Journal: Scientific reports

Article Title: The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling.

doi: 10.1038/s41598-020-64006-3

Figure Lengend Snippet: Figure 5. Co-localization of SLC10A7-mScarlet with STIM1, Orai1 and SERCA2 in HEK293 cells. HEK293 cells were seeded on coverslips and were transiently transfected with the SLC10A7-mScarlet (transcript variant v2) construct (red fluorescence). After 48 h, the cells were treated with 1 µM TG for 5 min and then the cellular co-localization with STIM1, Orai1 and SERCA2 was detected by immunofluorescence with respective anti- STIM1, anti-Orai1 and anti-SERCA2 antibodies (green fluorescence). Nuclei were stained with Hoechst 33342 (blue). Images represent maximum projections of z-stacks at 630-x magnification after deconvolution. Scale bars: 10 µm. Arrows: Co-localization of both proteins.

Article Snippet: Then, cells were incubated at 4 °C overnight with antibodies against STIM1 (1:800, D99E10, Cell Signaling), Orai1 (2 μg/ml, sc-377281, Santa Cruz Biotechnology, Heidelberg, Germany) or SERCA2 (1:250, sc-376235, Santa Cruz Biotechnology) in blocking buffer, followed by labeling with AlexaFluor 488-conjugated secondary antibodies (Invitrogen) and nuclear marker Hoechst 33342.

Techniques: Transfection, Variant Assay, Construct, Fluorescence, Immunofluorescence, Staining

Journal: Scientific reports

Article Title: The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling.

doi: 10.1038/s41598-020-64006-3

Figure Lengend Snippet: Figure 8. Schematic illustration of calcium signaling in non-excitable cells. Following the stimulation of G protein-coupled receptors (GPCR), phospholipase C (PLC) hydrolyses PIP2 to IP3. IP3 activates the IP3 receptor (IP3R) and triggers the depletion of ER Ca2+ stores. TG or ionomycin also cause the Ca2+ depletion from ER by different mechanisms. Declined ER Ca2+ levels are sensed by STIM1 proteins. STIM1 oligomerizes, migrates towards subplasmalemmal ER-PM junctions, and interacts with Orai1 to trigger store-operated calcium channel opening. Then, SERCA pumps Ca2+ back into the ER to refill the stores with Ca2+. SLC10A7 is hypothesized to negatively regulate STIM1, Orai1 and/or SERCA2 through protein interaction. TG, SERCA inhibitor; BTP-2, SOCE blocker.

Article Snippet: Then, cells were incubated at 4 °C overnight with antibodies against STIM1 (1:800, D99E10, Cell Signaling), Orai1 (2 μg/ml, sc-377281, Santa Cruz Biotechnology, Heidelberg, Germany) or SERCA2 (1:250, sc-376235, Santa Cruz Biotechnology) in blocking buffer, followed by labeling with AlexaFluor 488-conjugated secondary antibodies (Invitrogen) and nuclear marker Hoechst 33342.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1 α /XBP1s pathway

doi: 10.1016/j.apsb.2021.12.025

Figure Lengend Snippet: The substitution of SERCA2 C674 by S674 promotes PASMC proliferation by accelerating cell cycle. (A) The mRNA (left) and protein (right) levels of SERCA2 in PASMCs. mRNA, n = 5 ( Atp2a2a are not detectable in two samples); protein, n = 5. (B) The intracellular Ca 2+ level detected by Fluo-4 (green). Scale bar: 25 μm. (C) The percentage of EdU-positive proliferating cells (green). Nuclei are indicated by Hoechst 33258 (blue). Scale bar: 100 μm. (B) and (C), ∗ P < 0.05, SKI vs. WT, unpaired t -test, n = 5. (D) Cell proliferation. ∗ P < 0.05, SKI vs. WT, unpaired t -test, n = 10. (E) Representative cell cycle analyzed by flow cytometry and the relative percentage of cells in each phase. ∗ P < 0.05, G0/G1 phase or S phase, SKI vs. WT, ANOVA with Bonferroni correction, n = 3. (F) Cell cycle related proteins in PASMCs and in the remodeled SKI pulmonary vessels indicated by α -SMA staining (red). Scale bar: 50 μm. ∗ P < 0.05, SKI vs. WT, unpaired t -test, n = 5–6. Data were presented as mean ± SEM.

Article Snippet: PASMCs were incubated with specific antibodies for XBP1s, Ki67, SERCA2, or calnexin (Cat# 66903-1-Ig; Proteintech) for 12 h, followed by Alexa Fluor 488 goat anti-rabbit IgG (H + L) or Cy3-conjugated Affinipure goat anti-mouse IgG (H + L) for 2 h. Stained nuclei with DAPI or Hoechst 33258 for 10 min.

Techniques: Flow Cytometry, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1 α /XBP1s pathway

doi: 10.1016/j.apsb.2021.12.025

Figure Lengend Snippet: The substitution of SERCA2 C674 by S674 induces ER stress to promote PASMC proliferation. (A) Representative Western blot of ER stress related proteins in PASMCs and quantification of band intensities in graph. ∗ P < 0.05, SKI vs. WT, unpaired t -test, n = 5–6. (B) Representative Western blot of SKI PASMCs treated with ER stress inhibitor 4-phenylbutyric acid (PBA), and quantification of band intensities in graph. ∗ P < 0.05, PBA vs. solvent control (Ctrl), unpaired t -test, n = 5–6. (C) The expression of XBP1s (red) and the percentage of Ki67-positive (green) cells. Nuclei are indicated by DAPI (blue). ∗ P < 0.05, PBA vs. Ctrl in SKI PASMCs, unpaired t -test, n = 5. (D) Representative cell cycle analyzed by flow cytometry (left), and the relative percentage of cells in each phase (right). ∗ P < 0.05, all phases, PBA vs. Ctrl in SKI PASMCs, ANOVA with Bonferroni correction, n = 3. (E) Cell proliferation. ∗ P < 0.05, PBA vs. Ctrl in SKI PASMCs, unpaired t -test, n = 5. Data were presented as mean ± SEM.

Article Snippet: PASMCs were incubated with specific antibodies for XBP1s, Ki67, SERCA2, or calnexin (Cat# 66903-1-Ig; Proteintech) for 12 h, followed by Alexa Fluor 488 goat anti-rabbit IgG (H + L) or Cy3-conjugated Affinipure goat anti-mouse IgG (H + L) for 2 h. Stained nuclei with DAPI or Hoechst 33258 for 10 min.

Techniques: Western Blot, Solvent, Control, Expressing, Flow Cytometry