Total RNA was extracted with TRIzol reagent and then reversely transcribed into cDNA using a Pria Revert Aid First Strand cDNA Synthesis Kit (catalog number: #A5001, promega). Following the manufacturer’s protocols, Real-time PCR was performed using a SYBR Green Premix PCR Master Mix (catalog number: #DRR041B, TAKARA). Relative mRNA expression of CALM1 and EGFR was calculated using the 2−ΔΔCt method after being normalized to GAPDH, which served as internal loading control. PCR was performed with the following primer sets: CALM1 forward, 5′-GGTCAGAACCCAACAGAA-3′ and reverse, 5′-AGACTCGGAATGCCTCA-3′; and EGFR forward, 5′-AGGCACGAGTAACAAGCTCAC-3′ and reverse, 5′-ATGAGGACATAACCAGCCACC-3′. GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. All experiments were performed independently three times and shown was the representative one.

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