Figure 3.
ALCAM interacted with EGFR in myeloma cells. (A) Proximity ligation assay showing ALCAM and EGFR interaction in human MM cell lines (normal RPMI8226, MM1S cells, and ALCAM-KD cells) and primary MM cells (2 of 5) under fluorescence microscopy. The cell nucleus was stained using DAPI (blue). The white arrow points to the site with a positive signal (red spot). Staining EGFR and ALCAM in RPMI8226 cells alone as the negative control. (B) Left: immunoprecipitation of ALCAM and EGFR in RPMI8226 cells, followed by the western blotting of EGFR (top) and ALCAM (bottom), respectively; right: immunoprecipitation of ALCAM and EGFR in RPMI8226 cells, followed by the western blotting of ALCAM (top) and EGFR (bottom), respectively. (C) Confocal fluorescence microscopy analysis of immunofluorescently stained RPMI8226 cells. The y-axis indicates the value of integrated optical density, which could demonstrate the colocalization of ALCAM and EGFR or pEGFR. The data were obtained from 10 randomly choose fields and shown as the mean ± SD. Red, ALCAM; green, EGFR or pEGFR; blue, DAPI for nucleus. (D) Result quantification. (E) Similarly performed experiment as in panel C with RPMI8226-ALCAM- KD cells; results are summarized in panel F. The data were obtained from 3 independent experiments and shown as the mean ± SD. (G) The same immunofluorescence staining of ALCAM and EGFR in MM cells RPMI8226 with or without addition of recombinant EGF (10 ng/mL) for 1 hour. (H) Result quantification. (I) Structure simulation showing the interaction of EGF-EGFR extracellular domain (left) and ALCAM-EGFR extracellular domains (right). EGFR extracellular domain structure is shown in the black and white surface. The higher ZDOCK score indicates higher binding affinity. (J) Top: recombinant EGF (0.5 μg/mL) and EGFR (1 μg/mL) were dissolved in Tris-HCl buffer (pH = 7.4) in the presence of increasing doses of ALCAM-Fc (−, without ALCAM-Fc; +, ALCAM-Fc 0.5 μg/mL; ++, ALCAM-Fc 1 μg/mL), after 30 minutes’ incubation on ice, the mixtures were subjected for immunoprecipitation using αEGF antibody, followed by the western blotting of EGFR and EGF, respectively; bottom: ALCAM-Fc (0.5 μg/mL) and EGFR (1 μg/mL) were dissolved in Tris-HCl buffer (pH = 7.4) in the presence of increasing doses of recombinant EGF (−, without recombinant EGF; +, recombinant EGF 1 μg/mL; ++, recombinant EGF 2 μg/mL), after 30 minutes’ incubation on ice, the mixtures were subjected for immunoprecipitation using αALCAM antibody, followed by the western blotting of EGFR and ALCAM, respectively. All data in bar graphs were assessed by 2-tailed Student t test. *P < .05.

ALCAM interacted with EGFR in myeloma cells. (A) Proximity ligation assay showing ALCAM and EGFR interaction in human MM cell lines (normal RPMI8226, MM1S cells, and ALCAM-KD cells) and primary MM cells (2 of 5) under fluorescence microscopy. The cell nucleus was stained using DAPI (blue). The white arrow points to the site with a positive signal (red spot). Staining EGFR and ALCAM in RPMI8226 cells alone as the negative control. (B) Left: immunoprecipitation of ALCAM and EGFR in RPMI8226 cells, followed by the western blotting of EGFR (top) and ALCAM (bottom), respectively; right: immunoprecipitation of ALCAM and EGFR in RPMI8226 cells, followed by the western blotting of ALCAM (top) and EGFR (bottom), respectively. (C) Confocal fluorescence microscopy analysis of immunofluorescently stained RPMI8226 cells. The y-axis indicates the value of integrated optical density, which could demonstrate the colocalization of ALCAM and EGFR or pEGFR. The data were obtained from 10 randomly choose fields and shown as the mean ± SD. Red, ALCAM; green, EGFR or pEGFR; blue, DAPI for nucleus. (D) Result quantification. (E) Similarly performed experiment as in panel C with RPMI8226-ALCAM- KD cells; results are summarized in panel F. The data were obtained from 3 independent experiments and shown as the mean ± SD. (G) The same immunofluorescence staining of ALCAM and EGFR in MM cells RPMI8226 with or without addition of recombinant EGF (10 ng/mL) for 1 hour. (H) Result quantification. (I) Structure simulation showing the interaction of EGF-EGFR extracellular domain (left) and ALCAM-EGFR extracellular domains (right). EGFR extracellular domain structure is shown in the black and white surface. The higher ZDOCK score indicates higher binding affinity. (J) Top: recombinant EGF (0.5 μg/mL) and EGFR (1 μg/mL) were dissolved in Tris-HCl buffer (pH = 7.4) in the presence of increasing doses of ALCAM-Fc (−, without ALCAM-Fc; +, ALCAM-Fc 0.5 μg/mL; ++, ALCAM-Fc 1 μg/mL), after 30 minutes’ incubation on ice, the mixtures were subjected for immunoprecipitation using αEGF antibody, followed by the western blotting of EGFR and EGF, respectively; bottom: ALCAM-Fc (0.5 μg/mL) and EGFR (1 μg/mL) were dissolved in Tris-HCl buffer (pH = 7.4) in the presence of increasing doses of recombinant EGF (−, without recombinant EGF; +, recombinant EGF 1 μg/mL; ++, recombinant EGF 2 μg/mL), after 30 minutes’ incubation on ice, the mixtures were subjected for immunoprecipitation using αALCAM antibody, followed by the western blotting of EGFR and ALCAM, respectively. All data in bar graphs were assessed by 2-tailed Student t test. *P < .05.

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