stim1 redistribution  (MedChemExpress)

Journal: Advanced Science

Article Title: Calcium–Collagen Coupling is Vital for Biomineralization Schedule

doi: 10.1002/advs.202100363

Figure Lengend Snippet: Alteration of the Col1 biosynthesis caused consistent changes in ER Ca 2+ concentrations. A) Schematic illustration. Based on quantitative consistency of Ca 2+ and collagen, BMSCs underwent osteogenic induction by OM, and then the collagen expression was inhibited and recovered, followed by determination of Ca 2+ and collagen changes. Lv‐Col1 indicates lentiviral Col1 plasmids. B,C) The inhibitory effects of FT011 and the retrieval effects of Lv‐Col1 in dose‐dependent manner. N = 5. D) Fura‐2‐AM fluorescence changes. Scale bar = 10 µm. Histogram shows the fluorescence intensity (arbitrary units). N = 9. E) Cytosolic Ca 2+ changes tested by Fluo‐4‐AM (Fluo 4) (presented in Δ F/F ). Error bars represent ± SEM (control group, grey line, N = 30; inhibitory group, red line, N = 35; retrieval group, blue line, N = 30). Bar chart showing the area under the curves (AUC). N = 5. F) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. G) STIM1 localization. Arrowheads = STIM1 puncta. Scale bar = 5 µm. F P / F TOT is calculated as the ratio of fluorescence intensity in the peripheral region ( F P ) to the total cell fluorescence ( F TOT ). N = 5. H) STEM‐EDX elemental mapping and unstained immune‐TEM images. Insets are high magnification of selected areas. Scale bar = 500 nm and = 200 nm in insets. Unstained sections are pseudo‐colored. Arrows = Col1. I,J) The comparison of Col1 positive dots per cell and elemental compositions (at%) among regions containing ER. N = 6. K,L) The comparison of sectional size of dilated ER and the percentage of cells bearing dilated ER. N = 10. Cells in this figure are osteogenic induced for 3 days and then treated for 2h, with or without transfection. All of the experiments are performed at least three times. * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.0001, ns , no significant difference.

Article Snippet: [ ] To inhibit the expression of type I collagen, BMSCs were transfected with empty lentiviral vector and treated with the collagen inhibitor, FT011 (HY‐100495, MedChemExpress, USA) for 2 h. [ ] To stimulate STIM1 redistribution, 10 × 10 −6 m TG was used for 10 min. To chelate cytosolic Ca 2+ , 40 × 10 −6 m 1,2‐bis(o‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA, HY‐100168, MedChemExpress, USA) or an equivalent amount of DMSO was added to the OM solution.

Techniques: Expressing, Fluorescence, Control, Comparison, Transfection

Journal: Advanced Science

Article Title: Calcium–Collagen Coupling is Vital for Biomineralization Schedule

doi: 10.1002/advs.202100363

Figure Lengend Snippet: Knockdown of TRAM2 disrupted the connection between Ca 2+ and Col1. A) Schematic illustration. After bioinformatics analysis and preliminary screening, TRAM2‐knockdown BMSCs underwent osteogenic induction by OM, finding the inhibition and depletion of Ca 2+ and collagen, with the coupling disrupted. B) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. C) STIM1 localization. Arrowheads = STIM1 puncta. Scale bar = 10 µm. F P / F TOT calculation is performed as mentioned above. N = 5; one‐way ANOVA with Dunnett's multiple comparisons test. D‐E) Col1 and GAPDH (loading control) or CANX (loading control for ER protein) immunoblots of total and ER proteins. N = 4; two‐tailed Student's t ‐test. F) Col1 and GAPDH (loading control) immunoblots of cell (without extracellular matrix) and cytoplasm proteins. N = 4. G) Fura‐2‐AM fluorescence changes. Scale bar = 10 µm. Histogram shows the fluorescence intensity (arbitrary units). N = 9. H) Cytosolic Ca 2+ changes tested by Fluo4 (Δ F/F ) treated in the absence of Ca 2+ . Error bars represent ± SEM (control group, grey line, N = 40; knockdown group, red line, N = 35; + FT011 group, blue line, N = 40; retrieval group, green line, N = 45). Bar chart showing the area under the curves (AUC). N = 5. I) The D1ER fluorescence images. Scale bar = 10 µm. The relative basal FRET ratio (FRET/CFP) of D1ER. N = 6. J) ER Ca 2+ levels were monitored by D1ER in control group, N = 40; knockdown group, N = 35; + FT011 group, N = 40; retrieval group, N = 45, after the stimulation of 100 × 10 −6 m ATP. Error bars represent ± SEM. Histogram shows the average ER Ca 2+ levels in resting cells in each group. N = 10. Lv‐sh indicates TRAM2 knockdown cells. All of the experiments are performed at least three times. * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.0001, ns , no significant difference.

Article Snippet: [ ] To inhibit the expression of type I collagen, BMSCs were transfected with empty lentiviral vector and treated with the collagen inhibitor, FT011 (HY‐100495, MedChemExpress, USA) for 2 h. [ ] To stimulate STIM1 redistribution, 10 × 10 −6 m TG was used for 10 min. To chelate cytosolic Ca 2+ , 40 × 10 −6 m 1,2‐bis(o‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA, HY‐100168, MedChemExpress, USA) or an equivalent amount of DMSO was added to the OM solution.

Techniques: Knockdown, Inhibition, Fluorescence, Control, Western Blot, Two Tailed Test