Journal: The FASEB Journal

Article Title: The function of the calcium channel Orai1 in osteoclast development

doi: 10.1096/fj.202001921rr

Figure Lengend Snippet: FIGURE 3 Expression of osteoclast-related proteins in wild type and Orai1fl/fl-LysMcre bone marrow and in macrophages with and without RANKL differentiation. A, In RNA from unstimulated whole marrow wild type (WT) and Orai1fl/fl-LysMcre (Orai1−/−) cells, expression of mRNA for a bone protein, osteprotegerin (OPG), a bone produced osteoclast differentiation protein (RANKL) and bone resorption-related proteins of osteoclasts, cathepsin K, TRAP, and ATPa3, relative to GAPDH. In whole marrow, differences were statistically significant for TRAP and RANKL at P < .02. N = 4, mean ± SD. B, Expression of TRAP relative to GAPDH determined by qPCR analysis of mRNA from control (WT) and Orai1fl/fl-LysMcre (Orai1−/−) bone marrow cells, depleted of stromal cells, and cultured with mCSF without (left) or with RANKL (right) for 7 days (N = 3, mean ± SD). Because data showed a non-normal distribution, results were analyzed by non-parametric ANOVA (Kruskal-Wallis test) with Dunn's test for multiple comparisons; TRAP expression was significantly greater (P < .01, N = 3) in the RANKL-treated cells from Orai1fl/fl-LysMcre animals compared to cells from controls with and without RANKL. C, Expression of osteoclast markers in RANKL-treated cultured marrow cells from Orai1fl/fl-LysMcre (Orai1−/−) animals determined by qPCR; results expressed as fold change compared to controls (WT); mean ± SD, N = 3. DC-STAMP is significantly reduced in the absence of Orai1 (P < .001), while ATP6v0d2 shows a significant increase (P < .01)

Article Snippet: TRAP antibody: sections were blocked in PBS and 2% milk, and incubated 18 hours with rabbit ACP5 anti- TRAP polyclonal antibody (Proteintech, Rosemont, IL) at 1:100, washed, and incubated with AlexaFluor488- labeled goat anti- rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at 1:500.

Techniques: Expressing, Produced, Control, Cell Culture

Journal: The FASEB Journal

Article Title: The function of the calcium channel Orai1 in osteoclast development

doi: 10.1096/fj.202001921rr

Figure Lengend Snippet: FIGURE 4 In vitro osteoclastogenesis from bone marrow of Orai1fl/fl-LysMcre and WT mice. A, Histomorphology of TRAP+ cells differentiated in vitro in RANKL plus m-CSF. Representative results from independent experiments using cells from three pairs of Orai1fl/fl- LysMcre and WT mice. B, Histomorphology of TRAP+ cells differentiated in vitro in RANKL plus CSF-1 on mineralized matrix. Results are representative of independent experiments using cells from three pairs of Orai1fl/fl-LysMcre and WT mice. C, TRAP+ cells per mm2 in cultures described in A above. D, Mean TRAP+ cell size in cultures described in A above. Wild type plus RANKL is significantly greater than all other groups. E, Mean TRAP+ cell area in cultures described in A above. Wild type plus RANKL is significantly greater than all other groups. F, Resorption of plate matrix coating by bone marrow TRAP+ cells from Orai−/− and WT mice differentiated in vivo. Data show the percent of matrix area resorbed, n = 4, mean ± SD P < .001. G, Nuclei per cell in cultures. Marrow cell cultures were stimulated with m-CSF and RANKL for one week and then TRAP stained. The number of nuclei per TRAP+ cell was counted for Wild Type or Orai1fl/fl-LysMcre mononuclear cells. The average number of nuclei per cell in wild-type cells was approximately 5.5 and in the Orai1−/− the average was approximately 1 per cell. These numbers are statistically different (n = 48, mean ± SEM, P < .0001). H, Evaluation of cell numbers in marrow cultures from control and Orai1 conditional knock-out mice. Cell numbers (per well of 96-well plate) evaluated by Alamar blue assay; shown are cell numbers after 24 hours as percentage of baseline (t = 0) cell numbers. Results (mean ± SD, N = 4) are representative of 3 independent experiments; no significant differences were detected

Article Snippet: TRAP antibody: sections were blocked in PBS and 2% milk, and incubated 18 hours with rabbit ACP5 anti- TRAP polyclonal antibody (Proteintech, Rosemont, IL) at 1:100, washed, and incubated with AlexaFluor488- labeled goat anti- rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at 1:500.

Techniques: In Vitro, In Vivo, Staining, Control, Knock-Out, Alamar Blue Assay

Journal: The FASEB Journal

Article Title: The function of the calcium channel Orai1 in osteoclast development

doi: 10.1096/fj.202001921rr

Figure Lengend Snippet: FIGURE 5 Orai1−/− cells express osteoclast markers but do not multinucleate: Labeling of Orai1 and TRAP in WT and conditional Orai1−/− cells. In each case, WT is shown on the left and the LysM2 cre/ Orai1-floxed bone on the right. A, Moderate magnification (500 µm across) sections of bone labeled for TRAP (top) and in phase (bottom) showing large cells (osteoclasts) in the WT (marked with <) and small cells (mononuclear cells) in the conditional knockout. Higher powers are shown below for clarity. B, Higher magnification (150 µm) sections labeled for Orai1 (red) and nuclei (blue) showing multinucleated Orai1 expressing cells in the wild type (left) and Orai1−/− mononuclear cells attached to bone in the conditional knockout (middle) with a high power (75 µm) section for clarity (far right). C, TRAP (green) and nucleus (blue) labeled sections. A 200 µm field of the wild type (WT, left) shows multinucleated osteoclasts. A 100 µm field of the conditional knockout (Orai1−/−, middle) shows TRAP labeled mononuclear cells. A higher power section of the conditional knockout (25 µm) is shown for clarity (far right). D, The TRAP labeled surface area (Oc.S/BS equivalent) as a function of total bone surface was measured. The TRAP labeled surface was decreased in the conditional knockout (P < .01, n = 3). E, Osteoblast surface area (bone formation surface) (Ob.S/BS) estimated as cuboidal bone lining cell area. The difference was similar to the difference in TRAP labeled surface (P < .03, n = 3)

Article Snippet: TRAP antibody: sections were blocked in PBS and 2% milk, and incubated 18 hours with rabbit ACP5 anti- TRAP polyclonal antibody (Proteintech, Rosemont, IL) at 1:100, washed, and incubated with AlexaFluor488- labeled goat anti- rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at 1:500.

Techniques: Labeling, Knock-Out, Expressing