circ-calm4  (Genechem)

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

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

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

Techniques: Binding Assay, Western Blot, Staining

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 is a mediator that regulates miR-124-3p action. A , The target microRNAs (miRNAs) of circ-Calm4, as predicted by the miRanda Count, RNA hybrid Count and RegRNA 2.0 databases. B , Knockdown of circ-Calm4 by small-interfering RNA increased the miR-124-3p mRNA level in pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia for 24 h (n=6). C , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or mutant (Mut) circ-Calm4 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=5). D , Colocalization of circ-Calm4 and miR-124-3p in PASMCs. Scale bars=100 µm. MiR-124-3p probes were labeled with FAM (green), whereas circ-Calm4 probes were labeled with Cy3 (red). Nuclei were stained with DAPI (blue). Pearson coefficient is 0.77809, indicating a correlation. E , Downregulation of miR-124-3p in PASMCs exposed to hypoxia for 24 h relative to miR-124-3p expression in nontreated cells (n=8). F , Relative fluorescence of miR-124-3p in PASMCs exposed to hypoxia. Scale bars=100 µm. miR-124-3p probes were labeled with FAM (green). Nuclei were stained with DAPI (blue). 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. * P< 0.05, **P< 0.01, *** P< 0.001.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

Techniques: Small Interfering RNA, Luciferase, Construct, Mutagenesis, Labeling, Staining, Expressing, Fluorescence

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

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

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 inhibits pyroptosis in pulmonary arterial smooth muscle cells (PASMCs) through the miR-124-3p/PDCD6 (programmed cell death protein 6) pathway. A and B , Knockdown of endogenous circ-Calm4 abrogated upregulation of PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia (n=6). C and D , MiR-124-3p reversed but AMO-124-3p enhanced the upregulation of PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia (n=6). E and F , In PASMCs, knockdown of circ-Calm4 reduced PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia, whereas AMO-124-3p reversed the downregulation of PDCD6 protein and Pdcd6 mRNA expression (n=6). G and H , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of PDCD6 at the protein and mRNA levels (n=6). I , Circ-Calm4 expression was positively correlated with Pdcd6 mRNA expression in hypoxic mice. In hypoxic mice, circ-Calm4 expression was negatively correlated with miR-124-3p expression, and Pdcd6 mRNA expression was negatively correlated with miR-124-3p expression. 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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

Techniques: Small Interfering RNA, Expressing

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: A schematic diagram to illustrate the hypothetical model Circ-Calm4, as a competitive endogenous RNA, adsorbed miR-124-3p via a circular RNA sponging mechanism and abolished the suppression of Pdcd6 (programmed cell death protein 6) by miR-124-3p, leading to pyroptosis, proliferation, and apoptosis resistance. PASMC indicates pulmonary arterial smooth muscle cell.

Article Snippet: Cy3-labeled circ-Calm4 probes and FAM-labeled miR-124-3p probes were designed by GeneChem (Shanghai, China).

Techniques:

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

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

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

Techniques: Binding Assay, Western Blot, Staining

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 is a mediator that regulates miR-124-3p action. A , The target microRNAs (miRNAs) of circ-Calm4, as predicted by the miRanda Count, RNA hybrid Count and RegRNA 2.0 databases. B , Knockdown of circ-Calm4 by small-interfering RNA increased the miR-124-3p mRNA level in pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia for 24 h (n=6). C , HEK293 cells were cotransfected with a luciferase reporter construct carrying wild-type (WT) or mutant (Mut) circ-Calm4 and miR-124-3p or miR-124-3p-NC. Luciferase activities were measured via a dual luciferase assay (n=5). D , Colocalization of circ-Calm4 and miR-124-3p in PASMCs. Scale bars=100 µm. MiR-124-3p probes were labeled with FAM (green), whereas circ-Calm4 probes were labeled with Cy3 (red). Nuclei were stained with DAPI (blue). Pearson coefficient is 0.77809, indicating a correlation. E , Downregulation of miR-124-3p in PASMCs exposed to hypoxia for 24 h relative to miR-124-3p expression in nontreated cells (n=8). F , Relative fluorescence of miR-124-3p in PASMCs exposed to hypoxia. Scale bars=100 µm. miR-124-3p probes were labeled with FAM (green). Nuclei were stained with DAPI (blue). 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. * P< 0.05, **P< 0.01, *** P< 0.001.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

Techniques: Small Interfering RNA, Luciferase, Construct, Mutagenesis, Labeling, Staining, Expressing, Fluorescence

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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

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

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 inhibits pyroptosis in pulmonary arterial smooth muscle cells (PASMCs) through the miR-124-3p/PDCD6 (programmed cell death protein 6) pathway. A and B , Knockdown of endogenous circ-Calm4 abrogated upregulation of PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia (n=6). C and D , MiR-124-3p reversed but AMO-124-3p enhanced the upregulation of PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia (n=6). E and F , In PASMCs, knockdown of circ-Calm4 reduced PDCD6 protein and Pdcd6 mRNA expression induced by hypoxia, whereas AMO-124-3p reversed the downregulation of PDCD6 protein and Pdcd6 mRNA expression (n=6). G and H , Knockdown of circ-Calm4 by sh-circ-Calm4 countered the hypoxia-induced upregulation of PDCD6 at the protein and mRNA levels (n=6). I , Circ-Calm4 expression was positively correlated with Pdcd6 mRNA expression in hypoxic mice. In hypoxic mice, circ-Calm4 expression was negatively correlated with miR-124-3p expression, and Pdcd6 mRNA expression was negatively correlated with miR-124-3p expression. 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< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

Techniques: Small Interfering RNA, Expressing

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: A schematic diagram to illustrate the hypothetical model Circ-Calm4, as a competitive endogenous RNA, adsorbed miR-124-3p via a circular RNA sponging mechanism and abolished the suppression of Pdcd6 (programmed cell death protein 6) by miR-124-3p, leading to pyroptosis, proliferation, and apoptosis resistance. PASMC indicates pulmonary arterial smooth muscle cell.

Article Snippet: The RNA complex was pulled down by incubating the cell lysates with Biotin-labeled circ-calm4 probes (GeneChem Shanghai, China) at 37 °C for 3 hours and streptavidin beads for 30 minutes.

Techniques: