Hoechst 33258

HAECs were seeded into gelatin-coated 96-well plates and stimulated with 50 ng/mL TNFα or 10 ng/mL IL-1β for 5 h, 24 h, or 5 d. After the incubation, the cells were fixed with 4% PFA (Sigma-Aldrich) for 10 min. Membrane permeabilization was performed with 0.2% Triton-X 100 (Serva, Heidelberg, Germany) and blocking of unspecific binding sites with 3% goat serum (Abcam). The cells were stained with primary rabbit monoclonal antibody against ACE2 (1:100, #MA5-32307, Invitrogen) and goat anti-rabbit Alexa Fluor 647 (1:500, #A21245, Invitrogen) antibody. Nuclear counterstaining was performed by using 1 µg/mL Hoechst 33258 (Cayman Chemical, Ann Arbor, MI, USA). Fluorescent images were taken on an inverted Olympus IX71 microscope (Olympus, Shinjuku, Japan) with a 10× air objective using the 385 nm excitation line with a 447/60 blue emission filter (DAPI/Hoechst) and the 660 nm excitation line with a 692/40 Far red emission filter for ACE2. For quantification, background correction was performed, and ACE2 staining intensity was normalized to the DNA staining intensity.

Cells that had grown on top of cell culture inserts were quickly blotted on absorbent paper (from the bottom), and 500 µL of paraformaldehyde (PFA) 3.7% were immediately added to each insert hold in a 12-well plate and incubated at room temperature for 20 min. PFA was gently removed with dry tissue, and inserts were cut from the plastic chamber and placed on top of glass microscope slides. Then each 200 µL/insert was washed twice with PBS for 5 min. For blocking, the inserts were incubated for 30 min at room temperature in blocking buffer (PBS, 0.1% triton X, 1% BSA, and 10% FBS). Following blocking, primary antibody was prepared according to the concentrations in Table 1, in the same blocking buffer w/o triton X, and cells were incubated in 70 µL/insert of primary antibody mix overnight at 4°C in a humidified chamber. Following overnight incubation, samples were washed three times with PBS at room temperature (5 min/each 200 µL/insert). Tissue paper was used between washes for drying. Secondary antibody was prepared in blocking buffer and 2 μg/mL Hoechst 33,258 (Cayman chemicals) were added and cells were incubated in this mix for 1 h at room temperature in the dark. Finally, after two washes in PBS for 5 min/each in the dark, the samples were air dried, treated with glass slowfade (Thermo Fisher) and covered with a glass coverslip.

CCK-8 cell viability assay was performed, as described by the manufacturer (Dojindo Laboratories, Kumamoto, Japan). NSCLC cells were seeded into 96-well plates (1x10⁴ cells/well) and treated with various concentrations of crizotinib or DMSO for 24 h in the presence or absence of nivolumab. Similarly, the flow cytometry-based cytotoxicity was performed, as described protocol. Briefly, the target cells were labeled with CFSE (1 x 10⁶ cells in 1 ml PBS with 0.5 uM CFSE, 20 min, 37˚C in the dark) and washed twice with warm culture medium. CFSE-labeled 5 x 10⁴ tumor cells were incubated at various concentrations of crizotinib for 24 h with CIK cells pre-incubated with 20 μg/mL nivolumab or IgG 4 isotype (24 h) to perform redirected cytolysis assay at an E/T ratio of 10:1. Following 24 h of culturing, the cells were stained with Hoechst 33258 (Cayman Chemical, Hamburg, Germany) and were quantified using BD FACS Canto II. The absolute number of 3000 beads (Biolegend, San Diego, CA) was acquired by a BD Canto II cytometer. Then the absolute numbers of cells per uL were analyzed by FlowJo V10 software (Tree Star, Ashland, Oregon). The absolute number of cells were calculated according to Precision Count protocol provided by Biolegend Company as follows:

Cells were seeded in 96-well plates one day before starting an experiment to allow cells to attach to the plate (5000 cells per well). One plate was used for each day of the proliferation curve. In the plate for day 0, a standard curve of known cell number quantified with an automated cell counter (TC20, Bio-Rad) was prepared (0–40,000 cells per well). At a specific time of day, the media from the plate 0 were removed, cells were rinsed with PBS, PBS was removed and plates were stored at –80 °C. If necessary, a treatment was started in other plates at this point. A plate was collected every day at the same time as described above. At the end of the proliferation curve, DNA content was quantified using Hoechst 33258 (Cayman Chemical) as a proxy of cell number. Each well was incubated with 100 μL of H₂O for 1 h at 37 °C and afterward plates were placed at –80 °C until frozen. After thawing at 37 °C, a working dye solution (100 μL) was added to each well so that the final concentration was 1 μg/mL Hoechst 33258, 100 mM NaCl, 10 mM Tris-Cl, 10 mM EDTA (pH 7.4). Fluorescence was measured with the Tecan Infinite plate reader (excitation 350 nm, emission 455 nm). The blank value was subtracted and the proliferation curve was calculated using the standard curve from day 0.

Biological replicates of HNF1B WT and HNF1B R295C reprogrammed cells were washed with ice-cold PBS and fixed in 4% PFA (Santa Cruz Biotechnology, sc-281692) for 20 minutes at room temperature. Cells were permeabilized for 15 minutes in PBST (0.5% Triton X-100 in PBS) and blocked 2 hours with 5% normal goat serum (abcam, ab7481) and 1% BSA (MP Biomedicals, 0216006980) in PBST (blocking solution). Primary and secondary antibodies were incubated overnight and 2 hours, respectively. DNA of fixed cells was stained with 1:4000 dilution of Hoechst 33258 (Cayman, Cay16756-50) in PBS. Primary and secondary antibodies were diluted in the blocking solution. Antibodies and dilutions used for immunofluorescent staining are provided in Supplemental Table 9. Samples were mounted with an Ibidi mounting medium (Ibidi, 50001) and analyzed using a Leica DMI 6000 fluorescence microscope or an Olympus spinning disk for 3D cultures.

Sulfated proteoglycans and GAGs were quantified using the 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich, MA, USA) protocol [82 (link)]. The iPSCs and neurons were digested in a prepared papain solution (containing 1 mM EDTA, 2 mM DTT, and 0.3 mg/mL papain enzyme) (Sigma-Aldrich, MA, USA) at 60 °C for 1 h. Iodoacetic acid (Sigma-Aldrich, MA, USA) was then added to a final concentration of 10 mM, followed by the addition of 0.5 ml of 50 mM Tris/HCl (Sigma-Aldrich, MA, USA). Briefly, 20 μL of sample was mixed with 200 μL of DMMB reagent, and absorbance was read at 525 nm. A standard curve was established from chondroitin-6-sulfate (Sigma-Aldrich, MA, USA) to compare absorbance for the samples. The DNA content was measured using a fluorometric assay. Total GAG levels were normalized to total DNA content. Aliquots of the sample digestion were stained with 200 μL of Hoechst33258 (Cayman Chemical, MI, USA) working solution (2 μg/mL). The fluorescence intensities were then detected at 355 nm for excitation and 460 nm for emission. Both Hoechst-stained DNA and GAG contents were measured by a SpectraMax i3x Multi-Mode Microplate reader (Molecular Devices, CA, USA).