PMC

The reactions of the Oyster Reef network, the reaction flux graph and a representative flux configuration of 20 simulations with this network. In the reaction flux network on the left, each node represents a species as indicated by the labels. For example, FF is Filter Feeder, Mf is Meiofauna, etc. Each arrow represents a reaction, and its label is the reaction number. The thickness of each arrow is proportional to the reaction count. In the flux configuration graph on the right, each node is a reaction, and each arrow denotes the flux as the flow of the resource in the label. The thickness of each arrow is proportional to the flux strength.

Representative sorting gates used for the isolation of ES, Zscan4⁺ and 2‐cell‐like cells used throughout this study. Feeder cells were removed on the basis of their lack of far‐red fluorescence, which is higher in ES cells because of the presence of an H2B‐iRFP cassette. ESCs were defined as double negative for both the Zscan4 (Zscan4c::mCherry⁻) and the MERV‐L reporters (2C::tbGFP⁻). Zscan4⁺ cells were defined as positive for the Zscan4 reporter but negative for the MERV‐L reporter, and 2‐cell‐like cells were defined as positive for both reporters.Brightfield microscopy images indicating the confluency of the three populations shortly after plating in the Seahorse extracellular flux analyser plates. Representative images, in two magnifications, for the three independent biological replicates presented in Fig C and D are shown.Representative sorting gate for ES and 2‐cell‐like cells (left) used for the FACS‐assisted ROS measurements (right). Fluorescence intensity distributions for ES cells (blue) and 2‐cell‐like cells (green) in control (centre) and CellROX‐treated samples (right) are shown.

A. Determining biosynthetic rates of THP-1 cell metabolites. Cells were labeled with ¹³C₆-glucose, and label incorporation in metabolites of the CCM pathways was monitored at 0, 2, 5, 15, 30 and 60 minutes post label addition. Profiles for both ¹³C-incorporation (red line) and consumption of the naturally occurring isotopomer (blue line) for a representative set of intermediates are shown here (for values, n = 3, mean ±SD). B. Uninfected cells were maintained in complete RPMI media either in the absence (+G), or presence (+G+Rt) of rotenone. A parallel set of cells was maintained in glucose free media supplemented with 1 mM pyruvate as a feeder for the TCA cycle (-G+Pyr). Intracellular ATP levels were then determined by LC-MS/MS (n = 3, mean ±SD; * *p≤0.01) C. Maintenance of the mitochondrial membrane potential is regulated by the glycolytic flux. Uninfected cells were maintained either in complete RPMI media (+G) or in Glucose free media supplemented with Pyruvate as a feeder for the TCA cycle (-G+Pyr). At 24 hr after treatment, cells were stained with JC-1 and the mitochondrial membrane potential was determined in terms of the red/green fluorescence ratio by confocal microscopy (n = 3, mean ±SD, **p≤0.01). D. The proportion of cells undergoing apoptosis was also determined and the results are presented (n = 3, mean ±SD, **p≤0.01).

CDK4/6 Inhibition Impairs Proliferation of Primary AML Cells and Increases Median Survival In Vivo
(A) Impact of palbociclib on proliferation of primary AML blasts. Blasts were co-cultured on MSC feeder layers with and without 300 nM palbociclib for 72 hr. Mean ± SD; n = 3 for both t(8;21) and non-t(8;21) AML patient samples. ^∗p < 0.05 compared with no palbociclib.
(B) Phase contrast photographs showing primary t(8;21) AML blasts from patient sample LK111 in co-culture with MSCs with and without (CV) palbociclib. Standard bar, 200 μm.
(C) Proliferation of a t(8;21) AML sample from a relapsed patient on MS-5 feeders upon palbociclib treatment for 72 hr. Mean ± SD; n = 3 technical replicates.
(D and E) Cell cycle distribution (D) and changes in apoptotic subG1 cell fractions (E) of primary AML blasts obtained from two t(8;21) patients (patient samples LK19 and LK111) on MSC after 96 hr incubation with and without (CV) palbociclib.
(F) Clonogenic growth of three different t(8;21) patient samples after MSC co-culture for 96 hr. Indicated palbociclib concentrations were added either only to co-culture medium (Palbociclib in co-culture) or to both co-culture and semisolid medium (Palbociclib in co-culture & CFA). Colony numbers are relative to no palbociclib. Mean ± SD; n = 3. ^∗∗∗p < 0.001; ^∗p < 0.05 compared with no palbociclib.
(G) Bioluminescent images of RG mice transplanted with luciferase-expressing (luc⁺) Kasumi-1 cells after 21 days of treatment with control vehicle (CV) or palbociclib.
(H) Luminal flux of bioluminescence for CV (n = 8) or palbociclib-treated animals (n = 9). Treatment blocks are indicated at the bottom of the graph. ^∗∗p < 0.01; ^∗p < 0.05 compared with no palbociclib by one-way ANOVA using a D'Agostino and Pearson test; mean ± SEM.
(I) Survival curve for RG mice transplanted with luc⁺ Kasumi-1 cells. Significance was tested by log rank test.
(J) Percentage of Kit⁺ RUNX1/ETO9a (AE9a) GFP⁺ cells in mice after CV or palbociclib treatment as determined by fluorescence-activated cell sorting (FACS).
(K) Survival curve for Bl6 mice transplanted with KIT⁺ AE9a GFP⁺ cells. Significance was tested by log rank test. n = 5 for both groups with one censored animal in the palbociclib group due to death unrelated to palbociclib treatment.
(L) Model depicting the RUNX1/ETO-promoted G1 cell cycle progression and leukemic propagation by direct transcriptional activation of CCND2 and CDK6, which can be blocked by either CCND2 knockdown or by pharmacologic inhibition of CDK4/6-CCND complexes.
See also .