Ab76013

The organoids were then formalin fixed and paraffin embedded. Sections were deparaffinized with xylene, treated with antigen retrieval solution (1 g NaOH, 2.1 g citric acid in 1 L of H₂O) for 20 min in steam, cooled to room temperature, permeabilized with PBS-0.2% Triton for 15 min, and blocked with 10% donkey serum in PBS. Sections were stained with monoclonal rabbit antithyroid transcription factor 1 (TTF1, ab76013; Abcam, Cambridge, MA) or rabbit anti-CC10 (ab40873, Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 594-conjugated donkey antirabbit secondary antibody (ab150076, Abcam, Cambridge, MA, USA) at 1:500, goat anti-GFP (ab5450; Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 488-conjugated donkey anti-goat secondary antibody (ab150129, Abcam, Cambridge, MA, USA) at 1:500. Images were taken at 10×, 20×, and 40× with an Olympus FV1000 confocal microscope. Image analysis were performed using ImageJ software.

Lung adenocarcinoma tissue microarray analysis was purchased from the National Engineering Center for BioChips in Shanghai, China. A more complete description of the human specimens is included in Supplementary Table S11. The expression of phosphorylated GAC in the tissue was evaluated by immunohistochemical staining with an anti-GAC-pS314 antibody. The tissue microarray slide was deparaffinized, rehydrated, and subjected to an epitope retrieval step. Subsequently, 6% hydrogen peroxide was used to block endogenous peroxidase activity. The slide was washed three times in PBS and then incubated with the anti-GAC-pS314 antibody at 4 ℃ overnight. After three washes in PBS, the slide was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The stain was developed with either chromogen or haematoxylin solutions. The images of the tissue microarray were analyzed by ImageScope software. The immunohistochemical staining of tumors derived from the parental A549 and A549-GAC(S314A) cells was performed as indicated above. Anti-Ki67 and anti-TTF1 antibodies were purchased from Abcam (ab15580 and ab76013). Micrographs were obtained using an Olympus IX71 microscope.

At each differentiation stage, cells were fixed with 4% PFA for 15 min at room temperature then washed three times with PBS. The slides of cells were permeabilized with PBS + 0.5% Triton X-100 for 20 min and washed three times with PBS. Slides were blocked with 10% goat serum for 30 min and incubated with the primary antibodies at 4°C overnight (>16 h) diluted in PBS. Following incubation, the slides were rinsed with PBS and incubated with secondary antibodies at 37°C for 1 h. The slides were rinsed with PBS and the cell nucleus was stained with DAPI (Sigma Aldrich). The images were visualized using an Olympus BX41 fluorescence microscope. Dilutions and Catalog numbers for primary antibodies were as follows: Anti-Oct4 antibody (1:200 dilution) (Abcam, ab18976), Anti-Nanog antibody (1:200 dilution) (Abcam, ab80892), Anti-FOXA2 antibody (1:300 dilution) (Abcam, ab108422), Anti-SOX17 antibody (1:50 dilution) (Abcam, ab191699), Human/Mouse/Rat SOX2 antibody (1:200 dilution) (R&D Systems, AF2018), Anti-TTF1 antibody (1:200 dilution) (Abcam, ab76013), Anti-SOX9 antibody (1:200 dilution) (Abcam, ab26414). The secondary antibody: Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (1:500 dilution) (Abcam, ab150077), Rhodamine (TRITC)-conjugated AffiniPure Bovine Anti-Goat IgG (H+L) (1:100 dilution) (Jackson ImmunoResearch, 805-025-180).

Mice were anesthetized with isoflurane and perfused with PBS followed by 4% PFA at 11 am for SD and SD-Cont, and at 2 pm for RS and RS-Cont. Brains were fixed with 4% PFA overnight and placed into 30% sucrose until saturated. Thirty-micrometer cryosections were collected into PBS and stored in cryoprotectant at −20°C. For immunofluorescent staining, samples were stained using primary antibodies: anti-Nkx2-1 (TTF-1) (1:500, ab76013; Abcam) and secondary antibodies. To stain cFos, samples were stained with anti-cFos (1:1,000, 226003; Synaptic Systems) and universal biotinylated anti-mouse/rabbit IgG (Universal Elite ABC kit, PK-7200; Vector laboratories) antibodies with Universal Elite ABC kit and developed with Vector SG substrate kit peroxidase (SK-4700: Vector laboratories). The number of cFos-positive cells was quantified by visual scoring. Genotypes and conditions were blinded during the experimental procedures. For MAP2 immunofluorescence, 25-μm cryosections were stained using anti-MAP2 (1:100, ab5392; Abcam).