Journal: iScience

Article Title: The role of arachidonic acid metabolites in the subtype classification and pathogenesis of primary aldosteronism

doi: 10.1016/j.isci.2025.114598

Figure Lengend Snippet: Arachidonic acid promotes Ca 2+ uptake and increases CYP11B2 expression in adrenal cortical cells (A) Confirmation of the expression of CYP11B2 in primary adrenal cortical cells. Scale bars, 50 μm. (B) Cell viability of primary adrenal cortical cells treated with different doses of arachidonic acid (AA) ( n = 4). (C) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of AA ( n = 6). (D) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of Ang II (left) or endothelin-1 (right) along with AA ( n = 3). (E) Representative western blots showing levels of CYP11B2, CYP11B1, CYP17A1, and HSD3B2 in primary adrenal cortical cells treated with vehicle or AA. The quantitative results are shown on the right ( n = 3). (F) Changes of cytoplasmic Ca 2+ , labeled with Fura-2 AM, in primary adrenal cortical cells treated with 1 mM thapsigargin (TG) stimulation in a 1 mM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (G) Changes of mitochondrial Ca 2+ , labeled with Rhod-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with 200 μM ATP stimulation in a 300 μM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (H) Changes of endoplasmic reticulum (ER) Ca 2+ , labeled with Mag Fura-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with ATP stimulation in a Ca 2+ -free extracellular solution after preincubation with vehicle or AA ( n = 12). (I) Representative western blots showing levels of KCNJ5, Na + /K + ATPase alpha-1 subunit, NCX-1, Letm 1, MCU, VDAC, RyR2, and IP 3 R in primary adrenal cortical cells treated with vehicle or AA. (Left) Representative western blots showing levels of TRPC1, TRPC3, TRPC6, TRPV1, and TRPV4 in primary adrenal cortical cells treated with vehicle or AA. (Right) The quantitative results are shown on the left ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with 1 μM AA group by one-way ANOVA (B and C) and by Student’s t test (D–I).

Article Snippet: TRPC6 , Alomone , Cat#ACC-017; RRID: AB_2040243.

Techniques: Expressing, Western Blot, Labeling

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: Endogenous expression of TRPC6 in human IVD cells. (A) Basal mRNA expression of TRPC6 in primary IVD cells (n=17), presented as ΔCt values normalized to YWHAZ. (B) TRPC6 mRNA expression following 18 h treatment with Hyp9 (1 μM), expressed as fold change relative to vehicle control (red dashed line, n=17; Mann-Whitney test; #p < 0.0001). (C) Western blot analysis of TRPC6 (∼110 kDa) in cells treated with vehicle (0 μM) or Hyp9 (1 μM, 18 h); α-tubulin (52 kDa) serves as the loading control. (E) Densitometric quantification of TRPC6 protein levels across donors (n=4), presented as fold change relative to vehicle control (red dashed line, set to 1.0; Mann–Whitney test, p = 0.31). Data are presented as mean ± SEM; n denotes biological replicates.

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Expressing, Control, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: Hyp9-mediated TRPC6 activation induces dose-dependent Ca 2+ influx in human IVD cells. (A) Real-time Ca 2+ influx kinetics following acute Hyp9 stimulation (0.5–100 μM). (B) Peak Ca 2+ (ΔF/F 0 ) with nonlinear regression (EC 50 = 83.06 μM; R 2 = 0.95). (C) Total Ca 2+ influx quantified as area under the curve (AUC). (D) Time to peak Ca 2+ influx. All influx measurements were performed using the Fura-2 QBT assay on the FlexStation 3 platform, and values represent mean ± SEM (n = 3; biological replicates). Statistical significance (B-D) was assessed using Friedman test followed by Dunn’s multiple comparisons test, with comparisons made relative to DMSO control (*p < 0.05, **p < 0.01).

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Activation Assay, Control

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: TRPC6 activation induces and sustains inflammatory gene expression and protein secretion in human IVD cells. (A) Transcriptional profiling of inflammatory markers after 18 h of Hyp9 (1 μM) treatment. COX-2, IL-6, and IL-8 mRNA levels are presented as fold change relative (2 −ΔΔCt ) to vehicle control (dashed line, n= 17). (B-C) Time-dependent secretion of IL-6 (B) and IL-8 (C) protein into the culture media, quantified by ELISA and normalized to total DNA content presented as fold change relative to vehicle control (dashed line, n=5). (D-F) Longitudinal gene expression analysis of COX-2 (D), IL-6 (E), and IL-8 (F) over 18, 24, and 48 h, demonstrating sustained transcriptional activation (n=5, symbols matched with protein secretion). Data are presented as mean ± SEM, with individual donor points; n denotes biological replicates. Statistical significance (**p<0.01, #p<0.0001 vs. vehicle control) was determined by the Mann-Whitney test compared to vehicle control.

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Activation Assay, Gene Expression, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: TRPC6 activation elicits selective collagenolytic and aggrecan-associated transcriptional remodeling in human IVD cells. (A) Transcriptional profiling of Collagenolytic MMP markers after 18 h of Hyp9 (1 μM) treatment. MMP-1, MMP-2, MMP-3, and MMP-13 mRNA levels are presented as fold change (2 −ΔΔCt ) relative to vehicle control (dashed line, n= 17). (B-C) Time-dependent secretion of MMP-1 (B) and MMP-3 (C) protein into the culture media, quantified by ELISA and normalized to total DNA content, presented as fold change relative to vehicle control (dashed line, n=5). (D-F) Longitudinal gene expression analysis of MMP-1 (D), MMP-2 (E) MMP-3 (F), and MMP-13 (G) over 18, 24, and 48 h, demonstrating sustained transcriptional activation. (Symbols represent matched biological donors across time points; n = 5). (H) Aggrecan (ACAN) and aggrecanase gene expression following 18 h of Hyp9 treatment under serum-free conditions, including ACAN, ADAMTS4, and ADAMTS5, presented as fold change relative to vehicle control (n = 17). (I–K) Temporal transcriptional regulation of ACAN (I), ADAMTS4 (J), and ADAMTS5 (K) under serum-free conditions following Hyp9 treatment at 18, 24, and 48 h (n = 5). Data are presented as mean ± SEM with individual donor points; n denotes biological replicates. Statistical significance (**p<0.01, ***p<0.001, #p<0.0001 vs. vehicle control) was determined by the Mann-Whitney test compared to vehicle control.

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: TRPC6 activation modestly modulates neurogenic and angiogenic mediators in human IVD cells. (A) Transcriptional profiling of neuro-angiogenic markers after 18 h of Hyp9 (1 μM) treatment. NGF, BDNF, and VEGF mRNA levels are presented as fold change (2 −ΔΔCt ) relative to vehicle control (dashed line, n= 17). (B-C) Time-dependent secretion of NGF (B) and VEGF (C) protein into the culture media following Hyp9 treatment, quantified by ELISA under serum-replete (10%) conditions and normalized to total DNA content, presented as fold change relative to vehicle control (dashed line, n=5). (D-F) Longitudinal gene expression analysis of NGF (D), BDNF (E), and VEGF (F) over 18, 24, and 48 h, illustrating distinct temporal regulation patterns among neurotrophic and angiogenic mediators (n=5, symbols matched with protein secretion). Data are presented as mean ± SEM with individual donor points; n denotes biological replicates. Statistical significance (**p<0.01, #p<0.0001 vs. vehicle control) was determined by the Mann-Whitney test compared to vehicle control.

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: TRPC6-Mediated Ca 2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells

doi: 10.64898/2026.02.07.704609

Figure Lengend Snippet: TRPC6 activation recruits MAPK and NF-κB signaling pathways in human IVD cells. Human IVD cells were serum-starved and treated with Hyp9 (1 μM) or the positive control TNF-α (10 ng/mL) for 15 or 30 minutes. (A, D) Representative Western blot and densitometric quantification of ERK1/2 activation. (B, E) Representative Western blot and quantification of p38 MAPK activation, showing a robust increase in phosphorylation (p-p38/Total-p38). (C, F) Representative Western blot images and densitometric analysis of the NF-κB inhibitor IκB-α, showing rapid degradation following stimulation. (G) Representative immunofluorescence images showing the nuclear translocation of the NF-κB subunit p65 (green) following stimulation (Scale bar = 50 μm). Data are presented as fold change relative to vehicle control (dashed line); mean ± SEM, n=4; biological replicates. Statistical significance (∗p < 0.05 vs. vehicle control) was determined by Mann-Whitney test compared to vehicle control.

Article Snippet: Cells were blocked as described above and incubated with primary TRPC6 antibody (1:50, Alomone Labs, Jerusalem, Israel; ACC-120) overnight at 4 °C, followed by Alexa Fluor® 488-conjugated donkey anti-rabbit secondary antibody (1:500; Abcam, AB150073).

Techniques: Activation Assay, Protein-Protein interactions, Positive Control, Western Blot, Phospho-proteomics, Immunofluorescence, Translocation Assay, Control, MANN-WHITNEY