Journal: bioRxiv

Article Title: GIP receptor reduces osteoclast activity and improves osteoblast survival by activating multiple signaling pathways

doi: 10.1101/2022.07.02.498420

Figure Lengend Snippet: ( A ) Quantification of phosphorylated c-Src (p-Src) in osteoclasts exposed to vehicle, GIP, or GIP with GIP(3-30)NH 2 for 30 minutes measured by AlphaLISA. ( B ) Cartoon showing signaling pathways that may affect p-Src signaling. Inhibitors/ activators are in red. ( C-D ) Effect of pre-treatment with (C) the PKA inhibitor H-89 or (D) the adenylate cyclase activator forskolin (Fsk) on GIP-mediated p-Src responses in osteoclasts. p-Src was normalized to GAPDH in A, C, D. N=7 in A, N=10 in C, N=5 donors in D. Each point in A, C, D represents one donor, with mean or median shown in red. ( E ) Representative time-lapse images and ( F ) quantification of initiation of actin ring formation in 2 donors. Arrows indicate the actin ring. Each dot represents one cell measured with median shown in red. ****p<0.0001, **p<0.01, *p<0.05. Statistical analyses were performed by: Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test for 2A; one-way ANOVA with Sidak’s and Dunnett’s multiple comparisons tests, respectively, for panels 2B and 2C; nested t-test for 2F.

Article Snippet: Assays were performed in white 384-well plates (Optiplates, PerkinElmer) and AlphaLISA readings made on a Pherastar FS (BMG Labtech) plate reader.

Techniques: Protein-Protein interactions

Journal: bioRxiv

Article Title: GIP receptor reduces osteoclast activity and improves osteoblast survival by activating multiple signaling pathways

doi: 10.1101/2022.07.02.498420

Figure Lengend Snippet: ( A ) Quantification of phosphorylated Akt1/2/3 (p-Akt) generated in osteoclasts by vehicle (veh), GIP, or GIP with GIP(3-30)NH 2 measured by AlphaLISA. ( B ) Effect of pre-treatment with the PI3K inhibitor wortmannin. ( C ) Cartoon showing GIPR signaling pathways that may act on Akt. Inhibitors/ activators are in red. ( D-E ) Effect of pre-treatment with (D) the PKA inhibitor H-89 and (E) a Src inhibitor on p-Akt responses. N=9 in A and B, N=10 in D and N=6 donors in E. Each dot represents one donor measured with median shown in red in panels A, B, D, E. ( F ) Normalized mean fluorescence intensity ratio (340/380nm) of Fura-2-AM calcium imaging in an individual cell exposed to vehicle or GIP. Data are normalized to the 340/380 ratio at 0 seconds for each cell. ( G-H ) Maximal Ca 2+ I responses in all osteoclasts. G shows Emax of all cells measured. H shows average for each of the 4 donors. ( I ) Close-up of oscillations in a cell exposed to vehicle (left) and GIP (right). ( J-K ) Total number of Ca 2+ i oscillations and ( L-M ) amplitude of oscillations from all cells measured. K and M show averages for each of the 4 donors. Data for individual donors is shown in Figure S3. ( N ) Effect of pre-treatment with the calcium chelator BAPTA on p-Akt. N=6 donors. p-Akt was normalized to GAPDH in AlphaLISA assays. Each dot represents one cell measured with median shown in red. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Comparisons to vehicle-treated cells are labelled as a and to GIP-treated cells as b. Statistical analyses were performed using: Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test for panel 3A, 3B, 3D, 3E, 3N; Mann-Whitney test for panels 3G, 3J, 3L; unpaired t-test for panels 3H, 3K and 3M.

Article Snippet: Assays were performed in white 384-well plates (Optiplates, PerkinElmer) and AlphaLISA readings made on a Pherastar FS (BMG Labtech) plate reader.

Techniques: Generated, Protein-Protein interactions, Fluorescence, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: GIP receptor reduces osteoclast activity and improves osteoblast survival by activating multiple signaling pathways

doi: 10.1101/2022.07.02.498420

Figure Lengend Snippet: ( A ) Western blot analysis of phosphorylated p38 (p-p38) in osteoclasts exposed to vehicle or GIP for 30 minutes. (Right) Densitometry analysis of p-p38 relative to the calnexin loading control from 4 blots, each representing an independent donor. ( B ) Quantification of p-p38 measured by AlphaLISA. ( C ) Effect of pre-treatment with the calcium chelator BAPTA on p-p38 responses. ( D ) Effect of p38 inhibition on p-Akt in vehicle and GIP treated osteoclasts. ( E ) Quantification of phosphorylated p65 (p-p65), an NFκB subunit, in osteoclasts exposed to vehicle or GIP for 30 minutes measured by AlphaLISA. N=7 in B, N=6 donors in C-E. Data was normalized to GAPDH in AlphaLISA assays. ( F ) Representative images of p-NFκB in osteoclasts exposed to vehicle or GIP ± GIP(3-30)NH 2 for 60 minutes. DAPI was used to label nuclei and AlexaFluor488 to fluorescently label p-NFκB. ( G-H ) p-NFκB nuclear and cytoplasmic ratios in osteoclasts from N=4 donors. G shows ratios for all cells measured. H shows average for each of the 4 donors. Data for individual donors is shown in Figure S5. Comparisons to vehicle-treated cells are labelled as a and to GIP-treated cells as b. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Each dot represents one donor measured with mean or median shown in red in panels A, B, C, D, E, H. Statistical analyses were performed by: one-way ANOVA with Tukey’s multiple comparisons tests for panels 5A and 5B, with Dunnett’s test for panels 5D and 5G, and with Holm-Sidak’s test for panel 5H. Kruskal-Wallis one-way ANOVA with Dunn’s test was used for panels 5D and 5G.

Article Snippet: Assays were performed in white 384-well plates (Optiplates, PerkinElmer) and AlphaLISA readings made on a Pherastar FS (BMG Labtech) plate reader.

Techniques: Western Blot, Control, Inhibition

Journal: bioRxiv

Article Title: GIP receptor reduces osteoclast activity and improves osteoblast survival by activating multiple signaling pathways

doi: 10.1101/2022.07.02.498420

Figure Lengend Snippet: ( A ) Quantification of cAMP in osteoblasts exposed to vehicle (veh), GIP or GIP+GIP(3-30)NH 2 measured by LANCE assays. N=7 donors. ( B-C ) Quantification of phosphorylated Akt1/2/3 (p-Akt) in osteoblasts exposed to vehicle or GIP for 30 minutes measured by AlphaLISA. Cells were pre-treated with vehicle and (B) GIP(3-30)NH 2 , (C) the PKA inhibitor H-89 and PI3K inhibitor wortmannin. N=7 for B, N=6 donors for C. p-Akt concentrations were normalized to GAPDH in AlphaLISA assays. ( D ) Normalized mean fluorescence intensity ratio of Fura-2-AM. Data are normalized to the 340/380 ratio at 0 seconds for each cell. Data shows mean+SEM. N=4 donors. ( E-F ) Quantification of the maximal Ca 2+ I responses from data in panel D. E shows Emax of all cells measured. F shows average for each of the 4 donors. ( G ) Representative images of NFATc1 and ( H ) quantification of nuclear and cytoplasmic ratios in osteoblasts exposed to vehicle or GIP for 60 minutes. DAPI was used to label nuclei and AlexaFluor488 to fluorescently label NFATc1. N=5 donors. ( I ) Representative images of osteoblasts stained with TUNEL to detect apoptotic cells and DAPI to detect nuclei, with zoomed images of cells indicated by a yellow box shown below. ( J ) Quantification of the percentage of TUNEL-FITC positive cells. N=5 donors ( K ) Caspase-3/7 activity and ( L ) ATP generation, measured by CaspaseGlo and CellTiterGlo, respectively, in osteoblasts exposed to vehicle, GIP or GIP +/- inhibitors of signaling. N=6 donors. Comparisons to vehicle-treated cells are labelled as a, and to GIP-treated cells labelled as b, in panels J and K. ***p<0.001, **p<0.01, *p<0.05. Each dot represents one donor measured with mean or median shown in red in panels A-E, G-H. Statistical analyses were performed by: Kruskal-Wallis one-way ANOVA with Dunn’s test for panels 7A, 7B, 7E, 7F, 7H, 7K; one-way ANOVA with Dunnett’s multiple comparisons test for 7C and 7L; and unpaired t-test for 7J and 7M.

Article Snippet: Assays were performed in white 384-well plates (Optiplates, PerkinElmer) and AlphaLISA readings made on a Pherastar FS (BMG Labtech) plate reader.

Techniques: Fluorescence, Staining, TUNEL Assay, Activity Assay

Journal: iScience

Article Title: Characterization of adipose depot-specific stromal cell populations by single-cell mass cytometry

doi: 10.1016/j.isci.2022.104166

Figure Lengend Snippet:

Article Snippet: Anti-mouse c-Kit (2B8; Maxpar-ready) , Biolegend , Cat #105829; RRID: AB_2563710.

Techniques: Blocking Assay, Recombinant, Staining, Lysis, Expressing, Software