trpc1  (Alomone Labs)

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: TRPC1 , Alomone , Cat#ACC-010; RRID: AB_2040234.

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: 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: TRPV1 , Alomone , Cat#ACC-030; RRID: AB_2313819.

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: 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: 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

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: TRPV4 , Alomone , Cat#ACC-034; RRID: AB_2040264.

Techniques: Expressing, Western Blot, Labeling

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: The RUSH system applied to integrin α5. (A) Principles of the RUSH-α5 integrin. In all experiments, SBP-EGFP-ITGA5 (RUSH-α5) is co-expressed with streptavidin-KDEL (ER-hook). In the absence of biotin, this combined complex is retained within the ER. Biotin addition displaces the ER-hook and releases RUSH-α5 into the cytoplasm. (B) The number of contacts between EGFP and FN during simulations of the coarse-grained model. Left: simulation of EGFP being pulled towards the FN-binding site, starting when the C-terminus of the EGFP and the N-terminus of the integrin α5 are <1 nm apart, the linker included, leading to the formation of contacts . Right: simulation of a fully stretched EGFP, initially in close proximity to the FN-binding site, that is allowed to relax without a biasing force resulting in a spontaneous and rapid loss of contacts (<100 ns; ). The pulling process spanned 8 nm and 80 ns. The relaxation spanned 3200 ns. Contacts were calculated between EGFP and FN with a cutoff of 0.6 nm. (C and D) RUSH-α5 is expressed on the cell surface and forms a functional heterodimer with integrin β1. (C) Representative flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. (D) Representative immunoblots of GFP pulldowns performed in RUSH-α5 or control transfected cells ± biotin treatment for the indicated times and probed for endogenous integrin β1. The faster migrating band of immature integrin β1 is indicated by a green arrow and box and the slower migrating band of mature integrin β1 with a magenta arrow and box. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Binding Assay, Functional Assay, Flow Cytometry, Expressing, Western Blot, Control, Transfection

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery to the plasma membrane is spatially regulated by the ECM. (A) Model of RUSH-α5 (EGFP-integrin α5)–integrin-β1 heterodimer based on the structure of human α5β1-integrin bound to FN (PDB: 7NWL ) (see also ). (B) Representative immunofluorescence timelapse images of a U2OS cell expressing RUSH-α5 (SBP-EGFP-integrin α5) and plated on FN ± biotin treatment (single grayscale images for the indicated time points are shown; see ). (C) Representative immunofluorescence images of RUSH-α5 and active integrin α5β1 (SNAKA51 antibody) in RUSH-α5–expressing U2OS cells plated on FN or collagen + biotin (60 min). Grayscale single-channel images and merged images (white, colocalization; blue, nuclei [DAPI]) are shown. (D) Representative images of RUSH-α5 and RUSH-CD59 release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. Intensity line profiles generated across the yellow line are displayed relative to the position of the FN-coated micropattern lines. White insets represent regions of interest (ROIs) that are magnified for each channel. FN, fibronectin.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Expressing, Construct, Labeling, Generated

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of RUSH-α5–expressing U2OS cell plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 recruitment to adhesions is ligand-dependent. (A) Representative immunoblot of GFP pulldowns performed in RUSH-α5 or control transfected cells plated on FN or collagen and probed for endogenous integrin β1 and for GFP. Mature (magenta arrow) and immature (green arrow) integrin β1 are indicated. (B) Quantification of the relative fraction of mature to immature integrin β1 interacting with RUSH-α5 ± biotin treatment for the indicated times. N = 6 independent experiments; data are mean ± SD, One-way ANOVA, Dunn’s multiple comparison test, no significant difference between FN and collagen at all time points. (C) Representative images (see ) of U2OS cells co-expressing RUSH-α5 and RUSH-CD59 and plated on FN (top) or collagen (bottom) ± biotin treatment for the indicated times. Insets represent ROIs that are magnified. Scale bars: 20 µm. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Western Blot, Control, Transfection, Comparison, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS cells co-expressing RUSH-α5 (green) and RUSH-CD59 (magenta) and plated on FN (left, 10 µg/ml) or collagen (right, 10 µg/ml), biotin added after acquisition of time point 0 min. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery to FAs. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 and pmKate2-Paxillin plated on FN or collagen ± biotin treatment for the indicated times. Insets represent ROIs that are magnified and show paxillin-segmented adhesions (red outlines). (B) Quantification of the relative mean intensity of RUSH-α5 in segmented adhesions/cell ± biotin treatment for the indicated times. Data are mean ± SD; n = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments; One-way ANOVA, Holm-Šídák’s multiple comparison test; data distribution was assumed to be normal but this was not formally tested. (C) TIRF imaging of U2OS cells expressing RUSH-α5-pHluorin on a FN-coated surface after biotin release at T = 0. The arrows indicate exocytosis events. Exocytosis events were detected by performing a ratiometric analysis, which consisted of dividing each frame by the previous. All detected events before 19 min of release are indicated in red. The graph indicates the distance between the exocytosis events and the nearest FA segmented on the last frame of (37 min after release), compared to the distance of random dots to FAs, showing that the localization to FAs is not random. Individual measurements and the mean ± SD are represented. Unpaired T test. RUSH-α5-pHluorin spots n = 336 spots, random spots n = 116 spots from one experiment.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Comparison, Imaging

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery and localization following release. (A) RUSH-α5-pHluorin released in U2OS co-expressing Paxillin-mScarlet on FN- and anti-GFP antibody-coated surfaces. The intensity of RUSH-α5-pHluorin signal was quantified in and outside adhesions (paxillin positive, represented in the insets). Data are mean ± SD, N = 12 cells ( N = 6 cells from 1 experiment for T = 45 min), pooled from 2 independent experiments. Ordinary one-way Anova with Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested. Scale bars: 10 µm (main and insets). (B) High resolution imaging of RUSH-α5 after 15 min of release in U2OS. PDI (ER marker) or GM130 (Golgi marker) are co-stained. Arrows in the insets indicate RUSH-α5 positive vesicles. Scale bar: 10 µm (main), 5 µm (insets). (C and D) High-mannose integrin-α5 is delivered to the cell surface. (C) Flow cytometry analysis of high-mannose proteins at the cell surface detected with the fluorescent lectin PFL647 in U2OS cells expressing RUSH-α5, without release and 1 h after release. The left panel shows histograms of one experiment, the right panel shows the geometric fluorescence mean of the PFL647 signal for individual experiments ( N = 2 independent experiments). (D) U2OS expressing RUSH-α5 were labeled at their surface after 1 h release with a lectibody specifically recognizing high-mannose proteins. The lectibody was then pulled down by protein G beads. This Western blot shows GFP detection in the pull-down, indicating the presence of high-mannose RUSH-α5 at the cell surface after release. Representative of N = 3 independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Expressing, Imaging, Marker, Staining, Flow Cytometry, Fluorescence, Labeling, Western Blot

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse TIRF imaging of U2OS expressing RUSH-α5-pHluorin plated on FN-coated surface. Left: RUSH-α5-pHluorin. Right: ratiometric analysis, the exocytosis spots appear in yellow. Scale bar: 20 µm. One frame per 10 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Polarized delivery of newly synthesized integrin to the cell protruding edge. (A–C) RUSH-α5 intensity in U2OS cells plated on 9 µm-wide micropatterns coated with FN and anti-GFP or collagen and anti-GFP ± biotin treatment for the indicated times was analyzed at both cell edges (the predominantly protruding edge was denoted ROI1 and the other edge ROI2; and ). Representative intensity coded images (A) and quantification of RUSH-α5 release on FN (B; ) and collagen (C; ) (normalized first to the total intensity of the cell and then to 0 min biotin) are shown. Data are mean ± SEM. (D) Representative images and spatiotemporal track maps of cell edge contours over time in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Red insets represent protruding ROIs that are magnified. Blue insets represent retracting ROIs that are magnified. Spatiotemporal track maps: blue colors represent early time points and magenta colors represent late time points in the time-lapse series. (E) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas determined from spatiotemporal track maps). Data are mean ± SD. (B and C) N = 33 cells on FN and 38 cells on collagen, pooled from three independent experiments, two-way ANOVA, Holm-Šídák’s multiple comparison test. (E) N = 53 cells on collagen, 49 cells on FN, pooled from three independent experiments; one-way ANOVA, Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Synthesized, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS expressing RUSH-α5 plated on 9 µm-wide FN and anti-GFP-coated micropattern lines. Biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse imaging of spinning-disk confocal U2OS expressing RUSH-α5 plated on 9 µm-wide collagen and anti-GFP-coated micropattern lines. Biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early delivery of RUSH-α5 to the cell surface. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 (green) and the ER marker ERoxBFP (magenta) plated on FN (10 µg/ml) ± biotin treatment for the indicated times. Arrows indicate rapidly budding RUSH-α5–positive vesicles adjacent to cell protrusions (≤15 min after release) (see also ). (B) TIRF imaging of RUSH-α5 after release (0 min). The polarized delivery to the cell surface at the protruding area can be observed from 15 min after release (see also ). (C) Representative images of RUSH-α5 (green) and RUSH-CD59 (magenta) release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. White insets represent ROIs that are magnified for each channel. FN, fibronectin. (D) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. Representative histograms and quantification from two independent experiments of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Marker, Imaging, Construct, Labeling, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS cells co-expressing RUSH-α5 (green) and the ER marker ERoxBFP (magenta) plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing, Marker

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse imaging of U2OS cells expressing RUSH-α5 plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min, imaged by TIRF microscopy. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Golgi bypass early delivery of RUSH-α5 requires the integrin-α5 PDZ-binding motif. (A and B) Quantification of relative RUSH-α5 recruitment to protruding areas (A) or adhesions (B) in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times with or without Golgicide A (10 µM). (C) Representative immunoblot of GFP pulldowns from RUSH-α5 or GFP control transfected cells plated on FN and probed for GFP and endogenous GRASP65. N = 3 independent experiments. (D) Amino acid sequence of the integrin α5 tail highlighting the canonical PDZ-binding motif (SDA) and the two proline residues critical for the formation of the non-canonical PDZ-binding motif. The mutations of these sites used in our experiments are indicated below. (E) Representative streptavidin pulldowns of the indicated biotinylated recombinant integrin peptides incubated with cell lysates collected from CHO cells overexpressing GFP-GRASP65. A representative immunoblot probed for GRASP65 (note, two bands are present in the lysate: upper, GFP-GRASP65; lower, endogenous GRASP65; GFP-GRASP65 is apparent in the pulldown). N = 3 independent experiments. (F) Quantification of RUSH-α5 or RUSH-α5 ΔSDA recruitment to adhesions in U2OS cells ± biotin treatment for the indicated times. All data are mean ± SD. (A and B) One-way ANOVA with Tukey’s multiple comparison test for comparing time points, one-way ANOVA with Holm-Šídák’s multiple comparisons test for comparing untreated and Golgicide A, data distribution was assumed to be normal but this was not formally tested. (A) N = 26 cells RUSH-α5, N = 22 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (B) N = 24 cells RUSH-α5, N = 27 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (F) One sample t test to compare time points with T = 0, ordinary one-way ANOVA with Holm-Šídák’s multiple comparisons test to compare RUSH-α5 and RUSH-α5 ΔSDA, data distribution was assumed to be normal but this was not formally tested. N = 23 cells RUSH-α5, N = 23 cells RUSH-α5 ΔSDA, pooled from two independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Binding Assay, Expressing, Western Blot, Control, Transfection, Sequencing, Recombinant, Incubation, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early release of RUSH-α5 is adhesion dependent and polarized recruitment to protrusions is supported by endogenous integrin α5. (A–D) Validation of ITGA5 CRISPR-Cas9 KO U2OS cells. (A) Western blot analysis of WT and ITGA5 KO single cell clones showing the efficiency of the CRISPR-Cas9 ITGA5 KO in U2OS cells. (B) Genome sequence alignment of U2OS WT and ITGA5 KO clones with the ITGA5 WT sequence. The targeted exon and the gRNA used for CRISPR KO positions are indicated. (C) Representative flow cytometry analysis of cell surface integrin α5 in U2OS WT and ITGA5 KO clones. (D) Images of WT and ITGA5 KO U2OS clones stained for active integrin α5 (SNAKA51) and paxillin. Scale bar: 20 µm. (E) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in WT and ITGA5 KO U2OS cells transfected with RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test showed no significant differences between WT and ITGA5 KO. Data distribution was assumed to be normal but this was not formally tested. (F) Flow cytometry analysis of cell surface RUSH-α5 levels in adherent versus suspension U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP analyzed as in E are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test, data distribution was assumed to be normal but this was not formally tested. (G) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas) in WT and ITGA5 KO U2OS cells ± biotin treatment for the indicated times. One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal but this was not formally tested. Data are mean ± SD; N = 59 WT cells, 53 ITGA5 KO cells, pooled from three independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Biomarker Discovery, CRISPR, Western Blot, Clone Assay, Sequencing, Flow Cytometry, Staining, Transfection, Two Tailed Test, Suspension, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early release of RUSH-α5 is sensitive to GRASP silencing. (A) Immunoblot of GST pull-downs of recombinant integrin α5 WT or mutant (ΔSDA or PPAA) peptides incubated with GST alone or recombinant GST-GRASP65. No enrichment of GST-GRASP65 signal over background (no peptide beads) is detected with integrin α5 WT peptide, indicating that the integrin α5 peptides do not interact with recombinant purified GST-GRASP65. (B) ELISA assay detecting biotinylated recombinant integrin α5 WT or ΔSDA or PPAA mutant with HRP-streptavidin incubated on wells coated with GST alone or GST-GRASP65. No direct interaction between GRASP65 and integrin α5 WT peptide was detected. (C) Immunoblot of lysates collected from control-silenced or GRASP65 and GRASP55-silenced U2OS cells used in D, E, probed for GRASP65 and GRASP55. β-actin was probed as a loading control. (D) Representative immunofluorescence images of control-silenced or GRASP65 and GRASP55-silenced U2OS cells expressing RUSH-α5 and plated on dual-coated micropatterns (magenta dots, FN; non-fluorescent regions, collagen peptide GFOGER). (E) Relative recruitment of RUSH-α5 in control- or GRASP65- and GRASP55-silenced U2OS cells to FN dots within the cell boundary. Data are mean ± SD; n = 9 siCTRL cells, 11 siGRASP cells (36 and 44 dots, respectively) from one experiment. (F and G) Quantification of RUSH-α5 intensity in the four areas relative to signal intensity in the respective area 2.5 min prior to RUSH-α5 appearance in (F) new adhesions or (G) already existing adhesions (determined from the time-lapse images) on FN- and anti-GFP antibody-coated surfaces. Adhesions close to the cell edge and with a minimum lifetime of 15 min were analyzed and changes of RUSH-α5 intensity were plotted over time in the indicated areas ranging from distal to proximal to the cell body. Data are mean ± SD; One independent experiment 9 adhesions from 6 cells on 2 coverslips (F) and one independent experiment 9 adhesions from 5 cells on 2 coverslips (G). (H and I) Representative images (H) and (I) track maps related to . Red arrows indicate direction of adhesion growth. Scale bars: 20 µm. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Western Blot, Recombinant, Mutagenesis, Incubation, Purification, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 is delivered to the tip of adhesions and mediates adhesion growth. (A and B) Representative immunofluorescence image of U2OS cells expressing RUSH-α5 and pmKate2-Paxillin (white, colocalization) plated on FN (10 µg/ml) and anti-GFP (2.5 µg/ml; to trap cell surface RUSH-α5 at the point of delivery). Insets represent ROIs that are magnified. ROI2 is a FA demarcated into four equal areas for analysis and is further magnified in B. Scale bars 20 µm (whole cell image), 5 µm (ROI1), and 0.5 µm (ROI2 and B). (C) Representative image of an already established FA where RUSH-α5 delivery was quantified. Scale bar 0.5 µm. (D) Cartoon showing clutch model elements. Myosin motors pull on actin filaments with a speed v . This applies force to a substrate via integrins and adapter proteins (talin). The effect of force regulates the unbinding rates from integrins to the substrate ( k off ) and the folding/unfolding rates of talin ( k fold / k unfold ). When talin unfolds, adhesion reinforcement is assumed to happen, which is modeled by an increase in integrin density with value d add . Changes in integrin availability are modeled by changing the parameter d add . (E) Model prediction of adhesion growth with time for conditions in which integrin availability is low ( d add = 0.005 integrins/μm 2 ) or high ( d add = 0.01 integrins/μm 2 ). Adhesion growth (y-axis) is modeled through integrin density, which is plotted normalized to the starting value. (F) Quantification of adhesion growth in U2OS cells expressing RUSH-α5 and plated on FN or collagen ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (G) Quantification of adhesion growth in WT and ITGA5 KO U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (H) Quantification of cell spreading in WT and ITGA5 KO cells expressing RUSH-α5 ± biotin treatment for the indicated times. Data are mean ± SEM. (I) Quantification of the length of the longest protrusion (extending furthest from the initial plasma membrane localization during imaging) formed per cell after 45 min of biotin. Data are mean ± SEM. (J) Schematic depiction of the regulation of cell dynamics by transport of integrins through the biosynthetic pathway. Adhesion and cell spreading-dependent delivery of integrin from the ER is detected rapidly after release in cell protrusions. Canonical Golgi-dependent delivery is also polarized to cell protruding areas in an ECM-specific manner and contributes to FA growth and cell protrusion. (F–H) One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal, but this was not formally tested. (F) N = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments. (G) 57 WT cells and 52 ITGA5 KO cells, (H) 59 WT cells and 55 ITGA5 KO cells, pooled from three independent experiments. (I) Mann–Whitney test, N = 55 cells on FN, 66 cells on collagen, pooled from three independent experiments.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Clinical Proteomics, Membrane, Imaging, Comparison, MANN-WHITNEY