Among 18 cases of non-cancer, 7 cases were bronchitis, 7 cases tuberculosis, TPCA-1 chemical structure 3 cases pneumonia and 1 case brochiectasis. All patients had not received any anti-cancer therapy before receiving bronchoscopy. At least 5 biopsy specimens were obtained from one patient. One to two specimens were snap frozen

and stored at -80°C for RT-PCR analysis under the Small molecule library concentration condition of specimens were sufficient for routine diagnosis. The remaining specimens were fixed in buffered formalin for histopathological evaluation. This study was approved by the Guilin Medical University Review Board, and informed consent was obtained from all patients under the protocols prescribed by the Guilin University Ethics Committee. Semi-quantitative RT-PCR Total RNA was isolated from the biopsy tissue using Trizol reagent (TakaRa Bio Inc, Dalian, China) according to the manufacturer’s instructions. One μg of the mRNA was reverse transcribed to cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit (TakaRa). One μl of the cDNA was used in PCR for the amplification of β-actin and seven stem-cell-associated markers. The primers are presented in Table 1. The DNA thermal cycler conditions used were 94°C for 5 min (pre-denature), and 35 cycles of 94°C for 1 min, annealing for 30 s and extension at 72°C for

45 s, followed by a final extension Sapanisertib of 72°C for 2 min. Six μl of each PCR-amplified product were separated on a 2% agarose gel, which was then visualized by ethidium bromide staining using a JS-780 Gel Image Analysis System (Peiqing Sci Tech, Ltd, Shanghai, China). The ratio of integrated density of target genes over corresponding β-actin was normalized as relative mRNA expression levels of stem-cell-associated markers. Table 1 The primers and primary antibody used in this study Gene symbles Primers for RT-PCR   Antibodies for IHC         Primer sequences Annealing temperature (°C) Antibody sources Clone Dilution Bmi1 Reverse 5’-ATT GTC TTT TCC GCC CGC TT-3’

58.2 ProMab Biotechnologies Inc 3E3 1:800 Forward 5’-TGG CAT CAA TGA AGT ACC CTC-3’ CD44 Reverse 5’-TGC TAC TGA TTG TTT CAT TGC G-3’ 56.2 ProMab Biotechnologies Inc 8E2F3 1:30000 Forward 5’-GGA CCA GGC CCT ATT AAC CC-3’ CD133 Reverse GNA12 5’-AAA CAA TTC ACC AGC AAC GAG-3’ 54.1 ProMab Biotechnologies Inc 3 F10 1:400 Forward 5’-TAG TAC TTA GCC AGT TTT ACC G-3’ Sox2 Reverse 5’- GCT AGT CTC CAA GCG ACG AA-3’ 56.2 ProMab Biotechnologies Inc 10 F10 1:800 Forward 5’- TAC AGT CTA AAA CTT TTG CCC TT-3’ Nanog Reverse 5’-AGG CAA CTC ACT TTA TCC CAA-3’ 54.1 Cell signaling technology D73G4 1:300 Forward 5’-GAT TCT TTA CAG TCG GAT GCT T-3’ Oct-4 Reverse 5’-TGC AGA AAG AAC TCG AGC AA-3’ 56.2 Santa Cruz Biotechnology C-10 1:50 Forward 5’-CTC ACT CGG TTC TCG ATA CTG G-3’ Msi2 Reverse 5’-CAG ACC TCA CCA GAT AGC CTT-3’ 56.2 ProMab Biotechnologies Inc 2C11 1:1000 Forward 5’-TAC TGT GTT CGC AGA TAA CCC-3’ β-actin (217 bp) Reverse 5’GTG ACG TGG ACA TCC GCA AAG-3’ 60.