Fig. 1. BMI1 expression is inversely associated with C/EBPα expression in human adenocarcinoma cells.

(A) Left: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on experimental samples (n=3) from the human lung adenocarcinoma cell line H358 transfected with a rapamycin-inducible C/EBPα-expressing vector or a rapamycin-inducible empty vector (as control), and induced with rapamycin (for 36 hours). The mean expression is presented as a percentage of 18S RNA. The P values for each sample are indicated on the basis of a two-sided Welch’s t test. Right: Western blot analysis was carried out in H358 cells transfected with the rapamycin-inducible C/EBPα-expressing vector or the rapamycin-inducible empty vector by using anti-C/EBPα and anti-BMI1 antibodies. Loading was assessed, after complete stripping of the membrane, with an anti-actin antibody. The expected size in kilodaltons is indicated. (B) Left: Pie chart demonstrating BMI1 protein expression in patient-derived adenocarcinomas that are negative or low for C/EBPα (staining intensity, 0 or 1⁺). BMI1 was considered positive when staining was scored as 1⁺ to 3⁺. Right: Pie chart showing C/EBPα protein expression in patient-derived adenocarcinomas, subdivided as negative/low (staining intensity, 0or 1⁺) or positive (staining intensity, 2⁺ or 3⁺). (C) Representative examples of IHC data from three patients’ tissues (each enclosed in a box), which were independently and blindly scored by two pathologists. Staining intensity was scored as follows: 0 (no staining); 1⁺ (mild staining); 2⁺ (moderate staining); and 3⁺ (strong staining). Scale bar, 25 μm. (D) Overall survival curves for 490 patients from TCGA lung adenocarcinoma cohort (https://tcga-data.nci.nih.gov/tcga/). Only 490 of the 521 TCGA samples have both RNA-sequencing and survival data. Patients are stratified according to low or high C/EBPα expression, defined as log₂ expression < 9.0 or not, respectively. Survival probability is higher (P = 0.041) in BMI1^low patients, as compared to BMI1^high patients in the C/EBPα^low subgroup. The median expression of BMI1 used to define BMI1^low and BMI1^high patient subgroups was 10.2 in the C/EBPα^low group and 10.1 in the C/EBPα^high group. The method of Kaplan and Meier was used for graphical displays of overall survival. The log-rank test was used to assess differences in overall survival. A similar approach was also applied for C/EBPα^high samples. The P value and the sample size (n) for each subgroup are indicated on each plot. Among C/EBPα^low patients, 30 death events/109 patients presented with low BMI1 expression, and 39 death events/ 109patientspresentedwithhighBMI1expression. Among C/EBPα^high patients, 40 death events/136 patients presented with low BMI1 expression, and 39 death events/136 patients presented with high BMI1 expression. Asterisk indicates statistical significance.