Anti ttf1 8g7g3 1

Tissue isolation, fixation and staining procedures were performed as previously described in Shackelford et al., 2013. Briefly, the following antibodies were used: phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology, #2855 1:1600), cleaved caspase-3 (Asp175) (Cell Signaling Technology, #9661 1:200), anti-p63 (4A4) (abcam, #ab111449 1:100), anti-TTF-1 (8G7G3/1) (Dako, 1:200), anti-Ki67 (SP6) (Thermo Scientific, #RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), hexokinase II (Cell Signaling Technology, #2867 1:800), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, #2535 1:100), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (Cell Signaling Technology, #4370 1:400), phospho-GSKα/β (Ser21/9) (Cell Signaling Technology, #9331 1:50). Immunostained slides were digitally scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). A pathologist blindly evaluated the H&E stained sections for distribution of histological subtypes, verified by p63 and TTF-1 immunostaining, and calculated the percentage of positively stained cells for Ki67, cleaved caspase-3, phospho-S6, and hexokinase II. Digital slides were analyzed with the Definiens’ Tissue Studio (Definiens Inc. Parsippany, NJ) to determine adenocarcinoma tumor area and necrotic tumor area.

After the fixation step in 10% neutral buffered formalin overnight, lungs were transferred to 70% ethanol and further processing and embedding was done by the TPCL at UCLA. The following antibodies were used: anti-CK5 (EP1601Y) (abcam, ab52635 1:100), anti-TTF1 (8G7G3/1) (Dako, 1:1000), anti-Ki67 (SP6) (Thermo-Scientific, RM-9106-S1 1:200), anti-Glut1 (alpha-diagnostic, GT11-A, 1:400), anti-Cleaved Caspase 3 (CST, #9664, 1:1000). Slides were scanned onto a ScanScope AT (Aperio Technlogies Inc.). Digital slides were analyzed with Definiens and QuPath software.

Tissue isolation, fixation and staining procedures were performed as previously described (Momcilovic et al., 2017 (link); Momcilovic et al., 2015 (link); Shackelford et al., 2013 (link)). Briefly, lungs or tumors were fixed overnight in 10% buffered formalin, and then transferred to 70% ethanol. Tissues were processed and embedded by TPCL at UCLA. The following antibodies were used: phospho-4EBP1 (Thr37/46) (Cell Signaling Technology, #2855 1:800), anti-CK5 (EP1601Y) (abcam, ab52635 1:100), anti-TTF1 (8G7G3/1) (Dako, 1:1000), anti-Ki67 (SP6) (Thermo Scientific, RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), phospho-GSK3α/β (Ser21/9) (Cell Signaling Technology, #9331 1:50), c-Jun (Cell Signaling Technology, #9165 1:800), phospho-c-Jun (S73) (D47G9) (Cell Signaling Technology, #3270 1:200), anti-SLC1A5 (Sigma, HPA035240, 1:400), anti-GLUT1 (Alpha Diagnostic, GT11A, 1:400), anti-Myc (Y69) (abcam, ab32072, 1:100). Slides were scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). Digital slides were analyzed with QuPath software in order to determine percent positive cells for Ki67, GLUT1 and p4EBP1 stains.