Skip to main content
. 2020 May 7;78(3):477–492.e8. doi: 10.1016/j.molcel.2020.04.008

Figure 7.

Figure 7

Expression of Cell Surface G6B, a Cell Surface Protein, Identifies Mutant Clone-Derived HSPCs in Myelofibrosis

(A) Left: expression of 6 megakaryocyte markers from a panel of 20 HSPC and megakaryocyte cell surface antigens assayed by mass spectrometry time of flight (CyTOF) shows expression of G6B on CD34+ HSPCs from patients with primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (PET-MF), and post-polycythaemia vera myelofibrosis (PPV-MF) with either JAK2V617F (JAK2+) or calreticulin (mutCALR) driver mutations. Histograms show cell count (y axis) by expression level (x axis). Right: viSNE dimensionality reduction plots on a representative control and myelofibrosis sample for CD9 and G6B, illustrating more substantial differential expression of G6B than CD9 in myelofibrosis versus control cells

(B) Flow cytometric analysis of G6B expression on CD34+ Lin− HSPCs showing significant increase in G6B+ cells in myelofibrosis (% GFP+ cells, 28.8% ± 5.5% versus 2.4% ± 1.0%); chart shows mean + SEM (left) and example plot (right) shown, illustrating expression in both CD41+ and negative cells. ∗∗p ≤ 0.01 (t test). Controls (N = 8); myelofibrosis (N = 11).

(C) Immunohistochemical staining for G6B (diaminobenzidine, DAB brown) of bone marrow biopsy sections from controls and myelofibrosis patients with JAK2V617F and mutCALR-positive myelofibrosis showing marked expansion of G6B+ megakaryocytes and progenitors in myelofibrosis.

(D) Mononuclear cells from healthy donors and patients with JAK2V617F+ myelofibrosis were combined and 50 cell “mini-bulk” replicates were sorted from the G6B+ and G6B− fractions for Taqman qRT-PCR to quantify expression of JAK2V617F mutated and wild-type JAK2. Chart shows JAK2V617F relative to wild-type JAK2 expression for all mini-bulks from 3 replicate experiments.

(E) Internalization of a CD34 × G6B bi-specific antibody and isotype control antibody conjugated to a pH-sensitive cyanine CypHer5E dye that fluoresces at an acidic pH following internalization. Left: representative images show clear intracellular fluorescence for CD34 × G6B bi-specific but not isotype control. Right: mean fluorescence intensity of cells measured by flow cytometry 30 min after addition of antibody with/without two endocytosis inhibitors, Dynasore and Pitstop 2. Data shown using SET-2 cells, chart shows mean + SEM, ** - P < 0.01 n= 3. See also Figure S7.