Sd208

Primary astrocytes were transfected with the Nef or GFP plasmids and incubated with or without the inhibitor, SD208 from Tocris (5 μM, daily). Cells were collected at different time points (24 and 48 h). Extraction of RNA was done using the All Prep kit (Qiagen, Valencia, CA), and the quality of the RNA was verified and quantified using an Experion Automated electrophoretic workstation (BioRad). RNA was converted to cDNA using iScript cDNA synthesis kit from BioRad. TGFβ-1 and CCL2 amplification was performed using SYBR Green Super mix from BioRad. Primers were obtained from Qiagen. Beta actin amplification was used as an internal control. Relative expression was determined using the 2^−ΔΔC_T method normalized using beta actin. We reported the relative expression compared to the GFP group.

Contraction Assay Kit (Cell Biolabs) was used to assess contractile properties of cultured cells following the manufacturer’s protocol. Briefly, Lin^-gp38⁺ cells were cultivated with or without 10 ng/mL TGF-β for 72 h. Primary human cardiac fibroblasts were cultivated with or without 10 ng/mL TGF-β in addition to the inhibitors SD208 or A83-01 (both Tocris) for 72 h. Next, cells were detached by trypsinization and re-suspend in proper medium at 2–5 × 10⁶ cells/mL. Two parts of cell suspension and eight parts of cold Collagen Gel Working Solution composed of collagen, DMEM and neutralization agent were mixed in order to plate and 0.25 mL of the cell-collagen mixture per well in a 48-well plate. Collagen was allowed to polymerize for 1 h at 37 °C. After that 1 mL of culture medium was added atop each collagen gel lattice. Each condition was analysed in triplicates or quadruplicates. Images were taken at time 0, 24, 48, and 72 h after seeding. Areas of the gels were measured by ImageJ. Percentage of contraction of all conditions was measured compared to the average of unstimulated cells at time 0 set as 100%.

The following drugs were added to the respective culture media as summarised in Table 1: 10 ng/mL recombinant human TGF-β1 protein expressed from a CHO cell line (R&D Systems, 240-B)^{35 (link)}, 30 nM TGF-β receptor 1 inhibitor SD208 (Tocris, 3269)^{36 (link)}, 10 μM TGF-β receptor inhibitor SB431542^{34 (link)}.Table 1

Experimental combinations and outcomes for HCFs and iPSC-cFbs.

Cell type	Substrates (E)	Drug treatments	Treatment duration	Outcome
HCF	3 GPa, 25 kPa, 2 kPa	10 ng/ml TGF-β, 30 nM SD208, 10 μM SB431542	2–4 days	Proliferation: CyQUANT; RT-PCR: α-SMA, collagen 1; IF: α-SMA; western blot: α-SMA, collagen 1
hiPSC-cFb	3 GPa	10 ng/ml TGF-β, 30 nM SD208, 10 µM SB431542	2–4 days	Proliferation: CyQUANT; RT-PCR: α-SMA, collagen 1, collagen 3; IF: α-SMA

α-SMA alpha-smooth muscle actin, E elastic modulus, HCF human cardiac fibroblast, IF immunofluorescence, hiPSC-cFb human induced pluripotent stem cell-derived cardiac fibroblast, TGF-β transforming growth factor-beta, RT-PCR real-time reverse transcriptase polymerase chain reaction.

SD208 [1 μM], SIS3 [2 μM], (5Z)-7-Oxozeaenol (OXO) [1 μM] pharmacological inhibitors used in the project were purchased from Tocris Biosciences. To determine optimal non-toxic concentrations, we performed toxicity tests. CD14⁺ monocytes were incubated with 2-fold dilutions of inhibitors starting from 5 or 10 µM. Cells were incubated for 24h, stained with propidium iodide (Biolegend), and cytotoxicity was evaluated by flow cytometry. The highest non-toxic concentration was used in further experiments.

Small molecule inhibitors were purchased from SelleckChem, with the exception of SD208 (Tocris), ITD-1 (Adooq Bioscience), and were diluted in dimethylsulfoxide (DMSO). Inhibitors were added to cells at indicated concentrations with a final DMSO concentration of 0.5%. Lactacystin and chloroquine were purchased from Sigma Aldrich and diluted according to manufacturer’s instructions, and added to cells at a concentration of 1 μM, 10 μM and 30 μM, respectively. IC₅₀ calculations were obtained using 8–9 concentrations of compound and generated using the IC50 Calculator software by AAT Bioquest®. NLK, p38, or ALK5 for in vitro kinase assays were immunopurified from Kp53A1 cells. NLK activation was induced by incubating cells at 32° for 24-48 h, p38 by stimulating with IL-3, IL-6, SCF, and Epo at the same concentrations used for differentiating erythroid progenitors. IC₅₀ values for each kinase and compound were plotted along a horizontal bar with our observed value superimposed next to documented values (if available). IC₅₀ values were obtained for each kinase against each substrate (e.g., NLK phosphorylation of NLK, c-Myb, and Raptor), but as IC50 curves were virtually identical for each substrate, only one is presented.