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. 2023 Aug 8;4(8):101142. doi: 10.1016/j.xcrm.2023.101142

Figure 2.

Figure 2

Both cetuximab and osimertinib reduce proliferation and induce apoptosis of cells driven by L858R-EGFR, but only osimertinib inhibits cells expressing Del19-EGFR

(A) 11-18 (L858R-EGFR) and PC9 (Del19-EGFR) cells were seeded on six-well plates and on the next day they were treated for 48 h with cetuximab (Cetux., 10 μg/mL) or osimertinib (Osim., 50 nM for PC9 or 500 nM for 11-18 cells). Thereafter cells were treated with trypsin and counted. Five thousand (11-18) or 150 (PC9) cells were seeded in six-well plates to allow colony formation. Media (without drugs) were refreshed once every 3 days. After 14 days, cells were fixed and stained with crystal violet. For image quantification, five different fields were quantified per sample using ImageJ. Signals were normalized to the control wells. Values represent mean + SEM of three biological replicates. Significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparison test. ∗∗∗∗p < 0.0001; ns, not significant.

(B) H3255 and 11-18 cells were seeded on coverslips and treated for 48 or 72 h, respectively, with cetuximab (Cetux., 10 μg/mL) or osimertinib (Osim., 50 nM for H3255 and 500 nM for 11-18 cells). Cells were fixed in paraformaldehyde (4%) and incubated with an anti-KI67 antibody, followed by an Alexa Fluor 555-conjugated secondary antibody. DAPI (blue) was used to stain nuclei. Images were captured using confocal microscopy (40× magnification). The number of KI67-positive cells was normalized to the total number of nuclei. The signals shown in the graph bars are relative to Control. Scale bars, 20 μm. Values represent mean + SEM of three biological replicates. Significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparison test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(C) H3255, 11-18 and PC9 cells were treated for 48 h with cetuximab (Cetux., 10 μg/mL), erlotinib or osimertinib (Erlot. or Osim., 500 nM for 11-18, or 50 nM for the other cell lines). Protein extracts were blotted and probed with specific antibodies. Vinculin was used as the gel loading control. See also Figure S3.