Fig 2: Knockdown of FOP delays cell cycle re-entry. (A) RPE1 cells were transfected with siNC or FOP siRNAs followed by 48 h of serum-starvation. Cells were then serum re-stimulated for the indicated durations, with cells being labeled with EdU during the final 2 h before cell harvesting. The nuclei were stained with DAPI. Scale bar, 100 μm. (B) Quantification of the percentage of EdU positive cells described in (A). At least 500 cells per sample were analyzed in each experiment. (C) Immunoblotting analysis of the phosphorylation levels of Rb at Ser807/811 (pRb) and CDC2 at Tyr 15 (pCDC2) and the levels of Cyclin A and FOP in siNC and siFOP cells at 0, 6, 12, 18, and 24 h post serum re-stimulation. (D) Cell cycle profiles of siNC and siFOP treated cells at 0, 6, 12, 18, and 24 h after serum re-stimulation were determined by flow cytometry. At least 10,000 cells per sample were analyzed in each experiment. (E) Quantification of the percentage of S-M phases cells described in (D). Data are presented as mean ± SD from three independent experiments; ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001(Two-way ANOVA followed by Tukey’s multiple comparisons test).

Fig 3: Microcolonies exhibit a hypo-proliferative phenotype.(A) Functional annotation of genes downregulated in microcolonies/higher in macrometastases. GSEA msGDMIB for Hallmark and canonical pathways (p<0.05, 200 highest fold-change genes). (B) Abundance of transcripts in RNA-seq for genes associated with cell cycle arrest (Top) and positive regulation of mitosis (Bottom). (NµCol = 94, Nmacro = 111). Fraction of Ki-67-positive cells (C) and phosphorylated-histone H3-positive cells (D) in microcolonies and macrometastases as determined by immunostaining (n = 5 samples per group). (E) Representative confocal images of microcolonies and macrometastases from livers of 6419 c5 mice stained with anti-phospho-Rb (pRB; Ser807/811). Scale bar = 50 µm. (F) Fraction of pRB+ microcolonies and macrometastases, and (G) RB phosphorylation levels in pRB-positive cells normalized to average MFI in each mouse (n = 5 samples per group). (H) Fraction of p21+ cells in microcolonies and macrometastases as determined by immunostaining (n = 5 samples per group). (I) Schematic for ex vivo proliferation assay. Microcolonies and macrometastases are extracted via PIC-IT, seeded at 45 cells/96 wells and colony growth is monitored via microscopy over 72 hr. (J) Colony size distribution of metastatic cells over the first 72 hr after isolation. Left panel, colonies derived from microcolonies. Right panel, colonies derived from macrometastases (n = 3 independent experiments). (K) Fraction of isolated metastatic cells residing as solitary cells at 24, 48 and 72 hr post-isolation (n = 3 independent experiments). Bars represent mean ± SEM in all graphs. p-values were calculated by unpaired two-tailed Student’s t-test.

Fig 4: Reduced phospho-ERK and ACE2 expression by the combination of MEK inhibitor and remdesivir.(A) Effects of 5 μM remdesivir (RDV), RAF/MEK inhibitor VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression. HCT116 human colorectal cancer cells, H1975 human NSCLC cells and BEAS-2B normal human bronchial epithelial cells were treated with 5 μM VS-6766 or/and 5 μM Remdesivir for 48 hr. Remdesivir alone increased pERK protein expression in all three cell lines, which was completely depleted by VS-6766 treatment. Effects of 5 μM RDV, VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression in (B) normal human lung cells and (C–E) human lung cancer cells are shown. Serum-deprived cells were plated and cultured in medium containing 1% FBS for the indicated amount of time. For serum-stimulated cells, FBS was added to a final concentration of 10% for 24 hours (B–E, left 2 panels). Cells were plated in 10% serum for 16 hours, then media was removed and replaced with 1% FBS for serum-deprived cells. For serum-stimulated cells, FBS was added to a final concentration of 10% for 4 hours (E, right panel). ACE2 (PAB), ACE2 (CS), ERK1/2, and pERK were probed with Abnova PAB13444, Cell Signaling 4355, Cell Signaling 9102, and Cell Signaling 4370 antibodies. Ran was probed with BD Biosciences 610341 antibody as a loading control. (F) Modulation of pRb and ACE2 expression in H1299 cells with serum starvation, stimulation, and MEKi treatment. Control cells were grown with the normal 10% serum throughout the treatment. Serum starved cells were plated in 10% serum and incubated for 16 hours, then grown in media containing 0% serum for 72 hours. Serum stimulated cells were similarly starved for 72 hours, then stimulated with media containing 20% serum for 48 hours. All drug treated cells received 5 mM RDV, VS-6766, or the combination 48 hours before harvesting. (G) Modulation of ACE2 expression with serum starvation and stimulation. Four different cell lines (2 lung, 1 colorectal, and 1 breast cancer) were grown in 10% serum for 16 hours then were starved in 0% FBS for 0 (no starvation), 24, 48, or 72 hours. Cells were stimulated with 20% FBS and harvested either 0 (no stimulation), 12, or 24 hours later. An increase in pRb relative to total Rb was seen upon stimulation and this correlated with ACE2 levels. pERK correlation with ACE2 was heterogeneous, with a correlation seen in MCF7 cells but not H1299 or HCT-116 cells (three left-most panels). pRb relative to total Rb decreased upon starvation and increased upon stimulation, which correlated with ACE2 expression (H460 and HCT-116 in right-most panels).

Fig 5: Cell cycle progression is altered in DCT knockdown cells compared to control cells.(A) Cell lysates from control and DCT knockdown cells were harvested and subjected to western blotting on the day of the knockdown. pRb (Ser807/811) and actin levels are shown for three independent DCT knockdown experiments. (B) pRb band intensities of each lane were quantified and normalized first against the corresponding actin measurements and then against the normalized pRb levels in control samples. The bar graph shows the normalized pRb levels of DCT knockdown cells compared to their controls. (C) Representative cell cycle profiles for DCT knockdown and control cells as analyzed and visualized by FlowJo v10. (D) Cell cycle profiles of DCT knockdown and control cells were analyzed by PI staining and flow cytometer. The bar graph shows the mean value of the percentage of cells in G0/G1 phase for three independent experiments with six replicates each (one tailed t-test p = 0.0008).