To preliminarily explore the underlying mechanism of Shikonin against afatinib-resistant NSCLC in vivo, immunohistochemical analysis was performed and the result indicated that Shikonin decreased p-Akt and Bcl-2 expression, while increased cleaved caspase-3 and Bax expression (Figure 4D), confirming that Shikonin suppressed afatinib-resistant NSCLC xenograft tumor growth by regulating PI3K/Akt signaling pathway in vivo. The gene discussed is AKT1; the disease is non-small cell lung carcinoma.