Additional file 1 of MyD88 dimerization inhibitor ST2825 targets the aggressiveness of synovial fibroblasts in rheumatoid arthritis patients

Additional file 1: Fig. S1. Cell cycle analysis on hDF and OA SFs treated with ST2825. Representative histograms show cell cycle differences among different ST2825 concentrations and time points. G0/G1-phase cells, S-phase cells, and G2/M-phase cells are shown in the figure for hDF (A) and OA SFs (B). The y-axes represent the total count of cells, and the x-axes represent integrated intensity of DAPI measured by whole-well image cytometry. Fig. S2. Transcriptomic analysis revealed the upregulation of critical inflammatory mediators in RA SFs. (A) Description of OA and RA SFs used in RNA-seq experiments. (B) Volcano plot of up and downregulated genes in RA SFs compared OA SFs by 1.5 FC and p-value < 0.05 (C) Hierarchical clustering of the genes differentially expressed genes by Euclidean distance and centroid linkage method (D). Ingenuity Pathway Analysis (Qiagen) showing the top 5 canonical pathways predicted to be associated with the genes up and downregulated in RA SFs. (E) Heatmaps Log2 RPKM values of genes associated with canonical pathways identified in panel D. Fig. S3. Cell cycle analysis of RA-FLS treated with ST2825 at 48 and 72 h. Quantification of the percentage of RA SFs in various phases of the cell cycle upon ST2825 treatment at 48 (A) and 72 (B) h by imaging cytometry. One-way ANOVA and Dunnett’s multiple comparisons test were performed to determine statistical significance. *p<0.05, ****p<0.0001. Fig. S4. Cell cycle analysis on RA SFs treated with ST2825. Representative histograms show cell cycle differences among different ST2825 concentrations and time points. Apoptotic cells (yellow), G0/G1-phase cells (green), S-phase cells (blue), and G2/Mphase cells (purple) are shown in the figure. The y axes represent the total count of cells, and the x axes represents integrated intensity of DAPI measured by whole-well image cytometry. Fig. S5. Apoptosis analysis of RA-FLS treated with ST2825. Apoptosis was determined on RA SFs treated with 0, 5, and 10 μM of ST2825 at 0, 24, 48, and 72 h. No statistically significant differences were observed after 24 and 48 h of incubation with ST2825. The percentage of apoptotic cells significantly increased after 72 h of incubation with 10 μM of ST2825 (p=0.0476). Two-way ANOVA and Dunnett’s multiple comparisons test were used to determine statistical significane. *p<0.05. Fig. S6. Transcriptomic analysis identified DEGs and canonical pathways in RA SFs treated with ST2825. (A) Volcano plot of up and downregulated genes in RA SFs compared ST2825-treated RA SFs by 1.5 FC and p-value < 0.05. Enriched pathway analysis showing the top 10 canonical downregulated (B) and upregulated (C) pathways predicted to be associated with the genes up and downregulated in RA SFs treated with ST2825. Fig. S7. Transcriptomic analysis identified the effect of ST2825 on DEGs and canonical pathways in LPS-treated RA SFs. (A) Volcano plot of up and downregulated genes in LPS-stimulated RA SFs after treatment with ST2825 by 1.5 FC and p-value < 0.05. Enriched pathway analysis showing the top 10 canonical downregulated (B) and upregulated (C) pathways predicted to be associated with the genes up and downregulated in RA SFs. Fig S8. Raw data of western blot images. (A) Fig 5A and (B) Fig 5C. Table S1. Primer sequences for qRT-PCR. Table S2. Antibodies used for western blot

Additional file 3 of MyD88 dimerization inhibitor ST2825 targets the aggressiveness of synovial fibroblasts in rheumatoid arthritis patients

Additional file 3: Supplementary Data 2. Reactome Pathway Enrichment LPS-stimulated RA

Additional file 2 of MyD88 dimerization inhibitor ST2825 targets the aggressiveness of synovial fibroblasts in rheumatoid arthritis patients

Additional file 2: Supplementary Data 1. Reactome Pathway Enrichment

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

<div><p>Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.</p></div

Increased cartilaginous and bony matrix in remodeling fracture calluses of YF mice.

<p>For histological analysis, mid-sagittal sections of fractured femora were stained with Safranin O and fast green and quantitated by static histomorphometry. <b>A.</b> Representative sections at 7, 14, 21, and 28 days post-fracture. Bar charts show <b>B.</b> Total callus area <b>C.</b> Cartilaginous matrix area <b>D.</b> Bony matrix area. <b>E.</b> Ratio of cartilaginous matrix area over total callus area <b>F.</b> Ratio of bony matrix area over total callus area. n = 5–6, *p<0.05 vs. WT.</p

Fractured femora lacking Cbl-PI3K interaction are delayed in restoring mechanical strength.

<p>Torsion testing was performed on fractured femora during remodeling of the hard callus at 21 and 35 days post-fracture. Bar graphs show <b>A.</b> Peak torque and <b>B.</b> Work. n = 11 *p<0.05 vs. WT.</p

LCM Primer Sequences.

<p>LCM Primer Sequences.</p

MicroCT measurements showed increased total callus and bony volume in mice lacking Cbl-PI3K interaction.

<p>A. Fractured femora were harvested from WT and YF mice at 14, 21 and 28 days post-fracture, and scanned by MicroCT. Bar charts show B. Total callus volume C. Bony callus volume D. Ratio of bony over total callus volume. n = 5–6, *p<0.05 vs. WT.</p

Lack of Cbl-PI3K interaction results in increased osteoblast surface, incorporation of dynamic bone labels, and up-regulation of master transcription factor Osterix in remodeling fracture calluses.

<p>Mid-saggital sections of fractured femora were stained with Safranin O and Fast Green, and counterstained with Hematoxylin. <b>A.</b> Representative sections at 7, 14, 21, and 28 days post-fracture showing bony regions of fracture calluses. <b>B.</b> Osteoblast surface over bone surface (ObS/BS) measured by static histomorphometry. n = 6, *p<0.05 vs. WT. <b>C.</b> Mice were injected with Alizarin Complexone (red) 4 days prior to sacrifice, and Calcein (green) 1 day prior to sacrifice. Mid-sagittal sections of fractured femora were used to image incorporation of dynamic bone labels in the center of the fracture calluses at 14 and 21 days post-fracture. Cells from sections of calluses at 7, 14, and 21 days post-fracture were harvested by LCM, and expression of <b>D.</b> Runx2, and <b>E.</b> Osterix were quantified by qRT-PCR n = 6, *p<0.05 vs. WT.</p

Histomorphometric parameters of bone resorption.

<p>Mid-sagittal sections of fractured femora were TRAP stained and counterstained with Alcian Blue and Hematoxylin. Three sections/mouse and 3 mice/genotype at 14 and 21, days post-fracture were analyzed. Osteoclast Surface, erosion surface and bone surface as determined by histomorphometry are shown. Values shown are mean ± SD from WT mice n = 3, YF mice n = 3</p><p>* p<0.05 was considered statistically significant as compared to respective controls using analysis of variance with post-hoc analysis (ANOVA) with Bonferroni post-hoc test.</p><p>Histomorphometric parameters of bone resorption.</p