caspase 1  (Boster Bio)


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    Structured Review

    Boster Bio caspase 1
    Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), <t>caspase-1</t> ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p
    Caspase 1, supplied by Boster Bio, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 1/product/Boster Bio
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    caspase 1 - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Terazosin Stimulates Pgk1 to Remedy Gastrointestinal Disorders"

    Article Title: Terazosin Stimulates Pgk1 to Remedy Gastrointestinal Disorders

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23010416

    Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), caspase-1 ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p
    Figure Legend Snippet: Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), caspase-1 ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p

    Techniques Used: Western Blot

    Schematic presentation illustrating the possible pathways of UC induction and targets of ulcer protection by terazosin. Terazosin activates Pgk1, followed by stimulation of Akt signaling, finally downregulates Caspase-1 to block pyroptosis.
    Figure Legend Snippet: Schematic presentation illustrating the possible pathways of UC induction and targets of ulcer protection by terazosin. Terazosin activates Pgk1, followed by stimulation of Akt signaling, finally downregulates Caspase-1 to block pyroptosis.

    Techniques Used: Blocking Assay

    2) Product Images from "Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis"

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.120.315525

    Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Small Interfering RNA, Binding Assay, Activity Assay, Double Staining, Fluorescence, FACS, Staining

    MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P
    Figure Legend Snippet: MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Techniques Used: Transfection, Binding Assay, Western Blot, Activity Assay, Staining, Fluorescence

    Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P
    Figure Legend Snippet: Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Techniques Used: Small Interfering RNA, Binding Assay, Cotransfection, Activity Assay, Staining, Fluorescence, Transfection

    Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Binding Assay, Western Blot, Staining

    PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Luciferase, Construct, Expressing, Staining, Mouse Assay, Binding Assay, Activity Assay, Double Staining, FACS, Fluorescence, MANN-WHITNEY

    3) Product Images from "Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis"

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.120.315525

    Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Small Interfering RNA, Binding Assay, Activity Assay, Double Staining, Fluorescence, FACS, Staining

    MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P
    Figure Legend Snippet: MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Techniques Used: Transfection, Binding Assay, Western Blot, Activity Assay, Staining, Fluorescence

    Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P
    Figure Legend Snippet: Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Techniques Used: Small Interfering RNA, Binding Assay, Cotransfection, Activity Assay, Staining, Fluorescence, Transfection

    Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Binding Assay, Western Blot, Staining

    PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P
    Figure Legend Snippet: PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Techniques Used: Luciferase, Construct, Expressing, Staining, Mouse Assay, Binding Assay, Activity Assay, Double Staining, FACS, Fluorescence, MANN-WHITNEY

    4) Product Images from "Decreased expression of Rev-Erbα in the epileptic foci of temporal lobe epilepsy and activation of Rev-Erbα have anti-inflammatory and neuroprotective effects in the pilocarpine model"

    Article Title: Decreased expression of Rev-Erbα in the epileptic foci of temporal lobe epilepsy and activation of Rev-Erbα have anti-inflammatory and neuroprotective effects in the pilocarpine model

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-1718-7

    The application of SR9009 for 7 days reduced the expression of NLRP3 inflammasome-related proteins and inflammatory cytokines in the hippocampus after SE. a The expression of NLRP3, ASC, Caspase1, IL-1β, IL-18, IL-6, and TNF-α was detected by western blotting assay. b Densitometric analysis results of NLRP3, ASC, Caspase1, IL-1β, IL-18, IL-6, and TNF-α in the four groups. n = 5 for each group. The data presented as the mean ± SD. NS, not significant, * p
    Figure Legend Snippet: The application of SR9009 for 7 days reduced the expression of NLRP3 inflammasome-related proteins and inflammatory cytokines in the hippocampus after SE. a The expression of NLRP3, ASC, Caspase1, IL-1β, IL-18, IL-6, and TNF-α was detected by western blotting assay. b Densitometric analysis results of NLRP3, ASC, Caspase1, IL-1β, IL-18, IL-6, and TNF-α in the four groups. n = 5 for each group. The data presented as the mean ± SD. NS, not significant, * p

    Techniques Used: Expressing, Western Blot

    5) Product Images from "Protection of Anthocyanin from Myrica rubra against Cerebral Ischemia-Reperfusion Injury via Modulation of the TLR4/NF-κB and NLRP3 Pathways"

    Article Title: Protection of Anthocyanin from Myrica rubra against Cerebral Ischemia-Reperfusion Injury via Modulation of the TLR4/NF-κB and NLRP3 Pathways

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23071788

    Effect of PAEs from Dongkui Myrica rubra on protein expression: ( A ) the expressions of Toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), and β-actin; ( B ) the expressions of nuclear erythroid 2-related factor 2 (Nrf2), caspase-1, NOD-like receptor pyrin domain-containing 3 protein (NLRP3), IL-18, and β-actin. The quantifications of ( A ) and ( B ) are shown in ( C – H ). All of the data are shown as mean ± SD ( n = 10); * p
    Figure Legend Snippet: Effect of PAEs from Dongkui Myrica rubra on protein expression: ( A ) the expressions of Toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), and β-actin; ( B ) the expressions of nuclear erythroid 2-related factor 2 (Nrf2), caspase-1, NOD-like receptor pyrin domain-containing 3 protein (NLRP3), IL-18, and β-actin. The quantifications of ( A ) and ( B ) are shown in ( C – H ). All of the data are shown as mean ± SD ( n = 10); * p

    Techniques Used: Expressing

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    Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), <t>caspase-1</t> ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p
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    Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), caspase-1 ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Terazosin Stimulates Pgk1 to Remedy Gastrointestinal Disorders

    doi: 10.3390/ijms23010416

    Figure Lengend Snippet: Terazosin inhibits cell apoptosis and pyroptosis in the Caco-2 cells. Terazosin influence on the levels of proteins p-AKT ( A ), NF-κB p65 ( B ), p-IKBα ( C ), caspase-1 ( D ), and GSDMD ( E ) in Caco-2 cells were verified by Western blot analysis. All data are represented as Mean ± SEM, # p

    Article Snippet: Terazosin (TZ, purity ≥ 98%) and Dextran Sulfate sodium (DSS, MW~40kDa) were purchased from Aladdin Biotechnology Co., Ltd.(Shanghai, China); 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were from Solarbio Life Science (Beijing, China); Primary rabbit antibodies against Pgk1, caspase-1, GSDMD, p-AKT (T450), and β-actin were purchased from BOSTER Biological Technology Co., Ltd. (Beijing, China); Antibodies against NF-κB p65 (3033) and p-IKBα (5209) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot

    Schematic presentation illustrating the possible pathways of UC induction and targets of ulcer protection by terazosin. Terazosin activates Pgk1, followed by stimulation of Akt signaling, finally downregulates Caspase-1 to block pyroptosis.

    Journal: International Journal of Molecular Sciences

    Article Title: Terazosin Stimulates Pgk1 to Remedy Gastrointestinal Disorders

    doi: 10.3390/ijms23010416

    Figure Lengend Snippet: Schematic presentation illustrating the possible pathways of UC induction and targets of ulcer protection by terazosin. Terazosin activates Pgk1, followed by stimulation of Akt signaling, finally downregulates Caspase-1 to block pyroptosis.

    Article Snippet: Terazosin (TZ, purity ≥ 98%) and Dextran Sulfate sodium (DSS, MW~40kDa) were purchased from Aladdin Biotechnology Co., Ltd.(Shanghai, China); 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were from Solarbio Life Science (Beijing, China); Primary rabbit antibodies against Pgk1, caspase-1, GSDMD, p-AKT (T450), and β-actin were purchased from BOSTER Biological Technology Co., Ltd. (Beijing, China); Antibodies against NF-κB p65 (3033) and p-IKBα (5209) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Blocking Assay

    Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    doi: 10.1161/ATVBAHA.120.315525

    Figure Lengend Snippet: Circ-Calm4 small-interfering RNA (siRNA) inhibits pyroptosis in pulmonary artery smooth muscle cells (PASMCs). A and B , Circ-Calm4 siRNA reversed the increased protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) induced by hypoxia in PASMCs (n=6). C , Circ-Calm4 siRNA reversed the increased LDH (lactate dehydrogenase) activity in PASMCs subjected to hypoxia for 24 h. LDH release was evaluated with an LDH release kit (n=6). D , Knockdown of circ-Calm4 decreased the number of pyroptotic cells in PASMCs exposed to hypoxia. Cells were detected with annexin V-FITC/propidium iodide (PI) double staining using quantitative fluorescence-activated cell sorting (FACS) analysis. E , Knockdown of circ-Calm4 by siRNA reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). F , Circ-calm4 siRNA attenuated the fluorescence staining of Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph A-ASC and Caspase-1 were analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Article Snippet: Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C.

    Techniques: Small Interfering RNA, Binding Assay, Activity Assay, Double Staining, Fluorescence, FACS, Staining

    MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    doi: 10.1161/ATVBAHA.120.315525

    Figure Lengend Snippet: MiR-124-3p suppresses pyroptosis in pulmonary arterial smooth muscle cells (PASMCs). A and B , In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β); in contrast, AMO-124-3p increased these protein and mRNA levels (n=6; in Western blot, the image of IL-1β and IL-18 used the same actin.). C–E , Transfection of the miR-124-3p mimic ameliorated pyroptosis-related phenotypes upon hypoxia treatment, whereas AMO-124-3p exacerbated these phenotypes. LDH (lactate dehydrogenase) activity (n=6; C ). Propidium iodide (PI)-positive stained cells. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue) ( D ). Fluorescence staining for Caspase-1 and IL-18. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining ( E ). Scale bars=100 µm. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Article Snippet: Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C.

    Techniques: Transfection, Binding Assay, Western Blot, Activity Assay, Staining, Fluorescence

    Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    doi: 10.1161/ATVBAHA.120.315525

    Figure Lengend Snippet: Circ-Calm4 sponges miR-124-3p to mediate pulmonary arterial smooth muscle cell (PASMC) pyroptosis. A and B , Knockdown of endogenous miR-124-3p by AMO-124-3p abrogated the antipyroptotic effects of Circ-calm4 silencing by Circ-calm4-small-interfering RNA (siRNA) upon hypoxia exposure in PASMCs, as indicated by the protein and mRNA levels of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β; n=6). C , Cotransfection with circ-Calm4 siRNA and AMO-124-3p abrogated the antipyroptotic effects on PASMCs upon hypoxia exposure, as indicated by LDH (lactate dehydrogenase) activity (n=6). D , Knockdown of circ-Calm4 by siRNA reduced the positive propidium iodide (PI) staining induced by hypoxia in cells, whereas AMO-124-3p reversed the decrease in PI-positive PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). E , Circ-Calm4 knockdown blocked the fluorescence intensity of Caspase-1 and IL-18 under hypoxia exposure, and this decrease was abrogated after transfection of AMO-124-3p. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. * P

    Article Snippet: Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C.

    Techniques: Small Interfering RNA, Binding Assay, Cotransfection, Activity Assay, Staining, Fluorescence, Transfection

    Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    doi: 10.1161/ATVBAHA.120.315525

    Figure Lengend Snippet: Circ-Calm4 inhibits pyroptosis in a mouse model of hypoxia-induced pulmonary hypertension (PH). A and B , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment ),IL-18 (interleukin-18), and IL-1β (interleukin-1β) at the protein and mRNA levels (n=6; in Western blot, the image of NLPR3 and Caspase-1 used the same actin). C and D , Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxia-induced upregulation of Caspase-1 and IL-18 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Caspase-1 (green) and IL-18 (green), pulmonary smooth muscle stained with α-SMA (red), and DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction. The graph B-ASC was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Article Snippet: Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C.

    Techniques: Binding Assay, Western Blot, Staining

    PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

    doi: 10.1161/ATVBAHA.120.315525

    Figure Lengend Snippet: PDCD6 (programmed cell death protein 6) is a downstream target gene of miR-124-3p. A , The target genes of miR-124-3p, as predicted by the TargetScan, PicTar, StarBase, miRDB and miRanda databases. B , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or Mut Pdcd6 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=6). C , The expression of Pdcd6 in different cells (n=6). D , Upregulation of Pdcd6 in hypoxic pulmonary arterial smooth muscle cells (PASMCs) relative to normal cells. Scale bars=100 µm. Cells were stained for PDCD6 (green), and DAPI (blue) was used for nuclear staining. E , Upregulation of PDCD6 in hypoxic mice. Scale bars=100 µm. Lung sections were stained for PDCD6 (green), pulmonary smooth muscle was stained for α-SMA (red), and DAPI was used for nuclear staining. F and G , Si- Pdcd6 countered the hypoxia-induced upregulation of Caspase-1, NLRP3 (nucleotide-binding oligomerization segment-like receptor family 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment segment), IL-18 (interleukin-18), and IL-1β (interleukin-1β) expression at the protein and mRNA levels in PASMCs (n=6). H , Si-PDCD6 reversed the hypoxia-induced upregulation of LDH activity in PASMCs (n=6). I , Knockdown of Pdcd6 decreased the number of pyroptotic PASMCs upon hypoxia exposure. Cells were analyzed by annexin V-FITC/propidium iodide (PI) double staining using quantitative FACS analysis. J , Pdcd6 silencing reduced the positive PI staining induced by hypoxia in PASMCs. Scale bars=100 µm. Images of fluorescence staining with PI (red) and Hoechst 33342 (blue). K , Si-PDCD6 attenuated the fluorescence staining for Caspase-1 and IL-18 induced by hypoxia in PASMCs. Scale bars=100 µm. Cells were stained for Caspase-1 (green) and IL-18 (red), and DAPI (blue) was used for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means±SD. Statistical analysis was performed with 1-way ANOVA followed by Bonferroni correction and Student t test for 2 means. The graph D was analyzed by the Mann-Whitney U test and the graph F-IL-1β, the graph G- Ii-1β was analyzed by the Kruskal-Wallis test followed by Dunn post-test. * P

    Article Snippet: Cells were incubated with an anti-α-SMA antibody (4 µg/mL; BOSTER), an anti-Caspase-1 antibody (10 µg/mL; BOSTER), an anti-Caspase-3 antibody (5.4 µg/mL; Proteintech), an anti-Caspase-9 antibody (4 µg/mL Beyotime Biotechnology, Shanghai, China), anti-Ki-67 antibody (10 µg/mL; BOSTER), anti-Cleaved Caspase-3 (5 µg/mL;Cell Signaling Technology), and an anti-IL-18 antibody (7.6 µg/mL; Proteintech) overnight at 4 °C and were subsequently incubated with FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibodies for 2 hours at 37 °C.

    Techniques: Luciferase, Construct, Expressing, Staining, Mouse Assay, Binding Assay, Activity Assay, Double Staining, FACS, Fluorescence, MANN-WHITNEY