Journal: bioRxiv

Article Title: New Dual Inducible Cellular Model to Investigate Temporal Control of Oncogenic Cooperating Genes

doi: 10.1101/2024.02.23.581802

Figure Lengend Snippet: 293Tii cells were plated on 6-well plates at a density of 250,000 cells per well. After overnight adherence, cells were treated with titrating amounts of either: anhydrotetracycline at 0, 1, 5, 10, 25, or 50 ng/ml; or cumate at 0, 1, 5, 10, 25, or 50 µg/ml. Shown are representative images of cells imaged at 24 hours post-induction (hpi) on a Leica DMI microscope with a 10x objective for GFP fluorescence (HES3) or mCherry fluorescence (PAX3::FOXO1). Scale bar is 500 µm. ( A , B ) and then harvested for western blotting. Cells were lysed in RIPA buffer, and then 20µg of protein was loaded into each well of a 4-15% gradient gel. After transferring to a PVDF membrane, the membrane pieces were blotted with either a HES3 antibody ( C ), FOXO1 antibody that recognizes the PAX3::FOXO1 fusion and endogenous FOXO1 ( D ), or TUBULIN antibody ( C , D ). Each point represents a biological replicate (n=3 for each condition). The error bars represent the mean ± standard deviation. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated three times. Anhydrotetracycline p-values: 0ng vs 25ng, p=0.00081; 0ng vs 50ng, p=0.00008; 1ng vs 25ng, p=0.00112; 1ng vs 50ng, p=0.0001; 5ng vs 25ng, p=0.0108; 5ng vs 50ng, p=0.00075; 10ng vs 50ng, 0.00348. Cumate p-values: 0µg vs 5µg, p=0.0005; 0µg vs 10µg, p=0.00004; 0µg vs 25µg, p=0.00001; 0µg vs 50µg, p=0.000002; 1µg vs 5µg, p=0.0055; 1µg vs 10µg, p=0.0003; 1µg vs 25µg, p=0.00004; 1µg vs 50µg, p=0.00001; 5µg vs 50µg, p=0.008.

Article Snippet: Cells were then lysed with RIPA buffer and 1x protease inhibitor (PI78442, Fisher Scientific) for 2 hours on ice.

Techniques: Microscopy, Fluorescence, Western Blot, Transferring, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: New Dual Inducible Cellular Model to Investigate Temporal Control of Oncogenic Cooperating Genes

doi: 10.1101/2024.02.23.581802

Figure Lengend Snippet: 293Tii cells were plated on 6-well plates at a density of 250,000 cells per well. After overnight adherence, cells were treated with either 50ng/ml anhydrotetracycline (HES3 induction) or 50µg/ml cumate (PAX3::FOXO1 induction). Shown are representative images of anhydrotetracycline-induced cells at 24 hours post-induction (hpi) and 120hpi ( A ), and cumate-induced cells at 24hpi, 120hpi, and 192hpi ( B ). Cells were imaged on a Leica DMI microscope with a 10x objective. Scale bar is 500 µm. Cells were then harvested for western blotting. Cells were lysed in RIPA buffer, and 20µg of protein was loaded into each well of a 4-15% gradient gel. After transferring to a PVDF membrane, the membrane pieces were blotted with either a HES3 antibody ( C ), FOXO1 antibody that recognizes the PAX3::FOXO1 fusion and endogenous FOXO1 ( D ), or TUBULIN antibody ( C , D ). Relative expression of either HES3 ( C ) or PAX3::FOXO1 ( D ) was then quantified using ImageJ (relative to the uninduced cells). Each point represents a biological replicate (n=3 for each condition). The error bars represent the mean ± standard deviation. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated three times. HES3 reversibility p-values: uninduced vs 24hpi, p=0.00006; 24hpi vs 120hpi, p=0.00006; uninduced vs 120hpi, p=0.99. PAX3::FOXO1 reversibility p-values: uninduced vs 24hpi, p=0.00001; uninduced vs 120hpi, p=0.0003; uninduced vs 192hpi, p=0.11; 24hpi vs 120hpi, p=0.019; 24hpi s 192hpi, p=0.00009; 120hpi vs 192hpi, p=0.005.

Article Snippet: Cells were then lysed with RIPA buffer and 1x protease inhibitor (PI78442, Fisher Scientific) for 2 hours on ice.

Techniques: Microscopy, Western Blot, Transferring, Membrane, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: New Dual Inducible Cellular Model to Investigate Temporal Control of Oncogenic Cooperating Genes

doi: 10.1101/2024.02.23.581802

Figure Lengend Snippet: Post-sort 293Tii cells were plated on 6-well plates. After overnight adherence, cells were treated with either 50ng/ml anhydrotetracycline ( A ), 50µg/ml cumate ( B ), or both 50ng/ml anydrotetracycline and 50µg/ml cumate ( C ). Cells were imaged at 24 hours post-induction (hpi) ( A-C ) Cells were imaged on a Leica DMI microscope with a 10x objective. Scale bar is 500 µm. Treated cells were harvested for a western blot and were lysed in RIPA buffer, and then 20µg of protein was loaded into each well of a 4-15% gradient gel. After transferring to a PVDF membrane, the membrane pieces were blotted with either a HES3 antibody, FOXO1 antibody, or TUBULIN antibody ( D ). Relative expression of HES3 and PAX3::FOXO1 was then quantified. Each point represents a biological replicate (n=3 for each condition). The error bars represent the mean ± standard deviation. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated three times. HES3 expression p-values: uninduced vs tet-induced, p=0.00007; uninduced vs cumate-induced, p=0.9999; uninduced vs tet- and cumate-induced, p=0.0004; tet-induced vs cumate-induced, p=0.00007; tet-induced vs tet- and cumate-induced, p=0.28; cumate-induced vs tet- and cumate-induced, p=0.0004. PAX3::FOXO1 expression p-values: uninduced vs cumate-induced, p=0.000004; uninduced vs tet- and cumate-induced, p=0.000007; tet-induced vs cumate-induced, p=0.000006; tet-induced vs tet- and cumate-induced, p=0.00001.

Article Snippet: Cells were then lysed with RIPA buffer and 1x protease inhibitor (PI78442, Fisher Scientific) for 2 hours on ice.

Techniques: Microscopy, Western Blot, Transferring, Membrane, Expressing, Standard Deviation