Densitometry analysis of western blots results of proteins regulated by PIM and PI3K/mTOR pathway in MIA PaCa-2 GemR cells treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302 for 24 hours.

Each experiment was performed in triplicate and the results are shown as mean ±SD (N = 3). Densitometry analysis was performed using FIJI software [61]. Statistical analysis was performed using the Student’s test followed by an analysis of the normal distribution (Tukey’s test). *p (PDF)</p

AUM302 inhibits the cell signaling pathways regulated by PIM kinases and PI3K/mTOR pathway.

BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E) cells were treated with DMSO (vehicle) or TP-3654 (10 and 100 nM) or AUM302 (10 and 100 nM) for 24 hours.</p

AUM302 inhibits the proliferation of multiple pancreatic cancer cell lines.

The following pancreatic cancer cell lines, BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E), were treated with DMSO or TP-3654 (10 nM and 100 nM), or AUM302 (10 nM and 100 nM). Cell count was determined 24, 48, and 72 hours after treatment using a cell counter. The measurement of the control (cells with DMSO) was defined as 100%. Data represent mean ±SD (N = 6). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.</p

AUM302 and TP-3654 decrease the cell viability of MIA PaCa-2 gemcitabine-resistant (GemR) cell line.

MIA PaCa-2 GemR cell line was treated with 10 nM, 100 nM, or 1 μM of TP-3654 (A, B, & C) or AUM302 (D, E, & F) twenty-four hours after seeding. Cells were treated with test compounds for 72 hours and cell viability was measured using Cell Titer-Glo. Data represents mean ±SD (N = 6 and N = 4). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.</p

AUM302 inhibits the proliferation of MIA PaCa-2 gemcitabine-resistant (GemR) cells and activity of multiple signaling pathways.

(A) MIA PaCa-2 GemR cells were treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302. Cell count was determined 24, 48, and 72 hours after treatment using a cell counter. The measurement of the control (cells with DMSO) was defined as 100%. Data represent mean ±SD (N = 9). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA. (B—D) MIA PaCa-2 GemR cells were treated with DMSO (vehicle) or TP-3654 or AUM302 (10 nM, 100 nM, and 1 μM) for 24 (B), 48 (C), and 72 (D) hours and analyzed with western blot.</p

AUM302 changes the cell cycle profile of Hs766T pancreatic cancer cell line.

Cells were treated with DMSO or TP-3654 (100 nM) or AUM302 (100 nM) for 24 (A), 48 (B), and 72 hours (C). Cells were stained with propidium iodide and analyzed by FACS analysis. Data are represented as mean ±SD, N = 3, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.</p

Functional recovery after TKA surgery in rats with OA.

(A) Lateral knee radiograph (left), H&E (middle), and Sirius red (right) staining of the proximal tibia from a healthy rat. (B) Lateral knee radiograph (left), H&E (middle), and Sirius red (right) staining of the proximal tibia from an OA rat 4 weeks post-DMM surgery. (C) Incapacitance at 4 weeks after DMM or Sham surgery. *, p D-H, rats underwent TKA or Sham surgery 4 weeks after OA induction via DMM (OA + TKA) or Sham (Sham + Sham) procedures, respectively. (D) Incapacitance, (E) home cage locomotion, (F) rearing, (G) SFI, and (H) stride length. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs baseline, as determined by One-way ANOVA followed by Dunnett’s post-hoc test (n = 8).</p

TKA prostheses design.

Femoral and tibial prostheses were designed based on the dimensions of skeletally mature Sprague Dawley rats. (A) Measurements (mm) and schematic representation of the tibial and femoral prostheses. (B) Surface rendered model of femoral and tibial prostheses. (C) Photograph of the manufactured prostheses.</p

DRG transcriptome evaluation 24h after TKA surgery.

(A) Hierarchically clustered heat map comparing DRG transcriptomes between individual Sham and TKA rats (n = 6). (B) Volcano plot showing relative expression of DRG transcriptomes from TKA rats compared to Sham. The x-axis shows the log2 fold change and the y-axis shows the -log10 adjusted p values. Thresholds for significant changes in expression were set at ≥1.5 fold-change and adjusted p values C) Gene Set Enrichment Analysis (GSEA) plots for upregulated pathways in DRGs from TKA rats compared to Sham.</p

Analgesic efficacy after TKA surgery.

(A) Incapacitance on day 1 before and after vehicle (saline), morphine (1 mg/kg), ketorolac (10 mg/kg), combined ketorolac and morphine, or gabapentin (100 mg/kg). Dashed line indicates the mean incapacitance value of the Sham group. (B) Percent reversal of incapacitance for each rat in A. (C) Incapacitance on day 7 before and after vehicle, morphine, or ketorolac. Dashed line indicates the mean incapacitance value of the Sham group. (D) Percent reversal of incapacitance for each rat in C. (E) SFI on day 7 before and after morphine, ketorolac, or gabapentin. Dashed line indicates the mean SFI value of the Sham group. (F) Percent reversal of SFI for each rat in E. (G) Stride length on day 7 before and after morphine, ketorolac, or gabapentin. Dashed line indicates the mean stride length value of the Sham group. *, p < 0.05; **, p < 0.01; ***, p < 0.001, as determined by paired t-tests (n = 8).</p