Fig 1: (A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm2 vs. MCOPPB group, 30.6 ± 1.1 μm2, (p = 0.20, 3 plates/group). Representative images are shown (×200).
Fig 2: (A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm2 vs. MCOPPB group, 30.6 ± 1.1 μm2, (p = 0.20, 3 plates/group). Representative images are shown (×200).
Fig 3: (A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm2 vs. MCOPPB group, 30.6 ± 1.1 μm2, (p = 0.20, 3 plates/group). Representative images are shown (×200).
Fig 4: (A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm2 vs. MCOPPB group, 30.6 ± 1.1 μm2, (p = 0.20, 3 plates/group). Representative images are shown (×200).
Fig 5: (A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm2 vs. MCOPPB group, 30.6 ± 1.1 μm2, (p = 0.20, 3 plates/group). Representative images are shown (×200).
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