Hcs studio cell analysis software 6

Fibroblasts were platted in a 96-well, black-walled imaging plates (BD Biosciences, NJ, USA) at a density of 4 × 10³ cells/well. Fibroblasts were incubated for 5 days with or without treatment. Afterwards, plates were washed with PBS and cells were removed with 0.25 M ammonium hydroxide in 25 mM Tris for 15 min at 37 °C. The plate with the remaining extracellular matrix (ECM) was washed three times with PBS and fixed with 100% methanol for 30 min at −20 °C and finally washed three more times with PBS. ECM was immunostained with monoclonal mouse anti-Fibronectin antibodies conjugated with Alexa Fluor 488 (eBiosciences, Frankfurt am Main, Germany), polyclonal rabbit anti-Collagen Type I antibodies (Merck Millipore, Darmstadt, Germany) and polyclonal rabbit anti-Collagen Type III antibodies (Merck Millipore). See Supplementary Table 3 for more information about antibodies used in this experiment. Samples were analyzed using CX5-based automated quantification of extracellular matrix (Thermo Fisher Scientific; software HCS Navigator Version 6.6.1). Data were analyzed using HCS Studio Cell Analysis Software 6.6.1 (Thermo Fisher Scientific).

Human fibroblasts were cultured in 96-well plates. After 24 h, we differentiated fibroblasts into myofibroblasts by incubation with TGFβ for three days, with re-stimulation with TGFβ every twenty-four hours. We then treated the cells with CQ (10 µM, Biotrend) or 3-MA (5 mM, Sigma-Aldrich). Alternatively, we knocked down ATG7 and BECLIN1 (50 nM) (pre-designed, Invitrogen, CA, USA), via siRNA transfection using Viromer Blue transfection reagent (OriGene Technologies, Rockville, MD, USA), and continued the stimulation with recombinant TGFβ for two additional days. The control group (Fib) was pre-treated with vehicle for three days, then transfected with non-targeting siRNA and treated with vehicle for additional two days. The deactivated myofibroblast group (deact MyoFib) was pre-treated with TGFβ for three days, then transfected with non-targeting siRNA and treated with vehicle for two days. Afterwards, cells were fixed with 4% PFA and immunostained for stress fibers and αSMA as described above (immunohistochemistry and immunofluorescence staining section). Images were captured using CellInsight CX5 High-Content Screening (HCS) Platform (Thermo Fisher Scientific; software HCS Navigator Version 6.6.1). Data were analyzed using HCS Studio Cell Analysis Software 6.6.1 (Thermo Fisher Scientific).