Journal: iScience

Article Title: CCRR regulate MYZAP-PKP2-Nav1.5 signaling pathway in atrial fibrillation following myocardial infarction

doi: 10.1016/j.isci.2024.111102

Figure Lengend Snippet: PKP2 is an upstream regulator of TLR2/TLR4/Nav1.5 (A–C) The protein expressions of TLR2, TLR4 and Nav1.5 were detected by western blot. Compared with Sham group, the expressions of TLR2 and TLR4 were upregulated and the expression of Nav1.5 was downregulated in MI group. Compared with AAV-NC group, overexpression of AAV-PKP2 decreased the protein expression of TLR2 and TLR4, and restored the protein expression of Nav1.5 (A n = 7 mice/group, B n = 6 mice/group, C n = 5 mice/group). (D and F) The expression level of TLR2 in atrial tissue of mice overexpressing AAV-PKP2 was detected by immunofluorescence. (Sham, MI n = 21, +AAV-PKP2 n = 13; +AAV-NC n = 19 visions). Red represents TLR2, green represents α-actinin, and blue represents the nucleus. Scale bar = 10 μm. (E and G) The expression level of TLR4 in atrial tissue of mice overexpressing AAV-PKP2 was detected by immunofluorescence. (Sham n = 41, MI n = 21, +AAV-PKP2 n = 37, +AAV-NC n = 41 visions). Red represents TLR4, green represents α-actinin, and blue represents the nucleus. Scale bar = 10 μm. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ## p < 0.01, and ### p < 0.001. && p < 0.01 and &&& p < 0.001 (One-way ANOVA).

Article Snippet: The antibodies used were PKP2 (1:500, abs136897, Absin, China), TLR2 (1:2000, DF7521, Affinity, America), TLR4 (1:2000, 66350-1, Proteintech, America), MYZAP (1:2000, PA5-21116, Invitrogen, America), CX40 (1:1000, DF13633, Affinity, America), CX43 (1:1000, 26980-1, Proteintech, America), Nav1.5 (1:200, ASC-005, Alomone labs, Israel), Cav1.2 (1:200, ACC-003, Alomone labs, Israel), Kv4.2 (1:200, APC-023, Alomone labs, Israel), β-actin(1:3000, A00702, GenScript, America), β-tubulin(1:2000, WL01931, Wanleibio, China) and GAPDH (1:2000, BA002, Bio-Platform, China).

Techniques: Western Blot, Expressing, Over Expression, Immunofluorescence

Journal: iScience

Article Title: CCRR regulate MYZAP-PKP2-Nav1.5 signaling pathway in atrial fibrillation following myocardial infarction

doi: 10.1016/j.isci.2024.111102

Figure Lengend Snippet: Knockdown of PKP2 enhances inflammation after MI via TLR2/TLR4/Nav1.5 pathway (A and B) Western blot (A) and Real-time PCR (B) were used to detect the changes of protein and mRNA expression of PKP2 in atrial cardiomyocytes after transfection of si-PKP2 sequence (A n = 6/group, B n = 8/group). (C–F) The expression of TLR2 and TLR4 protein (C and E) and mRNA (D and F) increased after PKP2 knockdown (C and E n = 6/group, D and F n = 8/group). (G–J) The mRNA expression levels of IL-6, IL-1β, TNF-α and MCP1 increased after PKP2 knockdown in atrial cardiomyocytes (n = 8/group). (K) CCK8 detected a significant decrease in cell viability after PKP2 knockdown (n = 6/group). (L and M) Patch-clamp results showed that the loss of PKP2 resulted in a significant decrease in sodium current density (∗∗ p < 0.01 vs. Ctl, ## p < 0.01 vs. NC, n = 6/group). (N and O) Voltage-dependent steady-state activation (N) and inactivation (O) of Na + channel were not changed by PKP2 knockdown. (N, n = 6/group, O, n = 8/group). Data are presented as mean ± SEM. ∗∗ p < 0.01, and ∗∗∗ p < 0.001. # p < 0.05, ## p < 0.01, and ### p < 0.001 (One-way ANOVA).

Article Snippet: The antibodies used were PKP2 (1:500, abs136897, Absin, China), TLR2 (1:2000, DF7521, Affinity, America), TLR4 (1:2000, 66350-1, Proteintech, America), MYZAP (1:2000, PA5-21116, Invitrogen, America), CX40 (1:1000, DF13633, Affinity, America), CX43 (1:1000, 26980-1, Proteintech, America), Nav1.5 (1:200, ASC-005, Alomone labs, Israel), Cav1.2 (1:200, ACC-003, Alomone labs, Israel), Kv4.2 (1:200, APC-023, Alomone labs, Israel), β-actin(1:3000, A00702, GenScript, America), β-tubulin(1:2000, WL01931, Wanleibio, China) and GAPDH (1:2000, BA002, Bio-Platform, China).

Techniques: Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Sequencing, Patch Clamp, Activation Assay

Journal: iScience

Article Title: CCRR regulate MYZAP-PKP2-Nav1.5 signaling pathway in atrial fibrillation following myocardial infarction

doi: 10.1016/j.isci.2024.111102

Figure Lengend Snippet: PKP2 alleviates inflammatory response after MI by regulating the TLR2/TLR4/Nav1.5 pathway (A and B) Changes of TLR2 protein and mRNA expression after PKP2 overexpression in atrial myocytes (A n = 5/group; B n = 4/group). +PKP2: Hypoxia+PKP2, +NC: Hypoxia+NC. (C and D) Changes of TLR4 protein and mRNA expression after PKP2 overexpression in atrial cardiomyocytes (C n = 3/group; D Ctl n = 3; Hypoxia, +PKP2, +NC n = 4/group). (E) Co-IP results verified the protein interaction between PKP2 and TLR2, TLR4 (n = 3/group). (F–I) After PKP2 overexpression, the mRNA expression levels of IL-6, IL-1β, TNF-α and MCP1 were significantly decreased (n = 8/group). (J) Cell viability decreased after hypoxia, and PKP2 overexpression increased cell viability compared with NC group (n = 8/group). (K and L) Sodium current density was detexted by patch-clamp. Compared with Ctl group, the sodium current density of atrial cardiomyocytes was significantly decreased after 12 h hypoxia. Compared with the NC group, the density of sodium channel was significantly increased in the PKP2 group (∗∗∗ p < 0.001 vs. Ctl, ### p < 0.001 vs. Hypoxia, &&& p < 0.001 vs. NC, n = 8/group). (M and N) Voltage-dependent steady-state activation (M) and inactivation (N) of Na + channel were unaltered by PKP2 overexpression (n = 8/group). Data are presented as mean ± SEM. ∗∗ p < 0.01, and ∗∗∗ p < 0.001. # p < 0.05, ## p < 0.01, and ### p < 0.001. & p < 0.05, && p < 0.01, and &&& p < 0.001 (One-way ANOVA).

Article Snippet: The antibodies used were PKP2 (1:500, abs136897, Absin, China), TLR2 (1:2000, DF7521, Affinity, America), TLR4 (1:2000, 66350-1, Proteintech, America), MYZAP (1:2000, PA5-21116, Invitrogen, America), CX40 (1:1000, DF13633, Affinity, America), CX43 (1:1000, 26980-1, Proteintech, America), Nav1.5 (1:200, ASC-005, Alomone labs, Israel), Cav1.2 (1:200, ACC-003, Alomone labs, Israel), Kv4.2 (1:200, APC-023, Alomone labs, Israel), β-actin(1:3000, A00702, GenScript, America), β-tubulin(1:2000, WL01931, Wanleibio, China) and GAPDH (1:2000, BA002, Bio-Platform, China).

Techniques: Expressing, Over Expression, Co-Immunoprecipitation Assay, Patch Clamp, Activation Assay

Journal: iScience

Article Title: CCRR regulate MYZAP-PKP2-Nav1.5 signaling pathway in atrial fibrillation following myocardial infarction

doi: 10.1016/j.isci.2024.111102

Figure Lengend Snippet:

Article Snippet: The antibodies used were PKP2 (1:500, abs136897, Absin, China), TLR2 (1:2000, DF7521, Affinity, America), TLR4 (1:2000, 66350-1, Proteintech, America), MYZAP (1:2000, PA5-21116, Invitrogen, America), CX40 (1:1000, DF13633, Affinity, America), CX43 (1:1000, 26980-1, Proteintech, America), Nav1.5 (1:200, ASC-005, Alomone labs, Israel), Cav1.2 (1:200, ACC-003, Alomone labs, Israel), Kv4.2 (1:200, APC-023, Alomone labs, Israel), β-actin(1:3000, A00702, GenScript, America), β-tubulin(1:2000, WL01931, Wanleibio, China) and GAPDH (1:2000, BA002, Bio-Platform, China).

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Immunoprecipitation, SYBR Green Assay, Software, Sequencing

Journal: Innate immunity

Article Title: Mouse estrous cycle regulation of vaginal versus uterine cytokines, chemokines, α-/β-defensins and TLRs

doi: 10.1177/1753425912454026

Figure Lengend Snippet: Real time PCR primers were obtained from Applied Biosciences using TaqMan® expression assay pre-designed gene primer and probe sets. The table shows gene target and Applied Biosystems order identification. The relative expression of each gene is normalized to Beta Actin Control.

Article Snippet: 18 table ft1 table-wrap mode="anchored" t5 caption a7 Antimicrobials gene Applied Biosystems # Toll-like receptors gene Applied Biosystems # α-Defensin 1 Mm00655851_gH TLR1 Mm00446095_m1 α-Defensin 2 Mm00651548_g1 TLR2 Mm00440346_m1 α-Defensin 5 Mm00432803_m1 TLR3 Mm00446577_g1 β-Defensin 1 Mm00657074_m1 TLR4 Mm00445274_m1 β-Defensin 2 Mm0161469_m1 TLR5 Mm00546288_s1 β Defensin 3 Mm00731768_m1 TLR6 Mm01208943_s1 β-Defensin 4 Mm00441527_m1 TLR7 Mm00446590_m1 Beta Actin (Control) Mm00607939_s1 TLR8 Mm01157262_m1 TLR9 Mm00446193_m1 TLR11 Mm01701924_s1 TLR12 Mm01180208_s1 TLR13 Mm0123318_m1 Open in a separate window Real time PCR primers were obtained from Applied Biosciences using TaqMan® expression assay pre-designed gene primer and probe sets.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 5. oxLDL induces uPAR association with TLR4. A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Immunoprecipitation, Immunocytochemistry, Staining, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 6. oxLDL-induced cytokine expression is mediated by NF-κB. A. VSMC were lentivirus-infected to express Gaussia luciferase under control of NF-κB responsive promoter as described in the Materials and methods section. 24 h after infection cells were transfected with SiCo or other siRNA as indicated. 24 h after transfection cells were stimulated with 5 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. B. HEK 293 cells were lentiviral infected to express Gaussia luciferase under control of NF-κB responsive promoter. Expression of uPAR was achieved by lentiviral infection. TLR4 and CD36 expression was achieved by cell transfection. Cells were stimulated with 8 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. C. Cells were stimulated with oxLDL in the presence of proteasome inhibitors. G-CSF and GM-CSF expression was assessed by RT-PCR. D. oxLDL- induced G-CSF and GM-CSF expression in the presence of NF-κB inhibitor BAY-11-7085 was assessed by RT-PCR. E. Primary human monocytes and macrophages were treated with conditioned medium of SiCo- and uPARsi-transfected VSMC stimulated with 8 μg/ml oxLDL. MCP-1 release was followed using Human CCL2/MCP-1 Quantikine ELISA Kit.* indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Expressing, Infection, Luciferase, Control, Transfection, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 7. In vivo evidence for uPAR-mediated events. A. Images captured from sub-fibrous cap area of the plaque stained for CD36 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. B. Images captured from sub-fibrous cap area of the plaque stained for TLR4 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. Scale bar 100 μm. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: In Vivo, Staining