Fig. 2.

HK2 is required for Kras-driven lung tumor formation. a, b Protein level of HK2 was detected in KP2 and H23 cells expressing shRNAs for HK2 by WB. c, d Colonies were stained by crystal violet after 7 days of cell growth after knockdown of HK2 in KP2 and H23 cells. e–h Xenograft tumor growth of HK2 knockdown. Subcutaneously injected 1 × 10⁶ cells expressing scramble shRNA (CON) or shRNA for HK2 in the lower flank of NSG mice. Representative images of tumors 4 weeks after injection are shown (e). Quantification of tumors weight from tumors developed in NSG mice (P < 0.01) (f). g IHC staining of Ki67 from tumors developed in NSG mice carrying control or HK2-knockdown KP2 cells. h Quantification of Ki67 (P < 0.01) expression from representative images shown in G. i–p Rescuing HK2 assay. Protein level of HK2 was detected in three independent KRAS knockdown KP2 cell lines and one KRAS knockdown H23 cell line, all of which over-express HK2 (i, j). Then these cells were fixed and stained with crystal violet after 7 days (k, l). m–p Xenograft tumor assay of rescuing HK2 in Kras-knockdown KP2 cells. Subcutaneously injected 1 × 10⁶ KP2 cells expressing shRNA for Kras or Kras knockdown KP2 cells harboring up-regulating HK2 in the lower flank of NSG mice. Representative images of tumors at 4 weeks after injection are shown (m). Tumors weight (P < 0.01) from tumors developed in NSG mice (n). o, p IHC staining and quantitative analysis of cell proliferation marker Ki67 (P < 0.01)