNF-α (1:1000, 17590-1-AP, Proteintech), IL-37 (1:1000, ab278499, abcam), SOD (1:1000, ab183881, abcam), β-actin (1:1000, 20536-1-AP, Proteintech), GAPDH (1:10000, 10494-1-AP, Proteintech).

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Staining, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Frontiers in Pharmacology

Article Title: A full-spectrum Boswellia serrata extract with enhanced bioavailability, and its co-delivered system with curcumin alleviate pain and stiffness associated with moderate spondylitis: a randomized double-blind, placebo-controlled, 3-arm study

doi: 10.3389/fphar.2025.1577429

Figure Lengend Snippet: Mean change in (a) IL-1β and (b) NLRP3. Values are expressed as Mean ±SD, 2 × 3 repeated measures ANOVA was analysed to perform statistical significance.

Article Snippet: Serum concentrations of IL-1β (catalog no: E-EL-H0149) and NLRP3 (E-EL-H2557) were analyzed with the respective ELISA kits following the manufacturer’s instruction, (Elabscience, Biotechnology Co., Limited.

Techniques:

Journal: Diabetes & Vascular Disease Research

Article Title: Baicalin ameliorates atherosclerosis by inhibiting NLRP3 inflammasome in apolipoprotein E-deficient mice

doi: 10.1177/1479164120977441

Figure Lengend Snippet: Baicalin inhibited NLRP3 inflammasome activation in aortas: (a) Western blot analysis of NLRP3 and Caspase-1 p20, (b) relative mRNA levels of NLRP3 and caspase-1 were detected by qRT-PCR. Relative protein levels of NLRP3 (c), and caspase-1 (d) from western blot were quantified. Data were shown as mean ± SD. n = 7 for each group. * p < 0.05, ** p < 0.01 compared with the control group; # p < 0.05 compared with the AS group.

Article Snippet: Lentiviral vector expressing Nlrp3 shRNA or control shRNA was purchased from Origene (Beijing, China).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR

Journal: Diabetes & Vascular Disease Research

Article Title: Baicalin ameliorates atherosclerosis by inhibiting NLRP3 inflammasome in apolipoprotein E-deficient mice

doi: 10.1177/1479164120977441

Figure Lengend Snippet: Effects of NLRP3 silencing on the progression of atherosclerosis in ApoE −/− mice: (a) relative mRNA levels of NLRP3, caspase-1, ICAM-1, and VCAM-1 were detected by qRT-PCR, (b) IL-1β and IL-18 levels were measured by the ELISA assay, (c) mtROS and ROS levels were detected, and (d) atherosclerotic plaques area of aortic root was quantified. Scale bar = 50 μm. Data were shown as mean ± SD. n = 7 for each group. * p < 0.05, ** p < 0.01 compared with the control group; # p < 0.05 compared with the AS+sh-Control group. Mice of Control and AS+sh-Control group were treated with lentivirus expressing control shRNA, and mice of AS+sh-NLRP3 group were treated with lentivirus expressing NLRP3 shRNA.

Article Snippet: Lentiviral vector expressing Nlrp3 shRNA or control shRNA was purchased from Origene (Beijing, China).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, shRNA