Figure 2.

Suppression of lung tumorigenesis by Paf knockout

(A–C) PAF expression in proliferating lung cancer cells. (A) Co-immunostaining of Paf/Pcna and (B) Paf/Ki67 in KP mouse lung tumors; scale bars, 20 μm. (C) Quantification of Paf⁺ cells among Ki67⁺ and PcNa⁺ cell populations in KP lung tumors; data are from at least five fields; n > 200 tumor cells were analyzed. Error bars indicate SEM.

(D–F) Macroscopic analysis of lungs from KP and PKP mice at 3 or 4 months after Ad-Cre induction. (D) Extensive lung tumor nodules were visible on the surface of KP lungs, whereas no tumor nodules were found on the surface of PKP lungs; scale bars, 2 mm. (E) Hematoxylin and eosin (H&E) staining of lungs from KP (3 months) and PKP mice (4 months). Scale bars, 2 mm for whole-lung sections and 100 μm for magnified images. (F) Micro-CT images of lungs from KP (3 months; n = 6) and PKP (3 months; n = 6) mice; scale bars, 2 mm. H, heart. Asterisk denotes tumor lesion.

(G and H) Paf knockout (KO) suppressed lung tumor growth. (G) Quantification of tumor number and (H) tumor burden in KP and PKP lung samples. The middle sections of mouse lungs were analyzed to quantify tumor number. Tumor burden (percentage of total lung area) was calculated by measuring the tumor area in the middle sections of mouse lungs (KP 3 months, n = 6; PKP 3 months, n = 5, PKP 4 months, n = 4); error bars indicate SEM.

(I) Paf KO reduced lung tumor cell proliferation. Quantification of Ki67⁺ cells in KP and PKP lung tumors (see Figure S6C); at least 15 fields of 200× magnified images from KP (n = 4) and PKP (n = 9) lung tumors were analyzed; error bars indicate SD.

(J) Representative images of H&E-stained lung sections of surviving PKP mice at the end of observation (2 years); three of nine PKP mice (33.3%) survived until euthanasia.

(K) Paf KO extended mouse lifespan. KM survival curves of KP and PKP mice. KP median survival = 90 days (n = 10), PKP median survival = 353 days (n = 9). Log rank (Mantel-Cox) test.

(L–P) Paf KO suppressed Kras^G12D-driven lung tumorigenesis. Lung samples from Kras^LSL-G12D/+ (4 months after Ad-Cre induction, n = 4) and Paf^−/−; Kras^LSL-G12D/+ mice (4 months after Ad-Cre induction, n = 5) were analyzed. (L) Images of H&E-stained lung sections; scale bars, 2 mm for whole-lung sections and 100 μm for magnified images of tumors. (M) Quantification of tumor number from the middle sections of mouse lung samples. (N) Quantification of tumor burden calculated as in (H). (O) Cell proliferation analysis by quantification of Ki67⁺ cells; at least 15 fields of 200× magnified images from Kras^LSL-G12D/+ (n = 4) and Paf^−/−; Kras^LSL-G12D/+ (n = 5) lung tumors were analyzed; error bars indicate SD. (P) KM survival curves; Kras^LSL-G12D/+ median survival = 193 days (n = 6), Paf^−/−;Kras^LSL-G12D/+ median survival = 645 days (n = 7). Of seven Paf^−/−; Kras^LSL-G12D/+ mice, three (42.8%) survived until the end of observation (2 years). Log rank (Mantel-Cox) test.

Representative images are shown. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.