Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 1. Chronic hypoxia (CH) promotes proliferation and upregulates CaSR and TRPC6 expression in rat pulmonary venous smooth muscle cells (PVSMCs). (A–B) Intrapulmonary venous branches isolated from rat lung. Phase-contrast microscopy images of rat distal PVSMCs cultured for 3 (C) and 8 (D) days. (E) Dual immunofluorescence staining detection of α-smooth muscle actin (green) and CaSR (red) expression in PVSMCs; nuclei are stained with DAPI (blue). Effect of CH on PVSMC numbers (F) and viability (G). Representative images (I) and summarized data (H) showing the effect of CH on PVSMC DNA synthesis assessed by BrdU incorporation. (J) Real-time quantitative PCR results showing the effect of CH on CaSR mRNA expression in PVSMCs, with β-actin as an internal standard. Representative blots and summarized data (K) showing the effect of CH on CaSR protein expression relative to β-actin in PVSMCs, determined by western blotting. (L) mRNA expression of TRPC1 and TRPC6 in PVSMCs exposed to normoxia (Nor) or chronic hypoxia (CH), determined by real-time quantitative PCR. (M) Repre sentative western blots of TRPC1, TRPC6, and β-actin proteins in PVSMCs exposed to Nor and CH. **p < 0.01; ****p < 0.0001; ns, not significant vs. normoxic cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Expressing, Isolation, Microscopy, Cell Culture, Immunofluorescence, Staining, DNA Synthesis, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 2. Co-immunoprecipitation (Co-IP) Analysis of CaSR and TRPC6 Interaction in PVSMCs under hypoxia. (A) Co-IP of TRPC6 with CaSR. Protein lysates from cells were subjected to immunoprecipitation with anti-TRPC6 antibodies (IP: TRPC6) followed by immunoblotting (IB) with anti-CaSR antibodies (IB: CaSR). (B) Co-IP of CaSR with TRPC6. Protein lysates were subjected to immunoprecipitation with anti-CaSR antibodies (IP: CaSR) followed by immunoblotting with anti-TRPC6 an tibodies (IB: TRPC6).

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot

Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 3. Effects of specific CaSR modulators on CH-induced enhancement of PVSMC proliferation and TRPC6 expression. The effects of two inhibitors (NPS2143, NPS2390) and two agonists (spermine, R568) of CaSR on PVSMC number (A) and viability (B) under normoxic (Nor) or hypoxic (CH) conditions. Summarized data (C) and representative images (D) showing the effects of specific CaSR modulators on CH-induced increases in DNA synthesis of PVSMCs. (E) Real-time quantitative PCR showing the effects of CaSR modulators on CH-induced TRPC6 mRNA levels in PVSMCs. (F) Western blotting showing the effects of CaSR modulators on CH- induced TRPC6 protein levels in PVSMCs; representative blots and mean TRPC6 protein levels. CaSR modulators (NPS2143, 10 μmol/L; NPS2390, 20 μmol/L; spermine, 2 μmol/L; R568, 5 μmol/L). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Expressing, DNA Synthesis, Real-time Polymerase Chain Reaction, Western Blot

Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 4. Downregulation of CaSR attenuates CH-induced enhancement of PVSMC proliferation and inhibits TRPC6 expression. (A) Western blotting showing CaSR and β-actin protein levels in PVSMCs treated with transfection vehicle alone (Nor), siNT, or siCaSR. Effects of siCaSR on CH-induced increases in cell number (B) and viability (C). Representative images (D) and summarized data (E) showing the effect of siCaSR on CH-induced enhancement of PVSMC DNA synthesis assessed by BrdU incorporation assay. (F) Real-time quantitative PCR showing the effects of siCaSR on CH-induced upregulation of TRPC6 mRNA in PVSMCs. (G) Western blotting showing the effects of siCaSR on CH-induced upregulation of TRPC6 protein in PVSMCs, including representative blots and mean protein expression of TRPC6. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Expressing, Western Blot, Transfection, DNA Synthesis, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction

Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 5. Effects of siTRPC6 on CH-induced CaSR upregulation and PVSMC proliferation. (A) Representative Western blot images and mean protein expression of TRPC6 relative to β-actin in hypoxic PVSMCs treated with transfection vehicle alone (control), siNT, or siTRPC6. Effects of siTRPC6 on CH-induced increases in cell number (B) and viability (C). Representative images (D) and summarized data (E) showing the effect of siTRPC6 on CH-induced enhancement of PVSMC DNA synthesis assessed by BrdU incorporation assay. Effects of siTRPC6 on CH-induced upregulation of CaSR at the mRNA (F) and protein (representative blots and summarized data) (G) levels in PVSMCs. **p < 0.01; ****p < 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Western Blot, Expressing, Transfection, Control, DNA Synthesis, BrdU Incorporation Assay

Journal: Experimental cell research

Article Title: Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway.

doi: 10.1016/j.yexcr.2024.114363

Figure Lengend Snippet: Fig. 6. Effects of CaSR knockdown on hypoxia-mediated increases in SOCE and ROCE in PVSMCs, assessed by measuring [Ca2+] restoration. (A–C) Representative traces showing [Ca2+]i responses to restoration of extracellular [Ca2+] to 2.5 mM after perfusion with Ca2+-free KRB solution (0 Ca2+) containing 10 μM CPA and 5 μM nifedipine in PVSMCs treated with siNT or siCaSR under normoxic (Nor) or hypoxic (CH) conditions. (D) Average maximum increase in [Ca2+]i after restoration of extracellular [Ca2+] in normoxic siNT, hypoxic siNT, or siCaSR PVSMCs. (E–G) Representative [Ca2+]i traces in response to restoration of extracellular [Ca2+] to 2.5 mM after perfusion with Ca2+-free KRB solution (0 Ca2+) containing 100 μM OAG and 5 μM nifedipine in PVSMCs treated with siNT or siCaSR under normoxic (Nor) or hypoxic (CH) conditions. (H) Mean maximum increase in [Ca2+]I after restoration of extracellular [Ca2+] in normoxic siNT, hypoxic siNT, or siCaSR PVSMCs. ****p < 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal antibodies targeting CaSR (Cat #: ACR-004), TRPC1 (Cat #: ACC-010), and TRPC6 (Cat #: ACC-017) were supplied by Alomone Laboratories.

Techniques: Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Calcium oxalate crystals upregulate CaSR and SLC26A6 expression in vivo and in vitro . (A) Representative images of renal tissue pathological sections from each group of rats (HE staining, Pizzolato’s staining, and polarized light microscopy). (B) Western blotting detection of CaSR and SLC26A6 protein expression levels in NRK-52E cells after COM crystal intervention. (C,D) RT-qPCR (C) and Western blotting (D) detection of CaSR and SLC26A6 mRNA and protein expression levels in renal tissues of each group of rats. (E) Immunohistochemical analysis revealed the tubular localization patterns of CaSR and SLC26A6 in rat kidney tissue. Low-power field images demonstrated their widespread distribution within both the renal cortex and medulla. Precise cellular colocalization could only be assessed through high-power confocal analysis combined with segment-specific markers, representing a limitation of this study. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. NC group or Control group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, In Vivo, In Vitro, Staining, Light Microscopy, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Standard Deviation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: CaSR regulates SLC26A6 expression via the transcription factor FOXO4. (A) Western blotting detection of p-FOXO4 (Thr451) and SLC26A6 protein expression levels in NRK-52E cells treated with CaSR agonist (R568, 30 μM) or inhibitor (NPS-2143, 10 μM) in the presence of COM crystals. (B) Western blotting detection of SLC26A6 protein expression levels in NRK-52E cells treated with FOXO4 agonist (TNF-α, 20 ng/mL) or inhibitor (JY-2, 30 μM) in the presence of COM crystals. (C) Dual-luciferase reporter gene assay detecting the effect of FOXO4 on SLC26A6 promoter activity. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. COM group; #p < 0.05, ##p < 0.01 vs. specified group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, Western Blot, Luciferase, Reporter Gene Assay, Activity Assay, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: CaSR promotes FOXO4 phosphorylation via the PKA signaling pathway. (A) Western blotting detection of p-PKA substrate and p-FOXO4 (Thr451) protein expression levels in NRK-52E cells treated with CaSR agonist (R568, 30 μM) or inhibitor (NPS-2143, 10 μM) in the presence of COM crystals. (B) Western blotting detection of p-FOXO4 (Thr451) and SLC26A6 protein expression levels in NRK-52E cells treated with PKA agonist (8-Bromo-cAMP, 30 μM) or inhibitor (H-89 2HCl, 30 μM) in the presence of COM crystals. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. COM group; #p < 0.05, ##p < 0.01 vs. specified group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Phospho-proteomics, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Crystal deposition and tissue damage in the kidneys of rats from each group (A) Hematoxylin and eosin (HE) staining, polarized light microscopy, and Pizzolato’s calcium salt staining of kidney tissue from rats in the EG, CaSR-a, and CaSR-i groups. HE staining reveals tubular architecture and the extent of damage; polarized light microscopy reveals the birefringence of calcium oxalate crystals; Pizzolato’s staining stains calcium oxalate crystals black. (B) Hematoxylin and eosin (HE) staining, polarized light microscopy, and Pizzolato’s calcium salt staining of renal tissues from rats in the EG, PKA-i, and FOXO4-i groups. HE staining reveals the extent of tubular damage in each group; polarized light microscopy and calcium salt staining jointly confirm differences in crystal deposition among the groups.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Staining, Light Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Effects of intervening in the CaSR-PKA-FOXO4 signaling axis on urinary biochemical indicators and renal protein expression in rats. (A) Western blotting detection of p-PKA substrate, p-FOXO4 (Thr451), and SLC26A6 protein expression levels in renal tissues of each group of rats. (B) Concentrations of calcium ions and oxalate in 24-h urine of each group of rats. (C) Quantitative analysis of SLC26A6 protein expression from (A) . Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. E.G., group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Schematic diagram of the mechanism by which CaSR synergistically promotes calcium oxalate stone formation through dual signaling pathways. Under stimulation by calcium oxalate crystals (COM), the calcium-sensing receptor (CaSR) on the membrane of renal tubular epithelial cells is activated. The activated CaSR activates protein kinase A (PKA) via G proteins. PKA then functions through two parallel pathways: Phosphorylates Signal Transducer and Activator of Transcription 3 (STAT3), causing its nuclear translocation and upregulation of the tight junction protein Claudin-14 expression. Claudin-14 inhibits paracellular calcium reabsorption, leading to hypercalciuria; Phosphorylates Forkhead box protein O4 (FOXO4), causing its nuclear translocation and binding to the SLC26A6 gene promoter, upregulating its expression and promoting oxalate secretion into the lumen, leading to hyperoxaluria. The increased excretion of urinary calcium and oxalate collectively exacerbates the supersaturation of calcium oxalate in urine, ultimately promoting kidney stone formation.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Protein-Protein interactions, Membrane, Translocation Assay, Expressing, Binding Assay

Journal: Medicine

Article Title: Production of parathyroid-like cells from thyroid stem cells in co-culture environment

doi: 10.1097/MD.0000000000032009

Figure Lengend Snippet: Forward (F) and reverse (R) primer sequences employed in real-time PCR and expected amplicon lengths.

Article Snippet: For immunofluorescence imaging, to visualize cell-specific marker proteins, the following primary and secondary antibodies were used: rabbit polyclonal anti-calcium sensing receptor (CaSR) antibody (Abcam®, ab137408; 1:500), rabbit polyclonal anti-parathyroid hormone receptor 1 (PTH1R) antibody (Abcam®, ab75150; 1:500), rabbit polyclonal anti-thyroid stimulating hormone receptor (TSHR) antibody (Abcam®, ab202960; 1:500), rabbit monoclonal Anti-thyroid transcription factor 1 (TTF1) antibody [SP141] (Abcam®, ab227652; 1:25), mouse monoclonal anti-parathyroid hormone antibody [rPTH/911] (Abcam®, ab234415; 1 μg/mL), goat anti-mouse IgM cross-adsorbed secondary antibody (DyLight® 594) (Invitrogen®, SA5-10152; 1:200) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam®, ab150077; 1:1000).

Techniques: Real-time Polymerase Chain Reaction, Amplification, Sequencing

Journal: Medicine

Article Title: Production of parathyroid-like cells from thyroid stem cells in co-culture environment

doi: 10.1097/MD.0000000000032009

Figure Lengend Snippet: Immunofluorescence staining of proteins in parathyroid cells using confocal microscopy before differentiation protocol. (A) CaSR (membrane), (B) PTH (internal) and C) PTH1R (membrane), parathyroid cells stained in green for all proteins and counterstained in blue for nuclei. Objective 40 × original magnification, scale bars represent 20 µm. CaSR = calcium sensing receptor, PTH = parathormone, PTH1R= parathyroid hormone receptor 1.

Article Snippet: For immunofluorescence imaging, to visualize cell-specific marker proteins, the following primary and secondary antibodies were used: rabbit polyclonal anti-calcium sensing receptor (CaSR) antibody (Abcam®, ab137408; 1:500), rabbit polyclonal anti-parathyroid hormone receptor 1 (PTH1R) antibody (Abcam®, ab75150; 1:500), rabbit polyclonal anti-thyroid stimulating hormone receptor (TSHR) antibody (Abcam®, ab202960; 1:500), rabbit monoclonal Anti-thyroid transcription factor 1 (TTF1) antibody [SP141] (Abcam®, ab227652; 1:25), mouse monoclonal anti-parathyroid hormone antibody [rPTH/911] (Abcam®, ab234415; 1 μg/mL), goat anti-mouse IgM cross-adsorbed secondary antibody (DyLight® 594) (Invitrogen®, SA5-10152; 1:200) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam®, ab150077; 1:1000).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Membrane

Journal: Medicine

Article Title: Production of parathyroid-like cells from thyroid stem cells in co-culture environment

doi: 10.1097/MD.0000000000032009

Figure Lengend Snippet: Real time PCR results of the organoid groups after 28 days of incubation.

Article Snippet: For immunofluorescence imaging, to visualize cell-specific marker proteins, the following primary and secondary antibodies were used: rabbit polyclonal anti-calcium sensing receptor (CaSR) antibody (Abcam®, ab137408; 1:500), rabbit polyclonal anti-parathyroid hormone receptor 1 (PTH1R) antibody (Abcam®, ab75150; 1:500), rabbit polyclonal anti-thyroid stimulating hormone receptor (TSHR) antibody (Abcam®, ab202960; 1:500), rabbit monoclonal Anti-thyroid transcription factor 1 (TTF1) antibody [SP141] (Abcam®, ab227652; 1:25), mouse monoclonal anti-parathyroid hormone antibody [rPTH/911] (Abcam®, ab234415; 1 μg/mL), goat anti-mouse IgM cross-adsorbed secondary antibody (DyLight® 594) (Invitrogen®, SA5-10152; 1:200) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam®, ab150077; 1:1000).

Techniques: Real-time Polymerase Chain Reaction, Incubation, Gene Expression