Fig. 1

A Schematic presentation of approach for CRSPR/Cas9 based kinome knockout screening to discover the potential kinase responsible for 5FU resistance in OSCC. B, C Primary screening of 840 kinases was performed in H357 5FUR line with sublethal dose of 5FU (8 μM). The kinases (n = 506 nos) whose knockout alone induced significantly higher cell death (>30%) depicted in red were excluded. From the rest of the kinases (n = 334 nos) depicted in green, the survival fraction (5FU treated/Vehicle Control) was determined and top 60 candidates having lowest survival fraction were considered for secondary screening described in panel (C). D For secondary screening with top 60 kinases, four cell lines were considered i.e., H357 5FUR, SCC4 5FUR, SCC9 5FUR and H357CisR. After overlapping all three 5FUR cell lines, MINK1, SBK1, FKBP1A were found to be the common kinases among them. MINK1 was selected as a potential kinase target purely based on having the lowest survival fraction among all common candidates. E Heat map based on % of cell viability with or without treatment of 5FU or cisplatin followed by knocking out 60 individual kinases in H357 5FUR, SCC4 5FUR, SCC9 5FUR and H357 CisR cells. F The fluorescent images acquired from high content analyzer with indicated treated group in H357 5FUR lines during kinome screening. G Lysates were collected from indicated cells and immunoblotting (n = 3) was performed with indicated antibodies. H Protein expression of MINK1 was analyzed by IHC in chemotherapy- responder and chemotherapy-non-responder OSCC tumors. Scale bars: 50 μm. I IHC scoring for MINK1 from panel (H) (Q Score = Staining Intensity × % of Staining), (Median, n = 11 for chemotherapy-responder and n = 23 for chemotherapy-non-responder) *P < 0.05 by 2-tailed Student’s t test. J Protein expression of MINK1 was analyzed by immunohistochemistry (IHC) in pre- and post-TPF treated paired tumor samples from chemotherapy-non-responder patients. Scale bars: 50 μm. K IHC scoring for MINK1 from panel (J) (Q Score = Staining Intensity × % of IHC Staining). *P < 0.05 by 2-tailed Student’s t test.