Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental MDA-PCa-2b (MDA2b) and chronic IL-1 subline cells (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) were treated acutely for 3 days (A, C) or 6 days (B) with vehicle control (V), 25 ng/ml IL-1α (a), or 25 ng/ml IL-1β (b) and analyzed for cell viability using MTT (A, B) or protein accumulation by western blot (C). Acute IL-1 exposure reduces cell viability and proliferation, reduces full-length PARP (indicative of cell death activation), induces SOD2 and LCN2 protein accumulation (canonical IL-1-induced genes), and reduces AR and NKX3.1 (canonical AR target gene) protein accumulation in MDA-PCa-2b parental cells, but has little to no effect on the chronic IL-1 sublines. Thus, the IL-1 sublines evolved insensitivity to IL-1. Error bars, ± STDEV of 4 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. Fold MTT optical density (OD) is normalized to treatment control. β-actin is the western blot loading control.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Western Blot, Activation Assay

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: (A) MDA-PCA-2b parental (MDA2b) and chronic IL-1 subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels of the IL-1 receptor, IL-1R1 . Acute IL-1 exposure does not increase IL-1 receptor ( IL-1R1 ) mRNA levels in parental cells, suggesting basal IL-1R1 levels are sufficient to mediate IL-1 signaling. Furthermore, IL-1 does not show a differential effect on IL-1R1 mRNA levels in parental versus subline cells, suggesting subline insensitivity is independent of IL-1R1 levels. (B) Vehicle control treated cells were compared for basal mRNA levels of IL-1R1 and of canonical IL-1-induced genes, LCN2 , NOX1 , and SOD2 . IL-1R1, LCN2 , NOX1 , and SOD2 basal mRNA levels are comparable across the parental and subline cells, suggesting chronic IL-1 exposure does not induce constitutive activation of canonical IL-1 intracellular signaling. These data suggest that MDA-PCa-2b cell lines evolve insensitivity to exogenous chronic IL-1 exposure independent of IL-1R1 levels or constitutive activation of intracellular IL-1 signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line. For basal expression, mRNA levels are normalized to the parental cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR, Activation Assay, Expressing

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental (MDA-PCA-2b (MDA2b), LNCaP) and subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2, LNas1, LNbs1) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels (A, B, C). Acute IL-1 exposure increases LCN2 , NOX1 , and SOD2 mRNA levels in parental MDA-PCa-2b and LNCaP cells, but acute IL-1 exposure has attenuated or no effect on mRNA levels in the subline cells. Thus, both LNCaP and MDA-PCa-2b cell lines show conserved intracellular response to acute IL-1-induced changes mRNA levels and evolve chronic IL-1 insensitivity independent of constitutive canonical IL-1 intracellular signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR

Journal: Advanced Science

Article Title: Loss of SOCS1 in Donor T Cells Exacerbates Intestinal GVHD by Driving a Chemokine‐Dependent Pro‐Inflammatory Immune Microenvironment

doi: 10.1002/advs.202513735

Figure Lengend Snippet: Socs1 deficiency in T cells drives effector differentiation and enhances inflammatory responses in CD8 + T cells. (A) Experimental schematic. Splenocytes from WT (littermate control; Socs1 fl/fl ) and cKO (LckCre‐ Socs1 fl/fl ) mice were isolated and sorted by FACS for CD45 + cells and subjected to single‐cell RNA sequencing (scRNA‐seq, n = 5 per group). Alternatively, CD8 + T cells from the spleen of WT and cKO mice were sorted by FACS and subjected to bulk RNA‐seq, ATAC‐seq, and CUT&Tag analyses. (B) UMAP plot of 105 040 single cells from CD45 + splenocytes colored by annotated immune cell subsets. (C) UMAP visualization of CD45 + splenocytes, split by origin. (D) Comparison of the proportions of celltypes between WT and cKO groups. (E) UMAP plot of 19 490 T cells extracted from Figure and colored by annotated T cell subsets. (F) UMAP visualization of T cells, split by origin. (G) Heatmap of Ro/e (Ratio of observed to expected) scores for T cell subtypes in WT and cKO mice. The scores, calculated from scRNA‐seq cell counts, indicate the relative enrichment (red, Ro/e > 1) or depletion (white/light orange, Ro/e < 1) of each population within each genotype. Numerical values are presented alongside a semi‐quantitative summary. (H) UMAP plot of 9159 CD8 + T cells extracted from Figure and colored by annotated T cell subsets. (I) Comparison of the proportions of indicated CD8 + T cell clusters between WT and cKO groups. (J) Representative flow cytometry plots and frequencies of naive T cells (Tn; CD44 − CD62L + ), central memory (Tcm; CD44 + CD62L + ), and effector memory (Tem; CD44 + CD62L − ) in CD8 + T cells from peripheral blood (PB, left panel) and spleen (SP, right panel) (n = 5 per group). (K) Bar plots showing the expression of perforin, GZMB, TNF‐α, IFN‐γ, IL‐2, and CD107a in CD8 + T cells from WT and cKO mice, as measured by flow cytometry (n=5 per group). (L) UMAP visualization of integrated T‐cell transcriptomes from WT (left, 25 304 cells) and cKO (right, 21 588 cells) groups. Each point represents a single cell, colored by the frequency of its corresponding TCR clonotype, highlighting clonally expanded cells. (M) Quantification of overall TCR repertoire diversity. The Gini index (top) and Shannon entropy (bottom) were calculated for the entire T‐cell population from each mouse. (N) Distribution and clonal size of T cells across identified subsets. Barplot showing the absolute cell counts (left panels) and the clonal size composition (right panels) for each T‐cell subset from WT and cKO mice. Data represent one experiment out of two independent experiments. P values were determined using two‐sided Wilcoxon rank‐sum test (D, I, M) or unpaired two‐tailed Student's t‐test (J‐K). Data represent mean ± SEM (D, I, M) or mean ± SD (J‐K). ∗ p <.05, ∗∗ p <.01 and ∗∗∗∗ p <.0001.

Article Snippet: The following kits were used: IL‐1β (Proteintech, KE10003), IL‐6 (Proteintech, KE10007), and TNF‐α (Proteintech, KE10002).

Techniques: Control, Isolation, Single Cell, RNA Sequencing, Comparison, Flow Cytometry, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Loss of SOCS1 in Donor T Cells Exacerbates Intestinal GVHD by Driving a Chemokine‐Dependent Pro‐Inflammatory Immune Microenvironment

doi: 10.1002/advs.202513735

Figure Lengend Snippet: Evolution of small intestinal immune cell composition following transplantation of Socs1 cKO CD8 + T cells. (A) Experimental schematic. Lethally irradiated BALB/c recipient mice were transplanted with splenic T cells from WT mice (WT group) or cKO mice (cKO group), along with 5 × 10 6 TCD‐BM cells from WT mice. Survival was monitored daily. Body weight and GVHD score were assessed every five days. Immune cells in PB and small intestine from WT and cKO groups were assessed on Day 7, Day 16, and Day 24 by flow cytometry. (B) Survival analysis of recipients transplanted with 1 × 10 6 , 2 × 10 6, or 3 × 10 6 splenic T cells from WT or cKO mice (n = 10 mice per group). A control group received TCD‐BM only. Median survival times for cKO groups were 36 (1 × 10 6 ), 33 (2 × 10 6 ), and 26 days (3 × 10 6 ), respectively. In the corresponding WT groups, 9/10 mice in the 1 × 10 6 group survived to the end of the observation period, with median survival times of 35 days (2 × 10 6 ), and 42.5 days (3 × 10 6 ). Data were pooled from two independent experiments (n=10 mice/group). (C‐D) GVHD score (C) and body weight changes (D) in WT and cKO recipients transplanted with 1 × 10 6 splenic T cells. (E) Proportions of CD8 + T cells and monocytes in PB, and proportions of CD8 + T cells in the IEL and LP on Day 7, 16, and 24 post‐transplantation. (F) Boxplots showing the expression of TNF‐α, IFN‐γ, perforin, and CD107a in CD8 + T cells in IEL by flow cytometry on Day 7 and 16 post‐transplantation. (G) Experimental schematic of scRNA‐seq. Lymphocytes from IEL and LP were isolated and collected from 3‐4 mice, followed by FACS sorting for CD45 + donor‐derived (H‐2 b+ H‐2 d− ) cells respectively. Sorted cells from IEL and LP were mixed in a 1:2 ratio and subsequently subjected to scRNA‐seq. (H‐I) UMAP plot of all immune cells colored by samples (H) or annotated subsets (I). (J) UMAP plot showing annotated CD8 + T cell subsets (left), with barplot illustrating the proportion of each annotated subset in recipient intestines at indicated time points between WT and cKO groups (right). (K) Violin plots comparing the expression of cytotoxic molecules and cytokine receptor genes in CD8 + T cells on Day 26 post‐transplantation between WT and cKO groups. (L) UMAP plot showing annotated myeloid cell subsets (left), with barplot illustrating the proportion of each annotated subset in recipient intestines at indicated time points between WT and cKO groups (right). (M) Violin plots comparing expression of M1‐ and M2‐associated marker genes in myeloid cells on Day 26 post‐transplantation between WT and cKO groups. Data represent three independent experiments. P values were determined using chi‐squared test (B) or unpaired two‐tailed Student's t‐test (E, F) or two‐sided Wilcoxon rank‐sum test (K, M). Differences in GVHD scores and body weight between the WT and cKO groups at each time point were analyzed using multiple unpaired two‐tailed Student's t ‐tests (C, D). Data represent mean ± SEM (C‐E). ∗ p <.05, ∗∗ p <.01, ∗∗∗ p <.001 and ∗∗∗∗ p <.0001.

Article Snippet: The following kits were used: IL‐1β (Proteintech, KE10003), IL‐6 (Proteintech, KE10007), and TNF‐α (Proteintech, KE10002).

Techniques: Transplantation Assay, Irradiation, Flow Cytometry, Control, Expressing, Isolation, Derivative Assay, Marker, Two Tailed Test

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: High Relevance of CASP3 Activation and GSDME Cleavage in a Subset of EOPE Placentas. (A) Immunoblots of pyroptosis‐related proteins (GSDME‐FL, GSDME‐N, CASP3 and cleaved CASP3) in lysate of placenta villi from NC and EOPE groups. ERK2 was used as a loading control. (B) Relative quantification of GSDME‐N, and cleaved CASP3 in the placentas by Western blot (controls( n = 12), EOPE( n = 14)). Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, * p <0.05. (C) Pie chart showing the proportion of EOPE patients exhibiting increased levels of both GSDME‐N and cleaved CASP3 in placental villous lysates. (D) Representative Immunofluorescence pictures of GSDME, CK7 and DAPI in the normal pregnant placenta villi and decidua at the third trimester. Scale bars, 100 µm. (E)Representative immunostaining pictures of GSDME‐N and cleaved CASP3 in the placenta of NC ( n = 6) and EOPE ( n = 6) group. Scale bars, 50 µm. (F) Schematic diagram of ELISA for detecting blood samples at diagnosis (24–25 weeks). (G) The CASP3 and BAX levels in plasma from patients with EOPE ( n = 12) and controls ( n = 12) were detected by ELISA kits. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, *** p <0.0001. (H) The IL‐1β and IL‐18 levels in plasma from patients with EOPE ( n = 12) and controls ( n = 12) were detected by ELISA kit. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, *** p <0.0001. NC, control; EOPE, Early‐onset preeclampsia.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Control, Quantitative Proteomics, Immunofluorescence, Immunostaining, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Clinical Proteomics

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: Inhibition of GSDME or CASP3 reduced pyroptosis‐like phenotype and inflammation in trophoblast cells. (A) Immunoblots of GSDME and its relative quantification in sh CTRL and sh GSDME in HTR‐8/SVneo cells. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n ≥ 3, **** p < 0.0001. (B) Immunoblots of GSDME‐FL, GSDME‐N, CASP3, Cleaved CASP3 and HMGB1 in HTR‐8/SVneo cells (sh CTRL and sh GSDME ) treated with T/S. ERK2 was used as a loading control. (C) Phase‐contrast images of sh CTRL and sh GSDME HTR‐8/SVneo cells at 24 h after T/S treatment. Arrows indicated the swelling cells. Scale bars, 50 µm. Percentages of swelling cells after T/S treatment. Error bars, mean ± SEM, n = 3, duplicate each time, 3 fields of view were counted. The data were analyzed with a one‐way ANOVA, **** p <0.0001. (D) Flow cytometry of Annexin V and DAPI in sh CTRL and sh GSDME HTR‐8/SVneo cells treated with T/S. Percentages of Annexin V and DAPI double positive cells. Error bars, mean ± SEM, n ≥ 3. The data were analyzed with a one‐way ANOVA, **** p <0.0001. (E) Comparison of LDH release of HTR‐8/SVneo cells (sh CTRL and sh GSDME ) treated with T/S after 24 h. Error bars, mean ± SEM, n = 3, duplicate each time. The data were analyzed with a one‐way ANOVA, **** p <0.0001, *** p <0.001. (F) Relative levels of IL‐1β, IL‐18, MCP‐1, IL‐6 and IL‐23 in the supernatant of cultured sh CTRL and sh GSDME HTR‐8/SVneo cells using the LEGENDplex inflammation panel. Error bars, mean ± SEM, n ≥ 3. The data were analyzed with a one‐way ANOVA, * p <0.05, *** p <0.001, **** p <0.0001, ns, not significant. (G) Immunoblots of GSDME‐FL, GSDME‐N, CASP3 and Cleaved CASP3 in HTR‐8/SVneo cells treated with T/S and Z‐DEVD‐FMK. ERK2 was used as a loading control. (H) Phase‐contrast images of HTR‐8/SVneo cells at 24 h after T/S or Z‐DEVD‐FMK treatment. Arrows indicated the swelling cells. Scale bars, 50 µm. Percentages of swelling cells after T/S treatment. Error bars, mean ± SEM, n = 3, duplicate each time, three representative fields per group were analyzed. The data were analyzed with a one‐way ANOVA, **** p <0.0001, *** p <0.001. NC, control; T/S, SM164 and TNFα.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: Inhibition, Western Blot, Quantitative Proteomics, Control, Flow Cytometry, Comparison, Cell Culture

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: Pyroptotic trophoblasts drive pro‐inflammatory macrophage polarization within placenta villi organoids. (A) Schematic diagram of placenta villi organoids (PVOs) construction. (B) Haematoxylin and eosin (H&E) staining of placenta villi and PVOs. (C) Immunoblots of pyroptosis‐related proteins (GSDME‐FL, GSDME‐N, CASP3 and Cleaved CASP3) in lysate of PVOs treated T/S after 24 h. ERK2 was used as a loading control. (D) IL‐18 and IL‐1β level in the supernatant of PVOs treated T/S after 24 h using the ELISA kit. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n ≥ 3, *** p <0.001, ** p <0.01. (E) Representative Immunofluorescence staining images of iNOS, CD163, CK7 and DAPI of PVOs treated T/S after 24 h. Scale bars, 20 µm. Representative Immunofluorescence staining images of iNOS, CD163, CK7 and DAPI in the NC and EOPE placentas. Scale bars, 20 µm. The quantification of relative level of iNOS and CD163 in the PVOs treated T/S after 24 h. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 3, duplicate each time, three representative fields per slide were analyzed, **** p <0.0001. The quantification of relative level of iNOS and CD163 in the NC ( n = 6) and EOPE ( n = 6) placentas. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 6, three representative fields per slide were analyzed, **** p <0.0001. NC, control; T/S, TNFα and SM164.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: Staining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: Pyroptotic trophoblasts and pro‐inflammatory macrophages influence each other, promoting the inflammatory response. (A)Phase‐contrast images of TOs treated with T/S after 24 h. Scale bars, 50 µm. (B) Immunoblots of GSDME‐FL, GSDME‐N, GSDMD‐FL, GSDMD‐N and Cleaved CASP3 in TOs treated with SM164 and different TNFα concentrations (0, 10, 20, 40, 80 ng/mL) after 24 h. ERK2 was used as a loading control. (C) Cytotoxicity assay in TOs cell after T/S treatment based on the detection of released LDH. Error bars, mean ± SEM, n = 3, duplicate each time. The data were analyzed with a one‐way ANOVA, **** p <0.0001, ** p <0.01, * p <0.05. (D) Schematic diagram of TOs and macrophage (Mφ) co‐culture model construction. (E) Representative Immunofluorescence staining images of iNOS after co‐culture of macrophages in the basal state with pyroptotic and non‐pyroptotic TOs. Scale bars, 20 µm. (F) Schematic diagram of TOs and Mφ assembloid construction. (G) Composite z stack confocal images of TOs and Mφ assembloid at day 3 after reaggregation containing unstimulated or proinflammatory macrophages stained with the antibodies against CD68, CK7 and DAPI. Scale bars, 20 µm. (H) Immunoblots of pyroptosis‐related proteins (GSDME‐FL, GSDME‐N, CASP3 and Cleaved CASP3) in lysate of TOs treated T/S after 24 h and TOs and Mφ assembloid containing unstimulated or proinflammatory macrophages. ERK2 was used as a loading control. (I) IL‐18 and IL‐1β level in the culture supernatant of TOs treated T/S after 24 h and TOs and Mφ assembloid containing unstimulated or proinflammatory macrophages using the ELISA kit. Error bars, mean ± SEM, n ≥ 3. The data were analyzed with a one‐way ANOVA. **** p <0.0001. NC, control; T/S, TNFα and SM164.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: Western Blot, Control, Cytotoxicity Assay, Co-Culture Assay, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: Caspase‐3‐GSDME‐mediated placental pyroptosis plays a role in promoting systemic inflammation in vivo. (A) Schematic diagram of the T/S treatment protocol in mice. (B) Immunoblots of GSDME‐FL, GSDME‐N, CASP3 and Cleaved CASP3 in placentas of WT mice treated with TNFα plus SM164. ERK2 was used as a loading control. (C) Representative immunofluorescence staining of GSDME and cleaved CASP3 in placental tissue from T/S‐treated mice. The quantification of relative level of Cleaved CASP3 in placental tissue from T/S‐treated mice. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 3, duplicate each time, three representative fields per slide were analyzed, **** p <0.0001. Scale bars, 100 µm. (D) Representative immunofluorescence staining of MCT4, CD206, and CD68 in placental tissue from T/S‐treated mice. Scale bars, 50 µm. The quantification of relative level of CD206 and CD68 in placental tissue from T/S‐treated mice. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 3, duplicate each time, three representative fields per slide were analyzed, **** p <0.0001. (E) Immunoblots of GSDME‐FL, GSDME‐N, CASP3 and Cleaved CASP3 in placental tissue from T/S‐treated mice. ERK2 as the loading control. Relative quantification of GSDME‐N and cleaved CASP3 in placental tissue from T/S‐treated mice, as determined by Western blot. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n ≥ 3, ** p <0.01, * p <0.05. (F) Representative Immunofluorescence staining images of GSDME and Cleaved CASP3 and CD206, CD68 in placental tissue from T/S‐treated Gsdme −/− mice. Quantification of immunofluorescence signals for Cleaved CASP3 in placental tissue from T/S‐treated Gsdme −/− mice. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 3, duplicate each time, three representative fields per slide were analyzed, **** p <0.0001. Scale bars, 100 µm. (G) Representative Immunofluorescence staining images of MCT4, CD206 and CD68 in placental tissue from T/S‐treated Gsdme −/− mice. Scale bars, 50 µm. Quantification of immunofluorescence signals for CD206 and CD68 in placental tissue from T/S‐treated Gsdme −/− mice. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n = 3, duplicate each time, three representative fields per slide were analyzed, ns, not significant. (H) Immunoblots of GSDME‐FL, GSDME‐N and Cleaved CASP3 in placental tissue from T/S‐treated Gsdme −/− mice. ERK2 as the loading control. (I) Relative quantification of GSDME‐N, Cleaved CASP3 in placental tissue from T/S‐treated Gsdme −/− mice. Error bars, mean ± SEM. The data were analyzed by Student's t ‐test, n ≥ 3, ** p <0.01. (J) IL‐18 and IL‐1β level in the serum of T/S‐treated WT and T/S‐treated Gsdme −/− mice using the ELISA kits. Error bars, mean ± SEM. The data were analyzed with a one‐way ANOVA, n ≥ 3, **** p <0.0001, ns, not significant. NC, control; T/S, TNFα and SM164.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: In Vivo, Western Blot, Control, Immunofluorescence, Staining, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Caspase‐3/GSDME‐Mediated Trophoblast Pyroptosis and Reciprocal Macrophage Polarization Contribute to Inflammation in Early‐Onset Preeclampsia

doi: 10.1002/advs.202516948

Figure Lengend Snippet: Pyroptotic trophoblasts interact with and activate pro‑inflammatory macrophages, contributing to systemic inflammation. In trophoblast cells, stimulation with the apoptotic inducer T/S (TNFα + SM164) activated caspase‐3, which cleaved GSDME and triggered a switch from classical apoptosis to pyroptosis. This transition was characterized by cell swelling, membrane rupture, and the release of LDH, HMGB1, and proinflammatory cytokines IL‐1β and IL‐18. The secreted cytokines promoted the polarization of pro‐inflammatory macrophages, which in turn reinforced pyroptotic signaling in trophoblasts, amplifying systemic inflammation. Pharmacological inhibition of caspase‐3 with Z‐DEVD‐FMK or treatment with Vitamin D significantly attenuated pyroptosis associated inflammatory responses. CASP3, the activated (cleaved) form of caspase‐3.

Article Snippet: To measure human serum IL‐1β, IL‐18 and CASP3, the serum (50 μL) was extracted and detected with human human IL‐1β (Proteintech, Cat. KE00021), IL‐18 (abcam, Cat. ab215539), BAX (GILED BIOTECHNOLOGY, Cat. J20366) and CASP3 (GILED BIOTECHNOLOGY, Cat. J21110) following by the manufacturer's instructions.

Techniques: Membrane, Inhibition

Journal: Pharmaceuticals

Article Title: Aucubin from Eucommiae Cortex Alleviates Tendinopathy via an Estrogen Receptor β-Mediated Mechanism

doi: 10.3390/ph19020194

Figure Lengend Snippet: Aucubin attenuates H 2 O 2 -induced oxidative damage and inflammation in rat tenocytes, mechanisms involving the estrogen receptor pathway. ( A ) Viability of tenocytes after treatment ( n = 6). ( B ) Representative flow cytometry plots of apoptosis. ( C ) Quantitative analysis of apoptotic rates ( n = 3). ( D ) Representative micrographs of intracellular ROS levels detected by DCFH-DA staining. Scale bars: 100 μm. ( E ) Quantitative analysis of ROS fluorescence intensity ( n = 3). ( F – I ) Effects of AU on ( F ) malondialdehyde (MDA) content, ( G ) superoxide dismutase (SOD) activity, ( H ) IL-1β, and ( I ) TNF-α levels ( n = 3). Notably, the protective effects of AU on most parameters were mimicked by the estrogen agonist 17β-estradiol (E2) and antagonized by the estrogen receptor antagonist (R, R)-THC. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant were determined using specific Rat IL-1β (proteintech, Wuhan, China; Catalog: KE20005) and Rat TNF-α (proteintech, Wuhan, China; Catalog: KE20001) ELISA kits, respectively, strictly in accordance with the manufacturers’ instructions.

Techniques: Flow Cytometry, Staining, Fluorescence, Activity Assay

Journal: Pharmaceuticals

Article Title: Aucubin from Eucommiae Cortex Alleviates Tendinopathy via an Estrogen Receptor β-Mediated Mechanism

doi: 10.3390/ph19020194

Figure Lengend Snippet: Aucubin regulates the expression of proteases, oxidative stress, and cytokine markers in tendinopathy tissue. ( A , B ) Immunohistochemical (IHC) analysis of Matrix Metalloproteinase-3 (MMP-3): ( A ) representative images and ( B ) integrated optical density (IOD) quantification ( n = 6). ( C , D ) IHC analysis of Matrix Metalloproteinase-13 (MMP-13): ( C ) representative images and ( D ) IOD quantification ( n = 6). ( E , F ) IHC analysis of Cleaved Caspase-3: ( E ) representative images and (F) IOD quantification ( n = 6). ( G – J ) Effects of AU on the levels of ( G ) malondialdehyde (MDA), ( H ) superoxide dismutase (SOD) activity, ( I ) IL-1β and ( J ) TNF-α in tendon tissue ( n = 6). Scale bars: 100 μm ( A , C , E ). Data are presented as mean ± SD. ns, not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant were determined using specific Rat IL-1β (proteintech, Wuhan, China; Catalog: KE20005) and Rat TNF-α (proteintech, Wuhan, China; Catalog: KE20001) ELISA kits, respectively, strictly in accordance with the manufacturers’ instructions.

Techniques: Expressing, Immunohistochemical staining, Activity Assay

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic diagram of mice feeding and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . b–n Male AMPKα fl/fl mice and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 6 weeks were fed HFD with or without IL-1β neutralizing antibody (1 mg/kg, twice one week) to explore the obesity development. Immunofluorescent staining of IL-1β in BAT and ScWAT ( b ), body weight gain ( c , n = 5 mice), relative fat and lean mass ( d , n = 5 mice), the weight of Liver ( e , n = 5 mice), BAT ( f , n = 5 mice), and ScWAT ( g , n = 5 mice), representative H&E staining of the liver, BAT and ScWAT ( h ), insulin tolerance test ( i , n = 5 mice), the rectal temperature in cold exposure at 4 °C for different times ( j , k , n = 5 mice), immunohistochemical staining of UCP-1 in BAT ( l ), the proinflammatory genes of ScWAT ( m , Il1b, Tnfa, Nos2, Ccl2 and F4/80: n = 5 mice in each group, Il6: n = 4 mice in LysM-Cre, AMPKα fl/fl + IL-1β mAb group and n = 5 mice in other group), and the immunohistochemical staining of F4/80 in BAT and ScWAT ( n ). Data are presented as the mean ± SEM, groups were compared by two-way ANOVA followed by Fisher’s LSD test ( c – g , i – k , m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a–t Male LysM-Cre, IL-1β fl/fl mice, LysM-Cre, IL-1β fl/fl , AMPKα fl/fl mice, AMPKα fl/fl mice, IL-1β fl/fl mice, and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 8 weeks were fed HFD to explore the obesity and related phenotype. Body weight change ( a , n = 8 mice), body weight gain ( b , n = 8 mice), representative mice image ( c ), relative fat and lean mass ( d , n = 8 mice), the representative image of liver, BAT and ScWAT ( e ), the metabolic organ weight of liver ( f , n = 8 mice), BAT ( g , n = 8 mice) and ScWAT ( h , n = 8 mice), representative H&E staining of liver, BAT and ScWAT ( i ), insulin tolerance test ( j , n = 8 mice), the rectal temperature in cold exposure at 4 °C for different times ( k , l , n = 8 mice), immunohistochemical staining of UCP-1 in BAT ( m ), the proinflammatory genes of ScWAT ( n – s , n = 8 mice) and immunohistochemical staining of F4/80 in BAT and ScWAT ( t ). Data are presented as the mean ± SEM, groups were compared by one-way ANOVA followed by Fisher’s LSD test ( b , d , f – h , right of j , l , n – s ) or two-way ANOVA followed by Fisher’s LSD test ( a ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic representation of the potential substrate of AMPK in succinate formation, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . Co-immunoprecipitation analysis of the interaction of AMPKα with SUCLA2 ( b ) or SUCLG2 ( c ) in RAW 264.7 cells. d Immunoblot analysis of indicated proteins in BMDMs that are transfected with siRNA for 30 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. e Immunoblot analysis of indicated proteins in AMPKα fl/fl BMDMs (Flox) and LysM-Cre, AMPKα fl/fl BMDMs (MKO) that are transfected with siRNA for 18 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. f Immunoblot analysis of the expression of SUCLG1 and SUCLA2 after AMPKα was knocked down by siRNA for 48 h in RAW 264.7 cells. g The relative enzymatic activity of SUCLA2 (the direction from succinyl-CoA to succinate) was detected after AMPKα was knocked down by siRNA for 36 h followed by stimulation with 100 ng/mL LPS for an additional 12 h in RAW264.7 cells ( n = 3 biological replicates). h In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. i Venn diagram was used to integrate the phosphorylation site that was detected by mass spectrometry and predicted by GPS 5.0 ( http://gps.biocuckoo.cn/ ) or HPRD . j Protein sequence alignment indicated the conservation of SUCLA2 at Ser60 across multiple species. k Immunoblot analysis of indicated proteins in the in vitro kinase assay that mixed the purified His-CAMKKβ, His-AMPKα1β1γ1 with His-SUCLA2 (WT) or His-SUCLA2 (S60A). l Immunoblot analysis of indicated proteins in BMDMs after incubation with 200 µM A-769662 for 3 h. m In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 (WT) or His-SUCLA2 (S60A) in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2 ( n ) or S60A-SUCLA2 ( o ) after incubation with AMPK in vitro (n = 3 biological replicates). p The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2, S60A-SUCLA2, and S60D-SUCLA2 in the same protein content (n = 4 biological replicates). q–r The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2, S60A-SUCLA2 and S60D-SUCLA2 through lentivirus infection for 48 h followed the treatment by 100 ng/mL LPS for 6 h in the condition of glutamine replete ( q ) or deprived ( r ) condition (n = 4 biological replicates). The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2 with S60A-SUCLA2 ( s ) or S60D-SUCLA2 ( t ) through lentivirus infection for 48 h, the cells were treated with 200 µM A-769662 for 3 h in advance followed by 100 ng/mL LPS for 6 h (n = 4 biological replicates). Data are presented as the mean ± SEM, groups were compared by the unpaired two-tailed Student’s t test (g) or one-way ANOVA followed by Bonferroni’s multiple-comparisons test ( q , r ) or two-way ANOVA followed by Bonferroni’s multiple-comparisons test ( n , o , p , s , t ), representative data are shown from one of the three independent experiments ( b – f , h , k – m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay, In Vitro, Phospho-proteomics, Purification, Mass Spectrometry, Sequencing, Kinase Assay, Incubation, Gene Expression, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Immunofluorescent staining of CD68, pT 172 -AMPK, pS 60 -SUCLA2 and IL-1β in the subcutaneous adipose tissue from lean (BMI = 21.5), overweight (BMI = 25.7) or obese (BMI = 32.0) subjects, representative data are shown from one of the three independent experiments. b Model of how macrophage AMPK responds to glutaminolysis and regulates glutaminolysis-coupled IL-1β expression, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 .

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Expressing

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: The limit of detection and intra- and inter-assay coefficients (CV) of variation for the hormone and cytokine assays

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques: Inter Assay, Intra Assay

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Glycemia and serum hormone and cytokine levels at postnatal days (PND) 10, 21, 30, 50, 85, and 150 in male (M) and female (F) rats raised in litters (L) 4 or 12 pups

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques:

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Relative mRNA levels of cytokines in inguinal and perigonadal adipose tissue at postnatal days (PND) 10, 21, 30, 50, 85, and 150 in male (M) and female (F) rats raised in litters (L) 4 or 12 pups

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques:

Journal: Biology of Sex Differences

Article Title: Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens

doi: 10.1186/s13293-016-0079-5

Figure Lengend Snippet: Metabolic changes in males, females, and females that were androgenized (AF) on postnatal day (PND) 1. At PND10, there were significant differences in a the percentage of inguinal adipose tissue (IngAT) and serum b leptin, c adiponectin, and d interleukin (IL) 6 levels. At PND90, there were significant differences in e body weight, f glycemia and serum, g insulin, h leptin and i IL1β levels. The mRNA levels of j leptin, k adiponectin, l IL6, and m TNFα in perigonadal adipose tissue at PND90 were also affected. # p < 0.0001, ## p < 0.002, ### p < 0.0002, * p < 0.01, *** p < 0.005, @ p < 0.05. PND10: inguinal adipose tissue: N = 10; serum leptin and IL6 levels: N = 6; serum adiponectin: M and F: N = 8, AF: N = 9. PND90: body weight and glycemia: M: N = 8, F: N = 12, AF: N = 11; serum insulin, leptin, and Il1β levels: N = 6; mRNA levels for leptin, adiponectin, IL6, and TNFα: N = 6

Article Snippet: Assay-on-demand kits (Applied Biosystems) were used to assess the mRNA levels of leptin (Rn00565158_m1), adiponectin (Rn00595250_m1), IL6 (Rn01410330_m1), IL1β (Rn01336189_m1), and TNFα (Rn01525859_g1) quantitative real-time PCR according to the manufacturer’s protocol and analyzed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Techniques: