ALCAM suppressed EGFR-initiated hedgehog pathway activation. (A) Gene Set Enrichment Analysis showing hedgehog pathway gene enrichment in EGFRhigh MM cells. (B) Colony formation assay of CTR-KD or AL-KD RPMI8226, cocultured with BMSC in the presence of an EGFR inhibitor (gefitinib, 200 nM), an Erk inhibitor (U0126, 100 nM), or a Gli inhibitor (GNAT61, 5 μM). (C) Quantification of colony-forming assay. (D) Addition of recombinant EGF (10 ng/mL) in cultured CTR-KD and AL-KD RPMI8226 cells for 10 minutes, with the cell lysate used for western blotting. The results are quantified in the right panel. (E) Addition of ALCAM-Fc chimera (Al-Fc, 0.25 μg/mL) in cultured CTR-KD and AL-KD RPMI8226 cells for 1 hour, with the cell lysate used for western blotting. The results are quantified in the right panel. (F) RPMI8226 cells (CTR-KD and AL-KD) cocultured with BMSC-M for 48 hours or cultured in complete medium, and examined by western blotting. The results are quantified in the right panel. (G) RPMI8226-CTR cells cultured under normal conditions, or treated with recombinant EGF (10 ng/mL) for 10 minutes, or cocultured with BMSC-M for 48 hours, then examined the hedgehog pathway genes GLI1, PTCH1, and MYC by qPCR. (H) Performed similar experiment as in panel G with RPMI8226-ALCAM- KD cells. (I) RPMI8226 cells cultured with or without recombinant EGF (10 ng/mL) for 1 hour. The cells were examined by immunofluorescence staining followed by confocal florescence microscopy. The data were obtained from 10 randomly choose fields and shown as the mean ± SD. Red: Gli1; blue: DAPI for nucleus. The Gli1 distribution in cells are quantified in the right panel. (J) The same immunofluorescence staining of Gli1 in RPMI8226 cells (AL-KD) with or without addition of recombinant EGF (−, without EGF; +, EGF 10 pg/mL; ++, EGF 10 ng/mL). All data in bar graphs were assessed by 2-tailed Student t test. *P < .05, **P < .01; ***P < .001.