D9564

Cells were treated with anti-cancer drugs on day 3 for 72 h under co-culture conditions based on the microchannel chip. Gemcitabine (GEM), 5-fluorouracil (5-FU), and oxaliplatin (LOHP) were added at concentrations of 0, 0.1, 1, 10, and 100 μM and paclitaxel (PTX) at concentrations of 0, 0.01, 0.03, 0.1, 1, and 10 μM. To measure cell viability, cells were stained with 5 μM calcein AM (BDA-1000, BIOMAX, Seoul, Korea) diluted in culture medium to stain live cells, and then cell viability was measured by calcein AM intensity. Dose-response relationships were determined using an E_max model: $$\% \mathrm{Cell} \mathrm{viability}=\left(100-R\right)\times \left(1-\frac{{\left[D\right]}^m}{K_d^m+{\left[D\right]}^m}\right)+R$$
where (D) is the drug concentration, K_d is the concentration of drug that produces a 50% reduction of the maximum inhibition rate (E_max), m is a Hill-type coefficient and R is the residual unaffected (resistance) fraction (R = 100 − E_max). IC₅₀ was defined as the drug concentration required to reduce viability to 50% of the control (i.e., K_d = IC₅₀ when R = 0). The curves were fitted using GraphPad Prism 9.0. (GraphPad Software, Inc., San Diego, CA, USA). For measurement of invasion, cells were stained with rhodamine phalloidin (1:1000, R415, Invitrogen) and DAPI (1:1000, D9564, Sigma Aldrich) to stain F-actin and nuclei, respectively. Stained cells were observed using a confocal microscope, and images were analyzed.

KYSE30 and KYSE200 cells were seeded onto coverslips (801010, NEST) in 12-well plates. After treatment or transfection, the cells were fixed with methanol (−20 °C) for 10 to 15 min. The cells were washed three times with PBS, and blocked in 5% normal donkey serum (017-000-121, Jackson) for 1 h at 25 °C. The cells were incubated with anti-Flag (F3165, Sigma-Aldrich, 1:1000), and ATP1A1 (14418-1-AP, Proteintech, 1:100) overnight and stained with Alexa Fluor 488-conjugated AffiniPure donkey anti-mouse IgG (H + L) (1:200, 715-545-150, Jackson) or Alexa Fluor 594-conjugated AffiniPure donkey anti-mouse IgG (H + L) (1:200, 711-585-152, Jackson) secondary antibodies for 1 h at 25 °C. Then, the cells were washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D9564, Sigma, 1 μg/μl) at 25 °C for 10 min to visualize the nuclei. Images were obtained and processed by laser-scanning confocal microscopy (LSM800, Carl Zeiss).

Immunofluorescence was performed with spc- and cc10-specific antibodies, according to the following procedure. Paraffin sections (5 um) of lung tissue were pretreated for antigen-retrieval with Sodium Citrate buffer, pH 6, followed by cooling for 20 mins to room temperature. Subsequent to blocking with 2% bovine serum albumin in PBST, sections were incubated with antibodies against lung cell-type-specific marker proteins: rabbit Anti-Prosurfactant Protein C (pro SPC, 1:1000, AB3786 Millipore) and goat Anti-cc protein 10 (CC10, 1:400 Santa Cruz biotech., INC.). The antigen-antibody complexes were visualized by commercially available fluorescently labeled secondary antibodies: anti-rabbit IgG Alexa 647 (1:100, Invitrogen) and anti-goat IgG-Cy3 (1:400, LiStarFish). Sections were then incubated with DAPI (1:5000, D9564 Sigma-Aldrich), fixed with 2% PFA and mounted in mowiol mounting medium.

Frozen tissue sections were used for immunofluorescence assay to visualize the expression of caspase 3. Briefly, the cryostat sections were thawed at room temperature for 20 minutes and followed by incubation in blocking buffer (4% BSA) for 30 minutes. Subsequently, immunostaining was performed with an anti‐caspase 3 antibody (ab32351, Abcam) and an Alexa Fluor 594 WGA (Life Technologies). Nuclei were counterstained with 0.1 μg/mL DAPI (Sigma‐Aldrich; D9564). Images were captured using a Nikon Eclipse Ti‐U fluorescence microscope at ×400 magnification.

Cultured cells were washed once with Dulbecco's phosphate-buffered saline (DPBS; 8115028, Gibco) and fixed with ice-cold 4% paraformaldehyde (P6148, Sigma) for 10 min at 37 °C. Then treated by mixed solution of 0.5% TritonX -100 (T9284, Sigma) and 10% donkey serum (017-000-121, Jackson Immuno Reserach) for 30 min at room temperature for permeabilizing and blocking. The cells were then incubated with primary antibodies against vWF (1:50, 555849, BD) overnight in the dark at 4 °C. After three washes with DPBS, cells were incubated in 1% BSA (E661003, Sangon) in PBS containing secondary antibodies Alexa Fluor^® 555 (1:1000, A32727, Invitrogen). Nuclei were counterstained with DAPI (1:1000, D9564, Sigma). Images were acquired in an epifluorescence microscopy (Nikon Eclipse TE2000-U).

800 C3H/10T1/2 cells were seeded per well in a 96 well plate (glass bottom culture plates, MatTek, PBK96G-1.5-5-F) and kept at 37-degree Celcius for 24 h. Cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.2% digitonin (EMD Millipore, 300410) in PBS (Corning, 21-040-CV) for 10 min at room temperature. Next, the cells were incubated in 3% BSA (in PBS, Sigma, #9418) for blocking for 1 h at room temperature followed by incubation in primary antibody (ATRX, Santa Cruz, sc-15408, 1:200) at 4°C overnight, followed by secondary antibody (1:600, Invitrogen, A32754) incubation at room temperature for 1 h. Cells were washed three times with PBS for 5 min followed by DAPI staining (2 μg/ml in PBS, Sigma, D9564) for 5 min at room temperature. Mounting media (Vectashield, H-1000) was added immediately after DAPI staining. Cells were imaged using Widefield Microscope CellDiscoverer7 (CD7) automated widefield high-throughput system (Zeiss). Images were processed with ImageJ software (http://rsb.info.nih.gov/ij/). For the ATRX antibody, the ImageJ Brightness/Contrast was set as 30/112. For DAPI, the Brightness/Contrast was set as 37/123. All images were processed with the same parameter settings for each antibody.