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: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Expressing, Western Blot, Labeling

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: The expression of TRPC3 in adrenal gland is reduced by salt loading (A) Representative image of hematoxylin and eosin (H&E) staining (left) and immunofluorescence staining (right) for CYP11B2 (red) and TRPC3 (green) in peritumoral adjacent tissue (PAT, top) and aldosterone-producing adenoma (APA, bottom) section. Nuclei were labeled with DAPI (blue). Scale bars, 50 μm. (B) The amount of 24-h urine output (left), 24-h urinary concentrations of electrolytes (middle), and serum concentrations of aldosterone (right) in mice treated with normal-salt diet (NSD) and high-salt diet (HSD) ( n = 6). (C) The amount of 24-h urine output (left), 24-h urinary concentrations of electrolytes (middle), and serum concentrations of aldosterone (right) in rats treated with NSD and HSD ( n = 6). (D) Representative images of H&E staining (left), immunohistochemical staining for CYP11B2 (middle), and immunofluorescence staining for TRPC3 (red, right) in adrenal gland sections from mice treated with NSD and HSD for 2 weeks. Scale bars, 50 μm. (E) Representative images of H&E staining (left), immunohistochemical staining for CYP11B2 (middle) and immunofluorescence staining for TRPC3 (red, right) in adrenal gland sections from rats treated with NSD and HSD for 2 weeks. Scale bars, 50 μm. (F) Representative western blots showing levels of TRPC3 and CYP11B2 in the adrenal gland (left). The quantitative results are shown in the middle (mouse) and on the right (rat) ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with NSD group by Student’s t test (B–F). C, capsule; ZG, zona glomerulosa; ZF, zona fasciculata.

Article Snippet: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Expressing, Staining, Immunofluorescence, Labeling, Immunohistochemical staining, Western Blot

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 proliferation, migration, and aldosterone secretion of adrenal cortical cell in a TRPC3-dependent manner (A) 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, arachidonic acid (AA), pyr3, and/or GSK1702934A ( n = 12). (B) Changes of mitochondrial Ca 2+ , labeled with Rhod-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with 200 μmol/L ATP stimulation in a 300 μM extracellular Ca 2+ solution after preincubation with vehicle, AA, pyr3, and/or AA + GSK1702934A ( n = 12). (C) Changes of endoplasmic reticulum 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, AA, pyr3, and/or GSK1702934A ( n = 12). (D) Representative images of Hochest 33342 (blue) and EdU staining (red) (top), transwell migration assay (middle), and wound healing assay (bottom) in primary adrenal cortical cells treated with vehicle, AA, pyr3, and/or GSK1702934A. The quantification percentage of EdU-positive cells, migrated cell numbers measured by transwell migration assay, and migration area measured by scratch wound healing assay are shown on the right ( n = 6). (E) Levels of aldosterone in the cellular supernatant of primary adrenal cortical cells treated with AA and/or short hairpin (sh)-TRPC3 ( n = 6). (F) Representative western blots showing levels of TRPC3 and CYP11B2 in primary adrenal cortical cells treated with AA and/or sh-TRPC3. The quantitative results are shown on the right ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with the vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with the AA group by one-way ANOVA (A–F).

Article Snippet: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Migration, Labeling, Staining, Transwell Migration Assay, Wound Healing Assay, Western Blot