Fig. 6
From: AIF-regulated oxidative phosphorylation supports lung cancer development
Re-expressing WT or mitochondria-anchored AIF restores lung cancer sensitivity. a Schematic representation of the WT AIF (WT-Aif) and mitochondria-anchored mutant AIFΔ96-110 (MT-Aif). TMS, transmembrane sequence. The cathepsin/calpain cleavage site is indicated. b Knock-in targeting strategy to insert WT-Aif and MT-Aif transgenes into the ROSA26 locus. Exons are shown as 1, 2, and 3. HSV-tk = herpes simplex promoter; PGK-neo-PA = neomycin cassette for selection; AIF-FLAG = AIF (WT or mutated) with a 3 × FLAG tag, EcoR V = restriction sites for Southern blotting. c Kaplan Meier survival plots for Aif+/y KrasG12D (n = 11), Aiffl/y KrasG12D (n = 10), Aiffl/y WT ki KrasG12D (n = 10), Aiffl/y MT ki KrasG12D (n = 9), Aif +/y WT ki KrasG12D (n = 9), and Aif+/y MT ki Kras G12D (n = 8) mice. **P < 0.01; NS, not significant (log rank test). d Representative lung sections (H&E staining) of Aif+/y KrasG12D, Aiffl/y KrasG12D, Aiffl/y WT ki KrasG12D and Aiffl/y MT ki KrasG12D mice, analyzed at 12 weeks after Ad5-CMV-Cre infection. Scale bar, 2 mm. e Quantification of overall tumor burden in the indicated cohorts analyzed 12 weeks after Ad5-CMV-Cre inhalation (n = 5 for each genotype). Three planes from each lung were scored automatically by an algorithm programmed and executed using the Definiens software suite program. Data are shown as means ± SEM. **P < 0.01; NS, not significant (two-way ANOVA analysis, Dunnett’s multiple comparisons test). f Representative images of tumor spheroids derived from purified Aif+/y KrasG12D and Aiffl/y KrasG12D primary lung tumor cells. Images were acquired 4 days after cells were seeded in Matrigel (5000 primary tumor cells per well). The experiment was designed with 6 replicates for each condition and repeated with 3 different mice for each group. Scale bar, 1 mm. g Quantitative analysis (means±SEM) of tumor spheroid numbers described in f. ***P < 0.001; NS, not significant (Unpaired, two-sided t-test). h Representative images for BrdU staining of tumor spheroids derived from Aif+/y KrasG12D and Aiffl/y KrasG12D primary lung tumor cells seen as in f. BrdU labeling (10 μM/mL) was performed for 2 h. Experiments were performed with 6 replicates for each condition and repeated with 3 different mice for each genotype. Sections were counter-stained with DAPI. i Quantifications (means ± SEM) of BrdU+ cells within tumor spheroids shown in h. *P < 0.05; NS, not significant (Unpaired, two-sided t-test)
