Fig 2: NM23-H2 over-expression in HCC. (A) Specific immunoreactivity of NM23-H1 or NM23-H2 in cell lysates. Cells were transiently transfected with expression plasmids of NM23-H1 or NM23-H2. PC, parent cells; VC, vector control. (B) NM23-H2 over-expression in HCCs and surrounding matrix tissues according to Edmondson-Steiner grading (GI to GIII). (C) NM23-H1 was over-expressed in HCCs but not in surrounding matrix tissues. (D) Immunofluorescence assay showing ectopic expression is mainly localized in the cytoplasm and to a lesser extent in the nucleus of HEK293T and HepG2 cells. Hoechst 33258 staining of nuclei (blue). Immunoreactivity of NM23-H2 (red) is perfectively merged with GFP and NM23-H2 fusion proteins; bar, 20 µm.

Fig 3: The NMEP96S site directed mutant distinguishes 1-pHis and NDPK activities from suppression of motility in vitroA. MDA-MB-231T cells stably expressing a vector, wild type NME1 or its P96S or H118F (NME1P96S and NME1H118F) mutations were lysed and assessed for 1-pHis levels. Lysates were split into two, and one sample was heated (red). Blots were re-probed for NME1/2 and β-actin. B. 1-pHis and NME expression was also assessed by immunofluorescence in the above cells. Nuclei were visualized using DAPI (Blue). Images were captured at 65x magnification. C. Cells were tested for motility in Boyden chamber assay using 1% FBS as chemoattractant and motility quantitated E. D. Cells were tested for migration in scratch assay in 0.2% serum and migration quantitated F. G. The NDPK activity of lysates was quantitated using spectrophotometric assay. All experiments are representative of 4 replicates (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001).

Fig 4: Correlation of total and 1-phosphohitidine NME level in cell lysatesA. Cell lysates prepared from vector and NME overexpression (NME1, NME2 or mouse ortholog Nme1) in MDA-MB-231T cells were assessed for 1-phosphohistidine (1-pHis) level using anti-N1-phosphohistidine antibody on a basic gel with no heating. For specificity, replicate samples were heated at 95°C for 10 min and loaded next to each sample (red). B. MDA-MB-435 cells lysates from vector (C100) and NME overexpression (H1-177) were also assessed for 1-pHis in similar manner. Following stripping, both blots were re-probed with total NME and β-actin antibodies. C. For immunofluorescence, MDA-MB-231T cells with GFP co-expressing vector or NME overexpression were fixed in PFA and stained for 1-pHis using anti-N1-phosphohistidine antibody. Following 1-pHis primary incubation, cells were washed twice and were then stained with total NME1/2 antibody. Nuclei were visualized using DAPI (Blue). Images were captured at 65x magnification. Yellow arrows indicate vacuoles in 1-pHis staining highlighting acidic compartments.

Fig 5: MDA-MB-435 migrating cells have a low 1-phosphohistidine level that does not correlate with total NME levelA. Vector (C100) and NME1 (H1-177) overexpressing MDA-MB-435 cells were plated in chamber slides and treated as described in the legend for Figure 5A. B. After 24 hrs cells were fixed in PFA and immunofluorescence was performed for 1-pHis and NME1/2. Immunofluorescence staining was quantitated as total fluorescence intensity of 1-phosphohistidine (1-pHis) C. and total NME D. Both vector (C100) and NME overexpressing cells (H1-177) in the scratch area did not exhibit strong 1-pHis positivity. Nuclei were visualized using DAPI (Blue). Images were captured at 65x magnification in the scratch and confluent areas. Student's t-test was performed between Vector-Scratch vs NME-Scratch and Vector-Confluent vs NME-Confluent.