Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Effects of Klotho (100 pM) and selective TRPC6 inhibitor (BI-749327, 1 μM) on ionic currents of transient receptor potential canonical channel (I TRPC ) in human atrial fibroblasts. ( A ) The current tracings and I–V relationship of I TRP showed that BI-749327-treated fibroblasts (1 μM, 48 h, n = 13–15 from 4 independent experiments), Klotho-treated fibroblasts (100 pM, 48 h, n = 15 from 3 independent experiments), and combined Klotho and BI-749327 ( n = 15 from 3 independent experiments) had similarly lower peak inward currents and similarly lower peak outward currents compared with the control ( n = 13 from 4 independent experiments). ( B ) Western blots showed a similar expression of TRPC6 and total phospholipase C (PLC) between fibroblasts treated with Klotho (100 pM, 48 h) and control fibroblasts. However, the phosphorylated PLC (p-PLC) expression was lower in the fibroblasts treated with Klotho (100 pM, 48 h) ( n = 3 from 3 independent experiments). ( C , D ) Example photographs and average data ( n = 9 from 3 independent experiments) showed that human atrial fibroblasts treated with BI-749327 (1 μM, 48 h), Klotho (100 pM, 48 h), and combined Klotho and BI-749327 exhibited similarly less migratory capability than the control. Arrows indicate migrating fibroblasts in the wound space. GAPDH was used as a loading control. PLCβ3, phospholipase C beta 3; pPLCβ3, phosphorylated phospholipase C beta 3; TRPC6, transient receptor potential canonical channel 6. * p < 0.05, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Control, Western Blot, Expressing

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Effects of Klotho (100 pM) and selective TRPC6 inhibitor (BI-749327, 1 μM) on ionic currents of transient receptor potential canonical channel (I TRPC ) in human atrial fibroblasts. ( A ) The current tracings and I–V relationship of I TRP showed that BI-749327-treated fibroblasts (1 μM, 48 h, n = 13–15 from 4 independent experiments), Klotho-treated fibroblasts (100 pM, 48 h, n = 15 from 3 independent experiments), and combined Klotho and BI-749327 ( n = 15 from 3 independent experiments) had similarly lower peak inward currents and similarly lower peak outward currents compared with the control ( n = 13 from 4 independent experiments). ( B ) Western blots showed a similar expression of TRPC6 and total phospholipase C (PLC) between fibroblasts treated with Klotho (100 pM, 48 h) and control fibroblasts. However, the phosphorylated PLC (p-PLC) expression was lower in the fibroblasts treated with Klotho (100 pM, 48 h) ( n = 3 from 3 independent experiments). ( C , D ) Example photographs and average data ( n = 9 from 3 independent experiments) showed that human atrial fibroblasts treated with BI-749327 (1 μM, 48 h), Klotho (100 pM, 48 h), and combined Klotho and BI-749327 exhibited similarly less migratory capability than the control. Arrows indicate migrating fibroblasts in the wound space. GAPDH was used as a loading control. PLCβ3, phospholipase C beta 3; pPLCβ3, phosphorylated phospholipase C beta 3; TRPC6, transient receptor potential canonical channel 6. * p < 0.05, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Western Blot, Expressing

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: I TRP of human atrial fibroblasts in 4 different groups (control; Klotho 100 pM, 48 h; U73122 1 μM, 48 h; Klotho 100 pM plus U73122 1 μM, 48 h). The current tracings ( A ) and I–V relationship ( B ) of I TRPC6 of human atrial fibroblasts in 4 groups ( n = 16 from 5 independent experiments). ( C ) Fibroblasts in the control group had higher peak inward currents and outward currents than the other 3 groups ( n = 14–18 from 5–7 independent experiments). ( D ) Western blots showed similar expression of TRPC6 among the four groups. ( n = 14 from 3 independent experiments). GAPDH was used as a loading control. * p < 0.05, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Western Blot, Expressing

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Proposed mechanism of action of Klotho in the modulation of ion channels and intracellular calcium handling in human atrial fibroblasts. Klotho inhibits the activation of the PLC-IP 3 signaling and suppresses thapsigargin-induced ER Ca 2+ release, and subsequently attenuates SOCE by inhibition of TRPC6 currents. In addition, Klotho may also inhibit the PLC-DAG pathway and negatively modulate the inward currents of TRPC6. PIP 2 , phosphatidylinositol biphosphate; PLC, phospholipase C; DAG, diacylglycerol; IP 3 , inositol 1,4,5-triphosphate; IP 3 R, IP 3 receptor; ER, endoplasmic reticulum; STIM1, stromal interaction molecule 1; SERCA, sarcoendoplasmic reticulum calcium transport ATPase; TG, thapsigargin.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Activation Assay, Inhibition

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Effects of Klotho (100 pM) and selective TRPC6 inhibitor (BI-749327, 1 μM) on ionic currents of transient receptor potential canonical channel (I TRPC ) in human atrial fibroblasts. ( A ) The current tracings and I–V relationship of I TRP showed that BI-749327-treated fibroblasts (1 μM, 48 h, n = 13–15 from 4 independent experiments), Klotho-treated fibroblasts (100 pM, 48 h, n = 15 from 3 independent experiments), and combined Klotho and BI-749327 ( n = 15 from 3 independent experiments) had similarly lower peak inward currents and similarly lower peak outward currents compared with the control ( n = 13 from 4 independent experiments). ( B ) Western blots showed a similar expression of TRPC6 and total phospholipase C (PLC) between fibroblasts treated with Klotho (100 pM, 48 h) and control fibroblasts. However, the phosphorylated PLC (p-PLC) expression was lower in the fibroblasts treated with Klotho (100 pM, 48 h) ( n = 3 from 3 independent experiments). ( C , D ) Example photographs and average data ( n = 9 from 3 independent experiments) showed that human atrial fibroblasts treated with BI-749327 (1 μM, 48 h), Klotho (100 pM, 48 h), and combined Klotho and BI-749327 exhibited similarly less migratory capability than the control. Arrows indicate migrating fibroblasts in the wound space. GAPDH was used as a loading control. PLCβ3, phospholipase C beta 3; pPLCβ3, phosphorylated phospholipase C beta 3; TRPC6, transient receptor potential canonical channel 6. * p < 0.05, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Control, Western Blot, Expressing

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Cell migration, collagen production, and proliferation capabilities in human atrial fibroblasts treated with and without Klotho. ( A ) Example photographs and average data ( n = 10 from 3 independent experiments) showed that human atrial fibroblasts treated with Klotho at a high concentration (100 pM) exhibited less migratory capability than the control. Arrows indicate migrating fibroblasts in the wound space. ( B ) Example photographs and average data revealed that lower procollagen type IA1 expression ( n = 3 from 3 independent experiments) and lower procollagen type III expression ( n = 3 from 3 independent experiments) in human atrial fibroblasts treated with a high concentration of Klotho (100 pM). The expression of α-smooth muscle actin (α-SMA) was comparable in both groups ( n = 3 from 3 independent experiments). GAPDH was used as a loading control. ( C ) Treatment with both lower and high concentrations of Klotho (10 and 100 pM) for 48 h had no significant effect on the proliferation rate of human atrial fibroblasts. ( n = 6 from 3 independent experiments). GAPDH, glyceraldehyde 3-phosphate dehydrogenase protein. * p < 0.05.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Migration, Concentration Assay, Expressing

Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Cell migration and collagen production in human atrial fibroblasts in 4 different groups (control; Klotho 100 pM, 48 h; U73122 1 μM, 48 h; Klotho 100 pM plus U73122 1 μM, 48 h). ( A ) Example photographs and average data ( n = 9 from 3 independent experiments) showed the cell migration of human atrial fibroblasts in 4 different groups. Arrows indicate migrating fibroblasts in the wound space. ( B ) Example photographs and average data revealed the expression of procollagen type IA1, procollagen type III, total PLC, p-PLC, and α-SMA in human atrial fibroblasts in 4 groups ( n = 12–14 from 3 independent experiments). GAPDH was used as a loading control. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Migration, Expressing