Journal: BMC Biology

Article Title: An integrated in silico - in vitro approach for identifying therapeutic targets against osteoarthritis

doi: 10.1186/s12915-022-01451-8

Figure Lengend Snippet: In vitro validation of in silico predictions on chondrocyte phenotype changes. A Concept of in silico identification of potential drug targets. B Secreted ALP activity, relative to DNA quantity, positively linearly correlates with Col10a1 gene expression during hypertrophic differentiation with and without Ihh treatment. Results of one representative experiment. Each point is the average of 3 replicates and bars denote standard deviation. C Effect of PKA or SMAD3 activation as measured in silico and in vitro in ATDC5 ( N = 3 replicates, histograms show average fold change in ALP activity relative to control and bars are standard deviations, p -values are computed with one-tailed t -test and Welch’s correction) and human chondrocytes from OA donors (N= 4 donors with 3 replicates each, p -value is computed with one-tailed linear mixed effect model). In silico activation was performed by setting the variables to their maximum value (1.0), in vitro PKA (resp. SMAD3) activation was performed with Forskolin 1μM (resp. Activin 100ng/ml) for 24h. D Single and combinatorial drug screening in ATDC5 with selected conditions based on in silico predictions. Boxplot of the series of conditions across independent replicates ( z -scores of ALP activity fold change) with control conditions in purple. Conditions significantly lower than the control (combined p -value < 0.05) have dark grey borders and dots (Wilcoxon rank-sum test with BH correction and combined probabilities over independent runs). For each condition, dots are the average of biological triplicates, summary statistics are represented by a horizontal line for the median of independent experimental repetitions and a box for the interquartile range. The whiskers extend to the most extreme data point that is not >1.5 times the length of the box away from the box. Blue labels indicate potent conditions predicted by the in silico model, gray labeled conditions are added to the experimental set-up for information. CM stands for ‘control medium’, medium1 has 0.02% of DMSO and medium 2 0.035%. * Indicates in silico predicted conditions without significant decrease of ALP activity in vitro

Article Snippet: Cells were treated with one or a combination of the following compounds: Forskolin (1μM, Axon Medchem), Recombinant Human/Mouse/Rat Activin A Protein (100ng/ml, R&D Systems), Recombinant Mouse IGF-I/IGF-1 Protein (10ng/ml, R&D Systems), Transforming Growth Factor (TGF)β1 (10ng/ml, PreproTech), PD0325901 ( 1μM, Axon Medchem), PD161570 (1μM, Axon Medchem), ITSA1 (50μM, Chembridge), LDN-193189 (0.5μM, Axon Medchem), LY294002 (20μM, Axon Medchem) and IWP2 (2μM, Stem cell technology).

Techniques: In Vitro, Biomarker Discovery, In Silico, Activity Assay, Gene Expression, Standard Deviation, Activation Assay, Control, One-tailed Test, Drug discovery, Labeling

Journal: BMC Biology

Article Title: An integrated in silico - in vitro approach for identifying therapeutic targets against osteoarthritis

doi: 10.1186/s12915-022-01451-8

Figure Lengend Snippet: In-silico vs. In-vitro dose-response effect of PKA activation with FGFR1 inhibition. The most potent condition from the screening is investigated further for a potential dose effect. A Fold change (FC) in ALP activity, with respect to control, due to PKA activator (Forskolin, 1μM) or FGFR1 inhibitor (PD161560, 125nM, and 625 nM) or the combination of both. B A range of values for PKA and FGFR1 imposed activities is screened in silico with 0 meaning no activity and 1 being the max possible activity. The percentage of transitions remaining in the hypertrophic state or transitioning towards the healthy state is reported in the upper panels, the rest of the transitions go to the “None” state. In the middle panels, fold change (resp. inverse of fold change) in DNA-normalized ALP activity with respect to control DMSO in ATDC5 is reported for a range of Forskolin and PD161570 concentrations. The in-vitro situation without drugs (yellow rectangle) would correspond to the basal level of PKA and FGFR1 in in-silico hypertrophy but there is no one-to-one correspondence between the in silico and in vitro ranges. All in vitro results represent n=9 (3 bio-replicates in 3 independent experiments), p -values are computed on log-transformed data with a linear mixed-effect model, user-defined contrasts (only combination versus corresponding single doses were compared), one-sided test and adjustment for multiple comparisons with the Holm’s method. The combinatorial treatment effects were greater than the ones for either of the single treatment both in silico and in vitro , for all concentrations in the gradient of dose relationships investigated

Article Snippet: Cells were treated with one or a combination of the following compounds: Forskolin (1μM, Axon Medchem), Recombinant Human/Mouse/Rat Activin A Protein (100ng/ml, R&D Systems), Recombinant Mouse IGF-I/IGF-1 Protein (10ng/ml, R&D Systems), Transforming Growth Factor (TGF)β1 (10ng/ml, PreproTech), PD0325901 ( 1μM, Axon Medchem), PD161570 (1μM, Axon Medchem), ITSA1 (50μM, Chembridge), LDN-193189 (0.5μM, Axon Medchem), LY294002 (20μM, Axon Medchem) and IWP2 (2μM, Stem cell technology).

Techniques: In Silico, In Vitro, Activation Assay, Inhibition, Activity Assay, Control, Transformation Assay