Additional file 1: of A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers

Figure S1. HK1−HK2+ cancer cells are highly sensitive to HK2 knockdown-induced growth inhibition. Figure S2. DPI synergizes with HK2 knockdown or inhibition in HK1−HK2+ liver cancer cells. Figure S3. DPI synergizes with HK2 silencing/inhibition by targeting mitochondrial complex I in HK1−HK2+ liver cancer cells. Figure S4. HK isoform expression in Hep3B/shHK2DOX xenograft tumors with DOX and/or DPI treatments. Figure S5. Inhibition of fatty acid oxidation sensitizes HK1−HK2+ liver cancer cells to the HK2 inhibition/DPI combination. Figure S6. Modulation of HK1−HK2+ liver cancer cellular metabolism by the HK2i/DPI/PER combination. Figure S7. PER as a single agent does not have a significantly detectable effect on growth of subcutaneous Hep3B/shHK2DOX tumors. (PPTX 782 kb

Additional file 2: of A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers

Table S1. The list of 119 FDA-approved oncology drugs provided by the National Cancer Institute (NCI) tested for synergy with DOX treatment in Hep3B/shHK2DOX cells. Table S2. Synergy between HK2 inhibition and DPI. (DOCX 44 kb

Islets are highly uncoupled; mice islets more than human.

<p>A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.</p

Measuring islet oxygen consumption in a high throughput format.

<p>A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1^st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.</p

Measuring coupling efficacy.

<p>A) Oligomycin titration. The F1F0 ATP synthase inhibitor oligomycin was added after 20 mM glucose as indicated. Note that 5 µM oligomycin has a similar effect as 10 µM. B) Experimental setup to measure uncoupling. A few data points are recorded under basal conditions (3 mM glucose) followed by injection of oligomycin. The remaining OCR under oligomycin represents respiration that is not coupled to ATP synthesis (uncoupling). Data is shown both as absolute rates (left) and normalized to initial values (right). C) FCCP increase OCR under oligomycin. To verify the capacity of oligomycin to reveal uncoupling, islets were treated with FCCP (1 µM) prior to oligomycin. Note the increase in OCR under oligomycin representing maximal uncoupled respiration. D) Norepinephrine activated brown adipocytes show higher respiration under oligomycin. Basal measurement was followed by injection of control media or media containing 1 µM norepinephrine which activates uncoupling. Note the increase in OCR under oligomycin indicative of uncoupling. E) Oligomycin does not alter non-mitochondrial respiration. OCR was first measured with no drugs present followed by injection of only oligomycin (OM) or both oligomycin and rotenone/myxothiazol (Rot) (5 µM of both). Later, islets that were only injected with oligomycin at first also received rotenone/myxothiazol. Note that there is no difference in non mitochondrial respiration (under rotenone/myxothiazol) between the different treatments indicating that oligomycin does not alter non mitochondrial respiration.</p

Respiration of a small cohort of human Type 2 diabetic islets.

<p>Each graph represents islets from one patient (n = 4−5). Drugs added during the experiment are indicated. Type 2 diabetic donor: Male age 52, weight 100 kg, BMI 31.9, Caucasian. Non diabetic donor: Male age 32, weight 69 kg, BMI 24.7, Caucasian.</p