Fig 1: Schematic illustration of propionic acid-activated FFAR3 signaling and function in cardiac myocytes and in sympathetic neurons, and the inhibitory role of RGS4 in both tissues. See text for details. SNS: Sympathetic nervous system.
Fig 2: RGS4 and FFAR3 signaling in cardiac myocytes. (A) A 1 mM propionate-induced p38 MAPK phosphorylation/activation in the control (scrambled siRNA-transfected, Scr) or in RGS4-depleted (RGS4 siRNA) H9c2 cells. A representative blot is shown on the left and the densitometric quantitation on the right. Prop: Propionate. *, p < 0.05; n = 3 independent experiments performed in triplicate. (B,C) A 1 mM propionate-induced IL-1ß and IL-6 (B) or TGF-ß1 (C) protein synthesis in these cells. *, p < 0.05, vs. respective Scr; n = 3 independent measurements per condition per cell clone.
Fig 3: ßARs negatively regulate FFAR3 signaling via RGS4 and PKA in cardiac myocytes. (A) Gia activation in native H9c2 cells treated with 1 mM propionate, 10 µM isoproterenol (Iso), 10 µM salbutamol (Salb), or a combination thereof. *, p < 0.05, vs. basal (no treatment); NS: Not significant (vs. basal-No treatment) at p = 0.05; n = 3 independent measurements (in duplicate) per condition. (B) Gia activation in RGS4-depleted (RGS4 siRNA) or in native H9c2 cells pre-treated with 10 µM H89 (H89), in response to 1 mM propionate alone, or to 1 mM propionate in the presence of 10 µM isoproterenol (Iso) or 10 µM salbutamol (Salb). *, p < 0.05, vs. respective basal (no treatment); NS: Not significant (vs. respective propionate alone) at p = 0.05; n = 3 independent determinations (in duplicate) per condition per cell clone.
Fig 4: RGS4 and neuronal FFAR3-mediated NE release. (A) Levels of NE released into the culture medium from the control (infected with a lentivirus encoding for green fluorescent protein, LentiGFP) or RGS4-overexpressing (infected with a lentivirus encoding for full length RGS4, LentiRGS4) differentiated Neuro-2A (N2a) cells in response to 1 mM of propionate stimulation. No statistically significant difference in NE release was observed between the two cell clones under basal conditions, i.e., without propionate stimulation (data not shown). *, p < 0.05; n = 5 independent measurements per condition per cell clone. (B) ßAR density (Bmax) in plasma membranes isolated from H9c2 cardiomyocytes at 24 hrs post-stimulation with 1 mM propionate (Prop) or vehicle (Basal) of co-cultured control, wild-type (WT) or RGS4-overexpressing (RGS4 OE) N2a cells. The ßAR density measured with vehicle-treated RGS4-overexpressing N2a cells (Basal/RGS4 OE N2a) was similar to the one calculated with Basal/WT N2a cells (not shown). *, p < 0.05, vs. Basal/WT N2a; #, p < 0.05 vs. Prop/WT N2a; n = 3 independent determinations per condition per cell clone.
Fig 5: RGS4 and FFAR3-dependent Gi/o protein activation in cardiac myocytes. (A) Immunoblotting for RGS4 to confirm the efficiency of the siRNA-mediated knockdown of RGS4 in H9c2 cells at 48 hrs post-siRNA transfection. A representative blot, including for glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) as a loading control, is shown on the left, and the % protein reduction, based on densitometric analysis of 4 independent experiments, is shown on the right. Scr: Scrambled siRNA (control); *, p < 0.05; n = 4 independent experiments per condition. (B) A 1 mM propionic acid-induced Gia activation in control (scrambled siRNA-transfected, Scr), or in RGS4-depleted (RGS4 siRNA) H9c2 cells. *, p < 0.05; n = 4 independent experiments. (C) A 1 mM propionic acid-mediated inhibition of 10 µM forskolin (FSK)-induced cAMP accumulation in the same cells, expressed as a % of the inhibition observed in the control (Scr) H9c2 cardiac cells. Forskolin alone induced similar levels of cAMP accumulation in both cell clones (data not shown). *, p < 0.05; n = 4 independent experiments.
from Charles River for Lenti-CMV-RGS4v1, lentiviral particles
Titer: >10^8 IU/mL