Table 1.
Antibody information for western blots.
Table 2.
Primer sequences for RT-PCR.
Fig 1.
Analysis of G2/M cell cycle arrest under PTX in MDA-MB-157 & APC shRNA1 cells.
(A) Cell cycle analysis of untreated and PTX treated MDA-MB-157 and APC shRNA1 cells using flow cytometry. Nocodazole was used as a positive control because it is known to induce G2/M arrest. Representative flow cytometry graphs are included. (B) Graphical representation of the percentage of cells in the G2/M phase of the cell cycle in control and PTX treated cells. Data shown are the average of 4 independent experiments, and the STDEV is shown. * p < 0.05, ** p < 0.01 compared to control treated cells of the same genotype.
Fig 2.
Western blots of G2/M checkpoint proteins.
(A) Control (CTL) and paclitaxel (PTX) or cisplatin (CIS) treated cell lysates were probed for expression of G2/M checkpoint proteins. Representative western blots are shown for cyclin B1 and CDK1, including the inhibitory (Thr14 and Tyr15) and activating (Thr161) phosphorylation sites on CDK1. (B-F) Bar graph representation of G2/M transition protein quantification in treated and untreated MDA-MB-157 and APC shRNA1 cell lines. Bar graphs show levels of cyclin B1 and CDK1, Thr14, Tyr15 and Thr161 in these treated and untreated cells. For the phosphorylation sites, the value graphed was the ratio of the site expression over actin to the total CDK1 protein expression over actin (n = 3). Analysis indicated an increase in total CDK1 expression in all of the APC shRNA1 cells. * p < 0.05 as compared to all of the parental cells. Analysis indicated an increase in phosphorylated CDK1 at Thr14 in APC shRNA1 cells upon treatment with cisplatin. * p < 0.05 as compared to untreated APC shRNA1 cells, and *** p < 0.001 as compared to PTX treated APC shRNA1 cells.
Fig 3.
Western blot analysis of cellular fractions.
Protein lysates from CTL, PTX, or nocodazole (NOC) treated cells were fractionated and then probed with CDK1 (A and C) or cyclin B1 (B and D). HDAC and H3.3 were used as loading controls for the nuclear extract (NE) specifically, while actin was used as a widespread loading control. (A-B) Bar graph representation of protein quantification (n = 3). (C-D) Representative western blots are shown. Analysis indicated that CDK1 is preferentially located in the cytoplasm in the untreated parental and APC shRNA1 cells. Cyclin B1 shows no preferential localization. ** p < 0.01 as compared to corresponding cytoplasmic extract (CE). Westerns were run and membranes cut after transfer. For CDK1, the top portion was probed for HDAC (61kD), and the bottom portion was probed for CDK1 (34kD) and actin (42kD). For cyclin B1, the top portion was probed for cyclin B1 (55kD) and actin (42kD), and the bottom was probed for Histone H3.3 (15kD).
Fig 4.
Western blot analysis of canonical cell cycle proteins.
Untreated or PTX treated lysates were probed for expression of canonical cell cycle proteins. (A) Representative western blots are shown for cyclin A2, cyclin E1, cyclin D1, cyclin D3, CDK2, CDK4, CDK6, p18, p21, and p27. Actin was used as a loading control. (B-C) Bar graph of protein expression quantification in treated and untreated MDA-MB-157 and APC shRNA1 cell lines is shown (n = 3). (B) Analysis indicated that loss of APC resulted in a significant increase in CDK6 expression. * p < 0.05 compared to parental cells. (C) P27 expression decreased upon PTX treatment in both MDA-MB-157 and APC shRNA1 cells. * p < 0.05, ** p < 0.01 compared to control treated cells of the same genotype. No change was seen in the remaining proteins.
Fig 5.
RNA sequencing and validation of APC shRNA1 cells compared to parental controls.
(A) Hierarchical clustering and heat map of significantly differentially expressed genes associated with the cell cycle. Input data are the normalized expression values. The values in blue are up-regulated and those in red are down-regulated. (B) Real-time RT-PCR analysis for LBH and RGS4 in MDA-MB-157 and APC shRNA1 cells. Data were initially normalized by amount of GAPDH mRNA, and then further normalized to the parental cells. Data (AVG +/- SEM) depict changes in expression between parental and APC shRNA1 cells, with average of parental (n = 3) set to 1 and average of APC shRNA1 representing fold-change. (C) Western blot analysis for GLI1 and NUPR1 in MDA-MB-157 and APC shRNA1 cells with Actin as a loading control. Bar graph representation of protein quantification (n = 3 for GLI1 and n = 4 for NUPR1) in untreated and PTX treated cells. Analysis indicated that loss of APC increased GLI1 and NUPR1 in untreated cells. * p < 0.05, ** p < 0.01 as compared to parental cells.
Table 3.
Over-represented biological process ontologies of cell cycle/cell division associated with the transcriptome.