Fig. 5
S100A14 stabilizes kidney-type glutaminase in cultures of HCC cells. (A) Huh7 cells were transfected with one of two short hairpin RNAs against kidney-type glutaminase (shGLSKGA) or NTC, treated with sorafenib, then assessed for viability using the CCK-8 assay (n = 3). (B) Cells were transfected as described in panel A but also with empty expression vector (Vector) or plasmid expressing HA-tagged wild-type GLSKGA (GLSKGA WT) or a catalytically inactive mutant (GLSKGA S286A). Cell lysates were immunoblotted with antibodies against GLSKGA, HA, and GAPDH. (C) Cells were transfected as described in panel B, treated with sorafenib, and assayed for viability in the CCK-8 assay (n = 3). (D) Relative levels of GLSKGA mRNA in Huh7 cells that had been transfected with shS100A14 or NTC (n = 3). (E and F) (E) Western blotting of GLSKGA protein in cells transfected as described in panel D which were treated with cycloheximide (CHX) from 0 to 24 h and then (F) GLSKGA protein levels were quantified by ImageJ (n = 3). (G, H, I and J) Cells were transfected with shS100A14 or NTC to knock down S100A14 but overexpress HA-tagged wild-type or inactive GLSKGA, then (G) lysates were immunoblotted with antibodies against GLSKGA, HA, S100A14, and GAPDH, (H) cell viability under sorafenib treatment was measured using the CCK-8 assay, (I) production of ROS was assessed by staining cells with DCFH-DA probe followed by flow cytometry, and (J) levels of reduced glutathione (GSH) in lysates were assayed. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant