primescripttm iv 1st strand cdna synthesis mix  (TaKaRa)

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF effectively improves endothelial function (A) Hypoxia (1 % O 2 )-impaired HUVEC proliferation (CCK-8 assay) was rescued by PF (400 μM) at 48 h (n = 6). (B-D) Matrigel tube formation assay. (B) Representative images showing PF restored capillary-like structures under hypoxia. (C) Branching points and (D) total tube length quantified (n = 3). Scale bar: 100 μm. (E–K) PF modulated endothelial regulators in renal tissues. (E-G) eNOS, ANG-II and ET-1 mRNA by RT-qPCR. (H) Representative immunoblots and (I-K) quantification of eNOS, ANG-II and ET-1 protein (n = 5–6). (L–M) Serum levels of nitric oxide (NO, L) (n = 4) and ANG-II (M) (n = 6) detected by ELISA. Data presented as mean ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL/Hypoxia; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CRF/ Hypoxia + PF.

Article Snippet: The RNA was then reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Kit (6215A, TAKARA) according to the manufacturer's instructions.

Techniques: CCK-8 Assay, Tube Formation Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF attenuates Yoda1-induced Ca 2+ influx via functional modulation of Piezo1 channels (A) Immunofluorescence co-localization of Piezo1 (green) and PECAM-1 (red) in glomerular endothelium. Nuclei: DAPI (blue). Scale bar: 20 μm. (B-D) Renal Piezo1 expression analysis. (B) Piezo1 mRNA levels by RT-qPCR. (C) Representative Western blot bands. (D) Quantitative protein analysis normalized to GAPDH. (n = 5–6) (E–F) Intracellular Ca 2+ flux in HUVECs. Fluo-3 AM (5 μM)-labeled cells pre-treated with PF (400 μM, 1 h) or vehicle, then stimulated with Yoda1 (5 μM). PF significantly attenuated Yoda1-induced Ca2 + influx. Images captured via confocal microscopy (λex/λem: 488/530 nm). Scale bar: 20 μm. (G–H) Quantification of baseline (K) and Peak relative value (L) (n = 6). (I-J) Molecular docking of PF to Piezo1. (I) 3D structure showing hydrogen bonds (yellow dashes) with CYS977/PHE984/LYS988/LYS1154 and ARG1295. (J) 2D interaction map highlighting hydrophobic contacts and polar interactions. (K-L) Surface plasmon resonance (SPR) analysis. (K) Binding kinetics showing KD = 2.44 μM. (L) Sensorgrams of concentration-dependent binding.(M−Q) EndMT marker expression in HUVECs. (M) Western blot analysis of (N) eNOS, (O) VE-cadherin, (P) Vimentin, and (Q) TGF-β1. Quantification normalized to GAPDH (n = 3). Data presented as mean ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL/Yoda1; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CRF. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The RNA was then reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Kit (6215A, TAKARA) according to the manufacturer's instructions.

Techniques: Functional Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Confocal Microscopy, SPR Assay, Binding Assay, Concentration Assay, Marker

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF inhibits matrix stiffness-induced acceleration of EndMT through Piezo1 activation (A–F) Matrix stiffness-dependent protein modulation in HUVECs. HUVECs were cultured on polyacrylamide hydrogels with soft (1.00 ± 0.31 kPa) or stiff (40.40 ± 2.39 kPa) for 48 h, with or without PF. (A) Western blot analysis of (B) Piezo1, (C) VE-Cadherin, (D) eNOS, (E) Vimentin, and (F) TGF-β1. Quantification normalized to GAPDH. (n = 3; *p < 0.05, **p < 0.01 vs. soft; #p < 0.05 vs. stiff without PF). (G-L) Piezo1 knockdown reverses stiffness-induced EndMT. HUVECs transfected with Piezo1 siRNA (50 nM, 24 h) or scramble siRNA (control) were cultured on stiffness hydrogels (40.40 ± 2.39 kPa) ± PF (400 μM). (G) Western blot analysis of (H) Piezo1, (I) VE-Cadherin, (J) eNOS, (K) Vimentin, and (L) TGF-β1. Quantification normalized to GAPDH (n = 3; *p < 0.05, **p < 0.01 vs. scramble siRNA control; ns vs. Piezo1 siRNA without PF).(M) Schematic of co-culture model. HUVECs and NRK-49F fibroblasts were co-cultured on stiffness-tunable hydrogels using a transwell system (0.4 μm pore size) for 5 days to assess paracrine signaling. (N) TGF-β1 levels in HUVEC supernatants from the co-culture system were quantified by ELISA. (n = 3; *p < 0.05 vs. softness; #p < 0.05 vs. stiffness without PF). (O) Fibrotic gene expression in NRK-49F cells. RT-qPCR analysis of Fibronectin , COL1A1 , Vimentin , and TGF-β1 mRNA levels. Data normalized to 18 s ( n = 3). (P-S) Fibrotic protein expression in NRK-49F cells. (P) Western blot analysis of (Q) Fibronectin, (R) COL1, (S) Vimentin, and (T) TGF-β1. Quantification normalized to GAPDH (n = 3). (U–V) Immunofluorescence of Fibronectin (red) in NRK-49F cells. Nuclei stained with DAPI (blue). (U) Representative images. (V) Quantification of fluorescence intensity using ImageJ (n = 3). Scale bar: 20 μm. Data presented as mean ± SEM. *p < 0.05, **p < 0.01 vs . softness co-culture; # p < 0.05 vs . stiffness co-culture without PF . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The RNA was then reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Kit (6215A, TAKARA) according to the manufacturer's instructions.

Techniques: Activation Assay, Cell Culture, Western Blot, Knockdown, Transfection, Control, Co-Culture Assay, Pore Size, Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Advanced Research

Article Title: Direct pharmacological targeting of Piezo1 by Paeoniflorin: a novel therapeutic approach for renal fibrosis

doi: 10.1016/j.jare.2025.07.015

Figure Lengend Snippet: PF inhibits EndMT through the Piezo1-mediated HIF-1α signaling pathway (A–C) Renal HIF-1α expression analysis. (A) Representative RT-qPCR analysis of HIF-1α mRNA levels in kidney tissues. Data normalized to 18 s . (B-C) Western blot and quantification of HIF-1α protein expression in renal tissues. Data normalized to GAPDH (n = 5–6) . (D–H) Effects of PF or HIF-1α inhibitor BAY 87-2243 (10 μM, 24 h) on endothelial markers in HUVECs cultured with or without Piezo1 activation by Yoda1 (5 μM, 12 h). (D) Western blot analysis of (E) Piezo1, (F) HIF-1α, (G) VE-Cadherin, and (H) eNOS. Quantification normalized to GAPDH. Quantification showing Yoda1-induced Piezo1 upregulation and HIF-1α/VE-Cadherin/eNOS downregulation, reversed by PF or BAY 87-2243 (n = 3). (I-K) PF or BAY 87-2243 inhibits Yoda1-induced EndMT in HUVECs. (I) Western blot analysis of (J) Vimentin and (K) TGF-β1. Yoda1 increased Vimentin and TGF-β1, suppressed by PF or BAY 87-2243 (n = 3). (L) Schematic of HUVEC-NRK-49F co-culture. HUVECs pre-treated with/without Yoda1 (5 μM, 6 h) were co-cultured with NRK-49F fibroblasts for 48 h. (M) TGF-β1 levels in HUVEC supernatants from the co-culture system were quantified by ELISA. (N) RT-qPCR analysis of Fn1 , COL1A1 , and Vimentin mRNA in NRK-49F cells co-cultured with Yoda1-treated HUVECs. PF attenuated Yoda1-induced fibrotic marker expression (n = 3). (O-S) Western blot validation of (P) Fibronectin, (Q) COL1, (R) Vimentin, and (S) TGF-β1 in NRK-49F cells. PF reduced Yoda1-induced protein expression (n = 3). Data presented as mean ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL/control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CRF/Yoda1.

Article Snippet: The RNA was then reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Kit (6215A, TAKARA) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Marker, Biomarker Discovery, Control