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. 2021 Apr 15;81(8):1698–1714.e6. doi: 10.1016/j.molcel.2021.02.001

Figure 3.

Figure 3

PAF depletion induces quiescence and growth arrest of lung cancer cells

(A–E) Cell growth inhibition by PAF depletion. (A) Cell proliferation was assessed using crystal violet staining of control and Paf knockdown (KD) mouse lung cancer cells (KP); two shRNAs targeting Paf (shPaf #1 and #2) were used. (B) Cumulative population doublings of control and Paf-KD KP cells. (C) Cumulative population doublings of two different KP mouse lung cancer cell lines with Paf KD (KP836 and KP952 were established from two different Ad-Cre-induced KP mice). (D) Cumulative population doublings for Paf rescue experiment in Paf-KD KP cells. Cells were stably transduced with PAF. (E) Cell proliferation analysis of human LUAD cell lines with PAF KD; cumulative population doublings of five LUAD cell lines (A549, H23, H358, H1792, and H1355) that were stably transduced with lentiviruses encoding shPAF #1 or #2. Two-way ANOVA with Tukey post hoc test.

(F–J) G0/G1 arrest by PAF KD in mouse and human LUAD cells. (F) Cell-cycle distribution of control (shCtrl) and PAF-KD (shPaf) mouse lung cancer cells was assessed using propidium iodide (PI) staining followed by fluorescence-activated cell sorting (FACS) analysis (n = 30,000 cells). (G) Cell-cycle distribution of control (shCtrl) and PAF-depleted (shPAF) human lung cancer cells (A549 and H1792); PI staining with FACS analysis, n = 30,000 cells. (H) Analysis of cell-cycle distribution in synchronized control and Paf-depleted KP cells by thymidine double block and PI staining-FACS analyses. (I) Visualization of G0/G1 cells by nuclear localization of DHB-Venus in control and Paf-KD A549 and H1792 lung cancer cells stably transfected with shCtrl or shPAF with DHB-Venus; scale bars, 50 μm. (J) Quantification of nuclear localization of DHB-Venus in control and PAF KD A549 and H1792 cells; n > 500 cells in at least ten different fields were counted.

(K and L) Increase of cell quiescence (G0) by PAF KD. (K) Density scatterplots of control versus PAF-KD H1792 cells. G0 cells were assessed by pyronin Y and 7-aminoactinomycin D (7-AAD) double staining followed by FACS analysis; cells with low RNA content (low pyronin Y) in G0/G1 phase were considered G0 cells (Schmid et al., 2000). (L) Quantification of cell-cycle phases in H1792 cells (control and PAF KD).

Representative images are shown. Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.