Journal: The Journal of Biological Chemistry

Article Title: Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

doi: 10.1016/j.jbc.2024.107409

Figure Lengend Snippet: One copy of the endogenous oncogenic KRAS mutation causes an increase in cell proliferation. A , proliferation rates of the KRAS G12X/+ cells. Exponential growth curves were generated by fitting to three biological replicates using Prism (GraphPad). Y -axis is logarithmic. B , oncogenic KRAS.G12X mutations substantially reduce the doubling time. From the data presented in ( A ), the best-fit doubling time for each cell line was estimated using Prism (GraphPad). Error bars represent a 95% confidence interval (CI). C , distinct cell cycle profiles between the KRAS G12X/+ and the KRAS G12G(WT)/+ cells. Flow cytometry analysis for the DNA contents was conducted for the KRAS G12X/+ and KRAS G12G(WT)/+ cells. A summary result of the biological triplicates presented in Fig. S4 is presented to show the mean and SD values of the cell cycle populations for each sample. Considering the reduced doubling time of the KRAS G12X/+ cells, as shown in ( B ), the G1 phase of the cell cycle is estimated to be substantially reduced in the KRAS G12X/+ cells. D , the S phase population is increased in the oncogenic KRAS G12X/+ cells compared to the KRAS G12G(WT)/+ cells. The S phase population of the flow cytometry data presented in Fig. S4 is summarized, and the mean and SD values are presented. Ordinary one-way ANOVA followed by post hoc Dunnnett’s multiple comparisons test showed that the KRAS G12X/+ samples showed significant differences from the KRAS G12G(WT)/+ cells. E , the sub-G1 population was not increased in the oncogenic KRAS G12X/+ cells compared to the KRAS G12G(WT)/+ cells. The sub-G1 population of the flow cytometry data presented in Fig. S4 is summarized, and the mean and SD values are presented. Ordinary one-way ANOVA followed by post hoc Dunnnett’s multiple comparisons test showed that the sub-G1 population was comparable between the KRAS G12G(WT)/+ and KRAS G12V/+ or KRAS G12C/+ cells and was slightly decreased in the KRAS G12D/+ mutant cells.

Article Snippet: The following primary antibodies were used: anti-AKT (Cell Signaling Technology, 40D4 Mouse mAb #2920, dilution 1/2000), anti-pAKT (Cell Signaling Technology, rabbit Antibody #9271, dilution 1/1000),anti-ERK (Thermo Fisher Scientific: Invitrogen, mouse antibody #10221703, dilution 1/500), anti-pERK (Cell Signaling Technology, rabbit mAb #4370, dilution 1/2000), anti-KRas antibody (OriGene, mouse monoclonal #CF801672, dilution 1/1000), anti-Ras.G12V antibody (Cell signaling technology, rabbit monoclonal #14412, dilution 1/800), anti-γ-tubulin antibody (Sigma-Aldrich, mouse monoclonal, #T6557, 1/5000 dilution) and anti-pRb-Ser807/S811 antibody (Cell Signaling Technology, rabbit mAb #8516, 1/1000 dilution).

Techniques: Mutagenesis, Generated, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

doi: 10.1016/j.jbc.2024.107409

Figure Lengend Snippet: The KRAS G12X/+ cells show enhanced motility. A , the KRAS G12X/+ and the KRAS G12G(WT)/+ cells were cultured on collagen-coated plates, and the cell movement was recorded every 20 min for 31 time frames using ptychographic phase imaging. Each cell movement was tracked and analyzed using the Livecyte system (Phasefocus) as described in the . Data from three biological replicates from each cell line are presented. The positions of cell tracks are plotted on an X - Y axis, where the start of each track (time 0) is set to the center of the plot ((x, y) = (0, 0)). Each track is shown in a different color. B , the dataset presented in ( A ) was analyzed to deduce the velocity of each cell from one time frame to the next at every time point. The obtained values are plotted for each biological replicate and are analyzed with a nested one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test (Prism, GraphPad), which shows an increase in the KRAS G12X/+ cells. C , a decrease in the paxillin signals in the KRAS G12V/+ cells. The status of paxillin structures in the KRAS G12V/+ and the KRAS G12G(WT)/+ cells were visualized by immunofluorescence microscopy as described in the . The actin stress fiber and the nuclei were also counter-stained. The ratio of the area occupied by the paxillin structures and the cytoplasm was deduced for each image, as stated in Fig. S5 . For each biological replicate, 20 images were analyzed, and the data from three biological replicates were analyzed by a nested t test (Prism, GraphPad). The area occupied by the paxillin structures is significantly reduced in the KRAS G12V/+ cells.

Article Snippet: The following primary antibodies were used: anti-AKT (Cell Signaling Technology, 40D4 Mouse mAb #2920, dilution 1/2000), anti-pAKT (Cell Signaling Technology, rabbit Antibody #9271, dilution 1/1000),anti-ERK (Thermo Fisher Scientific: Invitrogen, mouse antibody #10221703, dilution 1/500), anti-pERK (Cell Signaling Technology, rabbit mAb #4370, dilution 1/2000), anti-KRas antibody (OriGene, mouse monoclonal #CF801672, dilution 1/1000), anti-Ras.G12V antibody (Cell signaling technology, rabbit monoclonal #14412, dilution 1/800), anti-γ-tubulin antibody (Sigma-Aldrich, mouse monoclonal, #T6557, 1/5000 dilution) and anti-pRb-Ser807/S811 antibody (Cell Signaling Technology, rabbit mAb #8516, 1/1000 dilution).

Techniques: Cell Culture, Imaging, Immunofluorescence, Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

doi: 10.1016/j.jbc.2024.107409

Figure Lengend Snippet: ERK and AKT activation and attenuation profiles upon EGF treatment in the KRAS G12X/+ and KRAS G12G(WT)/+ cells. Cells were serum-starved for 48 h before the EGF stimulation, as described in the . ERK ( A ) and AKT ( B ) phosphorylation status were monitored for 2.5 min, 5 min, 15 min, 30 min, and 45 min after the EGF stimulation by Western blotting. The ratios of phosphorylated ERK1/2 (pERK) and the internal control γ-tubulin ( A ), or the ratio of phosphorylated AKT (pAKT) and the total AKT ( B ), were quantitated using the Odyssey imaging system (LI-COR). Three biological replicates, presented in Figs. S6 and , were analyzed to plot the graphs that show the mean and the SD values. In the left graph, the results of the KRAS G12V/+ (three clones, 19–16, 19–29 and 65–16) and the KRAS G12G(WT)/+ cells are plotted, and in the right graph, the results of the KRAS G12C/+ and the KRAS G12D/+ cells are plotted, where the KRAS G12G(WT)/+ data were included as a comparison. EGF, epidermal growth factor; pERK, phospho-ERK.

Article Snippet: The following primary antibodies were used: anti-AKT (Cell Signaling Technology, 40D4 Mouse mAb #2920, dilution 1/2000), anti-pAKT (Cell Signaling Technology, rabbit Antibody #9271, dilution 1/1000),anti-ERK (Thermo Fisher Scientific: Invitrogen, mouse antibody #10221703, dilution 1/500), anti-pERK (Cell Signaling Technology, rabbit mAb #4370, dilution 1/2000), anti-KRas antibody (OriGene, mouse monoclonal #CF801672, dilution 1/1000), anti-Ras.G12V antibody (Cell signaling technology, rabbit monoclonal #14412, dilution 1/800), anti-γ-tubulin antibody (Sigma-Aldrich, mouse monoclonal, #T6557, 1/5000 dilution) and anti-pRb-Ser807/S811 antibody (Cell Signaling Technology, rabbit mAb #8516, 1/1000 dilution).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Imaging, Clone Assay, Comparison

Journal: Scientific Reports

Article Title: Ductal activation of oncogenic KRAS alone induces sarcomatoid phenotype

doi: 10.1038/srep13347

Figure Lengend Snippet: ( A ) The diagram of the Ela-CreERT and LGL-KRas G12V transgenes . Expression of the TAM-inducible Cre recombinase (CreERT) is driven by elastase I promoter/enhancer. KRAS G12V expression is blocked by a loxP-GFP-Stop-loxP cassette (LGL). A mouse BAC of 222-kb carrying the intact elastase I gene was used to provide the endogenous native context of elastase I gene . CreERT removes STOP cassette (LGL) through recombination, allowing the KRAS G12V expression. ( B ) Elastase I -driven recombination in mTmG and Rosa26R reporter mice ascertains ductal original. Green fluorescence was exclusively detected in the ductal cells of mTmG;Ela-CreERT reporter mice after vehicle ( panel a ) or TAM-feeding ( panel b ). Scale bar: 50 μm ( panel a ). β-galactosidase activity was observed in the SMG ductal cells of Rosa26R;Ela-CreERT mice after TAM-feeding ( panel d ) compared to control mice ( panel c ). Scale bar: 200 μm. ( C ) Characterization of SMGs of LGL-KRas G12V ;Ela-CreERT mice on day-24 post TAM-feeding. Gross anatomy revealed large ventrolateral cervical masses in SMGs of LGL-KRas G12V ;Ela-CreERT mice on day-24 ( panel b ). H&E and immunohistochemical (IHC) staining showed the microscopic abnormalities in the SMGs of TAM-fed LGL-KRas G12V ;Ela-CreERT mice on day-24 ( panels d, f, h) . Scale bars: 800 μm (in panels c and d ); 100 μm ( panels e–h ). ( D ) Wet weight of SMG from LGL-KRas G12V ;Ela-CreERT mice at 15-days post TAM-feeding (n = 13) or not (n = 7). *** p < 0.001. ( E ) Overall survival of mice after KRAS G12V induction is drastically reduced. Percent survival of Ela-CreERT versus LGL-KRas G12V ;Ela-CreERT mice after TAM administration. Median survival of LGL-KRasG12V;Ela-CreERT mice was 28-days. n = 13 per group; p < 0.0001 by Log-rank (Mantel-Cox) test. ( F ) KRAS G12V is activated in the SMG tumors of LGL-KRas G12V ;Ela-CreERT mice following TAM-gavage. Transgenic mice of indicated genotypes were gavaged with TAM. SMG samples were harvested on day-24 and whole tissue lysates (1 mg) from each sample were pulled down with Raf-1 RBD agarose beads. Pulldown reactions were resolved by SDS-PAGE (12%) and Western blotting was performed with an anti-KRAS antibody to detect active (GTP-bound) KRAS G12V .

Article Snippet: Equal amount of whole tissue lysates (1 mg) were pre-cleaned with glutathione S-transferase beads, then mixed with 20 μl of Raf1 RBD agarose beads, rotated at 4 °C for 1-hour, washed three times with lysis/wash buffer, boiled for 5-minutes in Laemmli buffer under reducing conditions, and resolved by 12% SDS-PAGE, followed by Western blotting with anti-KRAS antibody (ABD Serotec, Cat. #MCA3223Z).

Techniques: Expressing, Fluorescence, Activity Assay, Control, Immunohistochemical staining, Immunohistochemistry, Transgenic Assay, SDS Page, Western Blot