Fig 2: Decrease in mitochondrial enzymatic activities in Akap1 -/- mice exposed to hyperoxia. (A) Citrate synthase activity (milliunits/mL) in the mitochondrial lysates from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia, respectively. (B) Fumarase activity (milliunits/mL) in mitochondrial lysates from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia, respectively. (C) Aconitase activity (milliunits/mL) in the mitochondrial lysates from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia. Data represented as mean ± SEM. Two-way ANOVA was used for analysis. *p < 0.05 Akap1 -/- hyperoxia versus Wt hyperoxia control. **p < 0.01 Akap1 -/- hyperoxia versus Wt hyperoxia control. ***p < 0.001 Akap1 -/- hyperoxia versus Wt hyperoxia control. *p < 0.05 Wt hyperoxia versus Wt normoxia control.

Fig 3: AKAP1 loss induced Akt inactivation and increased Bax expression under hyperoxia. (A) Representative Western blot analysis of total Akt and phosphorylated Akt (pS473) in the lung lysates derived from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia. (B) Total Akt and pAkt/Total Akt quantitated after normalization with ß-Actin control. Data represented as mean ± SEM. n = 5 mice per group. Two-tailed unpaired Student’s t-test. *p < 0.05 Akap1 -/- normoxia to Wt normoxia. ***p < 0.001 Akap1 -/- normoxia versus Akap1 -/- hyperoxia. (C) Representative Western blot analysis of Bax in the lung lysates from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia. (D) Quantitation of Bax expression after normalization with ß-Actin control. Data presented as mean ± S.E.M. n = 5 mice per group. Two-tailed unpaired Student’s t-test. *p < 0.05 Akap1 -/- normoxia versus Wt normoxia, ****p < 0.0001 Wt normoxia versus Wt hyperoxia and ***p < 0.001 Akap1 -/- hyperoxia versus Akap1 -/- normoxia. (E) Representative IF image of Bax expression in the lung section of Wt and Akap1 -/- mice exposed to hyperoxia. White arrow shows positive Bax immunoreactivity. Nuclei stained with DAPI (blue). Magnification: ×400, scale bar: 50 µm. (F) Quantitative analysis of Bax expression is represented as CTCF. Bax immunoreactivity in the lung sections of Akap1 -/- hyperoxia versus Wt hyperoxia. Data represented as mean ± SEM. n = 5 mice per group. Unpaired two-tailed Student’s t-test. *p < 0.05 Akap1 -/- hyperoxia versus Wt hyperoxia. NO = normoxia. HO = hyperoxia.

Fig 4: Oxidative stress increases in Akap1 -/- mice exposed to hyperoxia. (A) Western blot analyses of SOD2 and HO-1 expression in the lysates from Wt and Akap1 -/- mice exposed to normoxia or hyperoxia. SOD2 and HO-1 expression were quantified after normalization with ß-Actin (loading control). **p < 0.01 Akap1 -/- hyperoxia versus Wt hyperoxia. ****p < 0.0001 Akap1 -/- hyperoxia versus Akap1 -/- normoxia. n = 6 mice per group. Two-tailed unpaired Student’s t-test. (B) Representative images from IF analysis shows increased expression of HO-1 (white arrows) in the lung section of Akap1 -/- hyperoxia versus Wt hyperoxia mice. Magnification: ×400. Scale bar: 100 µm. (C) Representative images from IF analyses of HO-1 (red) and CD68 (green) in lung sections derived from Wt and Akap1 -/- mice exposed to hyperoxia. Arrowheads indicates the HO-1 positive cells co-labelled with CD68 (Macrophage marker). Arrow shows HO-1 expressing cells without CD68 expression. Nuclei stained with DAPI (blue). Magnification: ×400, scale bar: 50 µm. (D) Quantitative analysis of HO-1 expression is represented as corrected total cell fluorescence (CTCF). HO-1 expression in the lung sections of Akap1 -/- hyperoxia versus Wt hyperoxia. n = 4 mice per group. Data represented as mean ± SEM. *p < 0.05 versus Wt hyperoxia. Unpaired Student’s t-test. n = 4 per group. (E) Quantitative analysis of CD68 + expression is represented as CTCF. CD68 + staining in the lung sections of Akap1 -/- hyperoxia versus Wt hyperoxia. n = 4 mice per group. Data represented as mean ± SEM. *p < 0.05 Akap1 -/- hyperoxia versus Wt hyperoxia. Unpaired Student’s t-test.

Fig 5: Differentially expressed genes in Akap1 -/- and Wt mice exposed to hyperoxia. (A) Venn diagram depicts upregulated genes unique and commonly shared between the Wt hyperoxia group and Akap1 -/- hyperoxia group and (B) Venn diagram depicts downregulated genes unique and commonly shared between the Wt hyperoxia group and Akap1 -/- hyperoxia group, respectively. (C) qRT-PCR analysis of transcripts (ETC and TCA) in the Akap1 -/- hyperoxia group relative to Akap1 -/- normoxia group (n = 6 mice per group). Data represented as mean ± SEM. Two-tailed unpaired Student’s t-test. *p < 0.05 Akap1 -/- hyperoxia versus Akap1 -/- normoxia control.