Rho kinase inhibitor y 27632

Human neoplastic organoid cultures were established from resected PDAC specimens. Specimens from pancreatic resections were digested enzymatically with collagenase/dispase dissociation and then plated in Matrigel to generate pancreas organoid cultures, in human complete medium [25 (link)]. PDAC organoids were then maintained in human complete medium. For the 50% of cell growth inhibition (IC₅₀) analysis, organoids were dissociated into single cells by first triturating them in media through a fire-polished glass pipette, and then by enzymatic dissociation with 2 mg/ml dispase dissolved in TrypLE (Life Technologies). Cells were counted and diluted to 10 cells/μl in a mixture of complete media, Rho Kinase inhibitor Y-27632 (10.5 μM final concentration, Sigma) and Growth factor-reduced Matrigel (GFR-Matrigel, 10% final concentration). 100 μl of this mixture (1000 cells/well) was plated in 96-well plates (Nunc), whose wells had been previously coated with a bed of GFR-Matrigel to prevent attachment of the cells to the bottom of the plate. Cell viability in response to the different drugs was measured using the CellTiter-Glo assay (Promega Corporation) and Synergy 4 reader (Biotek).

Infliximab (IFX), Etanercept (ETN), Certolizumab Pegol (CZP) were provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan), Takeda Pharmaceutical Company Limited (Osaka, Japan) and UCB Japan (Tokyo, Japan), respectively. Methotrexate and folic acid were obtained from Wako Chemicals (Osaka, Japan). Rho kinase inhibitor, Y27632, and PKH-26 were supplied from Sigma (St. Louis, MO, United States). TO-PRO-3 was from Thermo Fisher Scientific (Waltham, MA, United States). Rabbit anti-phospho JNK monoclonal antibody (mAb) and anti-JNK polyclonal antibody (Ab) were purchased from cell signaling technology (Danvers, MA, United States). Anti-GAPDH Ab was from Gene Tex (Irvine, CA, United States).

HaCaT cells were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/streptomycin at 37°C and 5% CO₂. HaCaT experiments were performed up to passage 30 in serum-free culture conditions using DermaLife K keratinocyte basal medium (Lifeline; SKU: LL-0007). Primary human keratinocytes were isolated from abdominal skin, foreskin, or actinic keratosis lesions collected at the Princess Alexandra Hospital Dermatology Centre (Brisbane, Australia) with patient consent and institutional approval (HREC/11/QPAH/477). Primary keratinocytes were cultured in DermaLife K keratinocyte basal medium supplemented with 10 μM Rho Kinase inhibitor Y-27632 (Sigma Aldrich; SCM075). Experiments on primary keratinocytes were performed up to passage eight. For S. aureus secretome stimulation assays, keratinocytes were seeded in 96-well plates and, unless otherwise stated, experiments were initiated at approximately 70% cell confluence. The keratinocytes were then incubated with sterile culture supernatant of S. aureus and bacterial medium control, diluted 1:40 in DermaLife medium, for 24 h. By end point, keratinocyte cultures reached confluence and cell supernatant was collected for cytokine quantification and the cell viability assessed as described below in 4.8 and 4.9, respectively.

786.0 [33] (link) and RCC10 [34] (link) were cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 7.5% CO₂. For inhibition of the RhoA pathway, cells were treated with the Rho inhibitor C3 transferase at a final concentration of 0.1 µg/ml (Cytoskeleton Inc., Denver, CO) or the Rho-Kinase inhibitor Y-27632 (Sigma-Aldrich, Steinheim, Germany) at a final concentration of 1 µM.

Primary human keratinocytes were isolated from abdominal skin and foreskin collected at the Princess Alexandra Hospital Dermatology Centre (Brisbane, Australia) with patient consent and institutional approval (HREC/11/QPAH/477). Primary keratinocytes were cultured in serum-free DermaLife K keratinocyte basal medium supplemented with 10 µM Rho Kinase inhibitor Y-27632 (Sigma Aldrich, Castle Hill, NSW, Australia; SCM075). Experiments on primary keratinocytes were performed up to passage eight. To explore the effects of S. aureus secreted compounds on human keratinocytes, the keratinocytes were cultured in S. aureus secretome, or DSM as control, diluted in antibiotic-free DermaLife medium. The MTT assay was used to establish the dose of S. aureus secretome that did not compromise cell viability and growth.