Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 1. Myeloid deficiency of Piezo1 re- duces AS development (A) Representative hematoxylin and eosin (H&E)- stained proximal aortas and quantification of atherosclerotic lesion area in aortic sinuses from lethally irradiated Ldlr/ mice transplanted with bone marrow (BM) derived from control (LysMCre+tdTomatoSTOPfl/+; BM WT) and myeloid- specific Piezo1-deficient (LysMCre+tdTomatoSTOPfl/+

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Staining, Irradiation, Derivative Assay, Control

Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 2. Piezo1 potentiation by chronic 7-KC in native mouse BMDMs (A) Mechanosensitive inward mean currents re- corded in the cell-attached configuration at holding potential 80 mV in bone marrow differentiated macrophages (BMDMs) from WT and KO mice (non-polarized, M0) and elicited by fast negative pressure pulses of D 10 mm Hg (bottom traces). After7daysofdifferentiationwithM-CSF,cellswere treated for 24 h in LDL-free medium with EtOH (vehicle, in black) or 10 mM 7-KC (in red). Right inset: histogram of peak current amplitude (maximal cur- rentat60mmHg)inEtOH(inblack)or7-KC(inred) condition, comparing WT and KO macrophages. (B) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at 80 mV in BMDMs polarized to M1. BMDMs M0 cells were first polarized to M1 in complete medium supplemented with LPS (10 ng/ mL) and IFN-g (20 ng/mL) cocktail for 24 h, and then treated with either EtOH (black traces) or 10 mM 7-KC (in red) for an additional 24 h in LDL- free medium. Left inset: M1 polarization was confirmed by TNFa gene expression and is repre- sented as relative expression to TOP1. Right inset: peak current amplitude in EtOH (in black) or 7-KC (in red) condition, comparing WT and KO macro- phages. (C) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at 80 mV in BMDMs polarized to M2. BMDM M0 cells were first polarized to M2 in complete medium supplemented with IL-4 (20 ng/ mL) and IL-13 (20 ng/mL) cocktail for 24 h, and then treated with either EtOH (black traces) or 10 mM 7-KC (in red) for an additional 24 h in LDL-free medium. Left inset: M2 polarization was confirmed by CD206 gene expression and is represented as relative expression to TOP1. Right insets: peak current amplitude in EtOH (in black) or 7-KC (in red) condition, comparing WT and KO macrophages. n, number of individual recordings are shown above the traces. N = 2, number of animals from each genotype used for isolation of bone marrow mesenchymal stem cells and differentiation to macrophages. Same mice as donor mice in Fig- ure 1. Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Membrane, Gene Expression, Expressing, Isolation, MANN-WHITNEY

Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 3. oxLDL and 7-KC enhance Piezo1 currents in transfected HEK Piezo1del cells (A) Mechanosensitive inward mean currents recorded in the cell-attached configuration at holding potential 80 mV in HEK Piezo1 knockout cells (HEK Piezo1del) exogenously transfected with Piezo1 together with mCherry and elicited by fast negative pressure pulses of D 10 mm Hg (bottom traces). Cells were chronically treated with either 50 mg/mL LDL (left panel, in black) or 50 mg/mL oxLDL (right panel, in red) and cultured for 24 h in LDL-free medium. (B) Pressure-effect curves of inward currents recorded at –80 mV in HEK Piezo1del from LDL-free (black symbols), LDL (empty symbols), and oxLDL (red symbols). P0.5 values (in mm Hg) are 66.9, 59.2, and 50.3, respectively. k values are 17.7, 15.5, and 17.1, respectively. n values are 10, 27, and 26, respectively. (C) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at 80 mV in HEK Piezo1del transfected with Piezo1 and mCherry. Cells were treated for 24 h in LDL-free medium with EtOH (vehicle, black traces) or 7-KC (10 mM in red). Inset: lack of a stretch-activated mean current confirmed in mCherry only transfected HEK Piezo1del and treated with 10 mM 7-KC for 24 h. (D) Pressure-effect curves of inward currents recorded at –80 mV in HEK Piezo1del from control EtOH-(in black) and 7-KC-treated cells (2.5 mM in orange, and 10 mM in red). P0.5 values (in mm Hg) are 44.1, 55.6, and 58.8, respectively. k values are 7.7, 11.6, and 18.7, respectively. n values are 47, 22, and 50, respectively. Data are represented as mean ± SEM. A Mann-Whitney U test was used for statistical analysis. *p < 0.05, ****p < 0.0001. n, number of individual recordings are shown above the traces. (E) Inside-out Piezo1 currents elicited by pressure pulses at a holding potential of 80 mV in the presence of EtOH or after a chronic treatment for 24 h with 7-KC (10 mM). (F) Lack of effect of acutely applied 7-KC (5, 10, and 25 mM), while Yoda1 (5 mM) enhances pressure-induced Piezo1 currents recorded at 80 mV in response to a repetitive 40 mm Hg pressure pulse (inset). (G) Membrane expression of Piezo1-GFP after a chronic treatment with either EtOH or 10 mM 7-KC for 24 h. Scale bars, 5 mm. (H) Membrane/total fluorescence in the presence of either EtOH or 7-KC. (I) Single Piezo1 channel current amplitude after a chronic treatment with either EtOH or 10 mM 7-KC for 24 h recorded at 80 mV without pressure stimulation (representative traces, left panel, and histogram, right panel). Mann-Whitney U test was used for statistical analysis. Data are represented as means ± SEM. The difference is not significant.

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Transfection, Knock-Out, Cell Culture, Membrane, Control, MANN-WHITNEY, Expressing

Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 5. Potentiation of Piezo1 by 7-KC is prevented by high cholesterol (A) Effect of membrane cholesterol enrichment (orange traces) on mean inward currents in RAW M0 cells recorded in the cell-attached patch configuration at 80 mV and elicited by increasing negative pressure pulses (bottom black traces). Cells were chronically treated for 24 h in LDL-free medium either with EtOH (vehicle) or 10 mM 7-KC and then incubated in the external solution (without serum) containing 120 mg/mL water-sol- uble cholesterol for 45 min at 37C. Cells were extensively washed 3 times before recording. (B) Same for HEK Piezo1del transfected with Piezo1. (C) Effect of membrane cholesterol depletion (MbCD, 5 mM, blue trace) on mean inward cur- rents in Piezo1 transfected HEK Piezo1del cells recorded in the cell-attached patch configuration at 80 mV and elicited by negative pressure pulse of 60 mm Hg (bottom black trace). (D) Peak current amplitude (I peak) and (E) time constants of inactivation (t) at 60 mm Hg pres- sure pulse in cell-attached configuration from HEK Piezo1del transfected with Piezo1. Two protocols for cell treatment were used: (1) 24 h in LDL-free medium with EtOH (black bars) or 7-KC (10 mM, red bars), (2) 45 min at 37C in external solution without serum (black bars, control) enriched either with water-soluble cholesterol (120 mg/mL, orange bars) or MbCD (5 mM, blue bars). Data are rep- resented as means ± SEM. A Mann-Whitney U test was used for statistical analysis. *p < 0.05, ***p < 0.001, ****p < 0.0001. n, number of individual recordings are shown above the traces.

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Membrane, Incubation, Transfection, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 6. 7-KC causes a decrease in membrane order Cells were incubated with 10 mM 7-KC/EtOH for 24 h (LDL-depleted serum) followed by NR12S staining and imaged using confocal microscopy. (A, C, and E) The GP maps of BMDMs, RAW, and HEK Piezo1del cells treated with EtOH (top panel) and 7-KC (bottom panel), respectively. Scale bars, 5 mm. (B, D, and F) The graphical representation of GP values (EtOH, black bars; 7-KC, red bars). The number of BMDMs analyzed were: M0 EtOH (n = 20), M0 7-KC (n = 31), M1 EtOH (n = 19), M1 7-KC (n = 31), M2 EtOH (n = 41), M2 7-KC (n = 43). The number of RAW macrophages analyzed were: M0 EtOH (n = 30), M0 7-KC (n = 32), M1 EtOH (n = 22), M1 7-KC (n = 20), M2 EtOH (n = 27), M2 7-KC (n = 28). The number of Piezo1 transfected HEK Piezo1del cells were: EtOH (n = 39) and 7-KC (n = 39). Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis with ****p < 0.0001.

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Membrane, Incubation, Staining, Confocal Microscopy, Transfection, MANN-WHITNEY

Journal: Cell reports

Article Title: Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol.

doi: 10.1016/j.celrep.2025.115542

Figure Lengend Snippet: Figure 7. Free DHA inhibits Piezo1 in the absence or in the presence of 7-KC (A) Typical recording of BMDMs M0 in the excised inside-out patch configuration at 80 mV and elicited by a fast negative pressure pulse of 40 mm Hg (bottom black trace). Control (black), acute application of DHA (green), washout (gray). (B) Same from RAW M0. Inset: histograms of peak current amplitude (DHA at 10 mM, for RAW and BMDMs, respectively). (C) Same for HEK Piezo1del expressing Piezo1 either in the absence of 10 mM 7-KC (left panel) or in the presence of 7-KC (middle panel, red trace). Histogram on the right panel shows peak current amplitude in the different experimental conditions, including 7-KC and DHA, as indicated. In the 7-KC condition (in red, without DHA) three values (1,523; 1,102, and 843 pA) are off scale. (D) Kinetic of current inhibition by acutely applied DHA (1, 3, 5, and 10 mM) in a HEK Piezo1del transfected cell with Piezo1 and recorded in the excised outside-out patch configuration at a holding potential of +60 mV and in response to a +30 mm Hg pressure stimulation.

Article Snippet: This was followed by incubatingwith Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1- AP) in PBS-HS for 2 h at 37 C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBSHS for 1 h at 37 C. In control experiments only the secondary antibody was included.

Techniques: Control, Expressing, Inhibition, Transfection

Journal: The Journal of surgical research

Article Title: Diosmetin protects against ischemia/reperfusion-induced acute kidney injury in mice.

doi: 10.1016/j.jss.2017.02.067

Figure Lengend Snippet: Fig. 3 e Diosmetin suppresses the NF-kB signaling pathway after renal I/R injury. (A) Representative images of immunohistochemical staining of p65 in kidneys from different groups. Magnification: 4003. Bar [ 100 mm. Red arrow denotes p65 staining of a nucleus. (B) Quantitative assessment of p65 expression in the kidney. (C) Western blotting showed protein levels of p65, phosphorylated-p65 (p-p65), IkBa, and phosphorylated-IkBa (p-IkBa). GAPDH was used as a loading control. (D-G) Quantification of NF-kB pathway-related proteins. (H) Real-time qPCR analysis of inflammatory cytokines following I/R. Values are expressed as the mean ± SE (n [ 6 mice each group).*P < 0.05 versus sham; #P < 0.05 versus I/R. SE [ standard error; qPCR [ quantitative polymerase chain reaction; GAPDH [ glyceraldehyde-3-phosphate dehydrogenase. (Color version of figure is available online.)

Article Snippet: After incubationwith the primary antibody against NFkB p65 (Abcam, Cambridge, MA) at 4 C overnight, the sections were washed and then incubated with horseradish peroxidase-conjugated antirabbit secondary antibody for 30 min at room temperature.

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction