Figure 4 from ATF6 Promotes Colorectal Cancer Growth and Stemness by Regulating the Wnt Pathway
Authors: Rodvold Jeffrey J. , Grimmer Matthew , Ruiz Karen , Marsters Scot A. , Oikonomidi Ioanna , Tan-Aristy Eileen , Pham Victoria C. , Sarkar Tamal , Harnoss Jonathan M. , Shatz-Binder Whitney , Modrusan Zora D. , Wu Thomas D. , Lill Jennie R. , Villemure Elisia , Rudolph Joachim , de Sousa e Melo Felipe , Ashkenazi Avi 
Published: October 2024

<p>ATF6 disruption perturbs Myc and Wnt signaling and decreases colorectal cancer cell seeding capacity. <b>A,</b> Effect of ATF6 knockdown on Wnt signaling genes. Colo201 shATF6.1 cells were incubated for the indicated time with Dox (0.5 μg/mL) and analyzed by RT-qPCR for expression of <i>ATF6</i> and Wnt signaling genes (<i>AXIN2</i>, <i>LGR5</i>, and <i>TCF7</i>) mRNA (<i>n</i> = 3). <b>B,</b> Gene expression of CCK81 shATF6 cells as treated and analyzed in <b>A</b> (<i>n</i> = 3). <b>C,</b> Effect of ATF6 knockdown on LGR5 surface expression. Colo201 shATF6.1 cells were treated with or without Dox (0.5 μg/mL) for 3 days and analyzed by flow cytometry for LGR5 surface expression (<i>n</i> = 3). <b>D,</b> LGR5 expression of CCK81 shATF6 cells as in <b>C</b> (<i>n</i> = 2). <b>E,</b> Raw luminescence units of TOP.FLASH reporter Colo201 shATF6.1 cells after 2-day treatment of Dox (0.5 μg/mL) to report Wnt signaling activity (<i>n</i> = 4). <b>F,</b> TOP.FLASH expression of CCK81 shATF6 cells as in <b>E</b> (<i>n</i> = 3). <b>G,</b> Effect of ATF6 knockdown on Myc signaling genes. Colo201 shATF6.1 cells were incubated for the indicated time with Dox (0.5 μg/mL) and analyzed by RT-qPCR for expression of Myc signaling genes (<i>MYC</i>, <i>CDCA7</i>) mRNA (<i>n</i> = 3). <b>H,</b> Gene expression of CCK81 shATF6 cells as in <b>G</b> (<i>n</i> = 3). <b>I,</b> Effect of ATF6 knockdown on Myc expression. Colo201 shNTC, shATF6.1 and shATF6.2 cells and CCK81 shNTC, shATF6 cells were incubated for the indicated time with Dox (0.5 μg/mL) and analyzed by immunoblotting. Cells treated without (−) Dox were harvested on day 4. <b>J,</b> Effect of ATF6 knockdown on seeding potential. Colo201 shNTC and shATF6.2 cells were seeded at decreasing concentrations of 96-well and grown in the presence or absence of Dox (0.5 μg/mL) for 7-day. Cell abundance determined by raw luminescence units detected from CellTiterGlo assay (<i>n</i> ≥ 3). <b>K,</b> Seeding potential of CCK81 shNTC and CCK81 shATF6 cells as in <b>J</b> (<i>n</i> ≥ 3). <b>L,</b> Viability of Colo201 shATF6.2 cells seeded at high (1.25e4 cell/well) or low (2e2 cell/well) densities, as described in <b>J</b> (<i>n</i> ≥ 3). <b>M,</b> Viability of CCK81 shATF6 cells seeded at high (1.25e4 cell/well) or low (2e2 cell/well) densities, as described in <b>L</b> (<i>n</i> ≥ 3). <b>N,</b> Fluorescent microscopy images of Colo201 shATF6.1 cells seeded at high (1.25e4 cell/well) or low (4e2 cell/well) densities in ULA wells after 7 days growth in absence or presence of Dox (0.5 μg/mL) as visualized by nuclei signal (red). <b>O,</b> Fluorescent microscopy images of CCK81 shATF6 cells, as described in <b>N</b>. MFI, Median Fluorescence Intensity; RLU, raw luminescence unit. **, <i>P</i> < 0.005; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001.</p>