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Extended Data Fig. 4 | Consideration of combined AET dosing on the basis of
its effect on mouse models. a, Top, effect of different dosages of epigenetic
modifiers on the viability of LLC1, HNM007 and 4T1 cells in vitro (72 h, Cell
Counting Kit-8). Graphs show the mean of 3 independent experiments; two-
sample, two-sided t-tests compared with mock. Bottom, effect of low-dose
5-azacytidine (100 nM) plus entinostat (50 nM) on the proliferation of LLC1,
HNM007 and 4T1 cells in vitro. A total of 1 × 10^5 viable cells was plated per well.
Cells were collected at 24, 48 and 72 h and counted using a cell counter (Bio-
Rad) after Trypan blue exclusion. Graphs show the mean of 3 independent
experiments; significance at 72 h was determined by one-way ANOVA followed
by Tukey’s test for multiple comparisons. b, The effect of low-dose
5-azacytidine (100 nM) plus entinostat (50 nM) on the viability of MDSCs from
bone marrow (day 3) of LLC mice (top) and HNM007 mice (bottom) in vitro
(Cell Counting Kit-8). Graphs show the mean of 3 independent experiments.
c, The effect of low-dose 5-azacytidine (100 nM) plus entinostat (50 nM) on the
apoptosis of MDSCs from bone marrow (day 3) of LLC and HNM007 mice
in vitro. Cell apoptosis was measured by FACS at 48 h. The bottom right
quadrant (annexin-V+/ 7-A A D−) and top right quadrant (annexin-V+/ 7-A A D+)
represent early and late apoptotic cells, respectively. Graphs show the
percentage of total apoptosis (early and late apoptosis) in mock and treatment
groups (n = 3 biological replicates). d, Tumour growth and body weight of NSG
mice bearing LLC tissue that were treated with different doses of entinostat
plus 5-azacytidine. Significance at day 12 (top) and day 14 (bottom) was
determined by one-way ANOVA followed by Tukey’s test for multiple
comparisons. e, Summary table of tumour growth, body weight and treatment-
related death of NSG mice bearing LLC tissue. Regimens in red indicate dosages
with no effect on tumour growth, weight loss or treatment-related death.
f, Tumour growth and body weight of NSG mice bearing HNM007 tissue that
were treated with 5-azacytidine at 0.5 mg kg−1 d−1 plus entinostat at 5 mg kg−1 d−1
or vehicle. Significance at day 14 was determined by two-sample, two-sided t-
test. g, h, The effect of low-dose AET on the proliferation (g) and apoptosis (h)
of donor-derived CD45.1+ MDSCs from bone marrow in CD45.2 LLC mice.
Proliferation and apoptosis of immature (MHC-II−) and mature (MHC-II+)
CD45.1+ cells were measured by FACS at 36 h after transfusion (day 2). Graphs in
g show the percentage of Ki67+ cells (n = 3 mice per group). Graphs in h show the
percentage of total apoptosis (early and late apoptosis) in mock-treated and
low-dose-AET groups (n = 3 mice per group). In b, c, g, h, two-sample, two-sided
t-tests were used. All bars show mean ± s.e.m. *P < 0.05.