Nano thermometry measure of muscle efficiency

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1 SUPPORTING INFORMATION Nano thermometry measure of muscle efficiency Suvra S. Laha, Akshata R. Naik, Eric R. Kuhn, Maysen Alvarez, Alyson Sujkowski, Robert J. Wessells, Bhanu P. Jena* Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA Equal contribution *Correspondence: MATERIALS AND METHODS QDs conjugation to Drosophila melanogaster in presence of ATPase inhibitors D. melanogaster flight muscles were incubated with 2 ul of CdTe QDs for 15 minutes before the addition of 100 ul of the respective inhibitor. We used 1nM bafilomycin (VH + - ATPase inhibitor) 1 and 250 um N-benzyl-p-toluenesulfonamide (BTS) 2, individually for each experiment (n=3 for each inhibitor). After incubating for 15 minutes with the inhibitor, we imaged using a Axiovert 200 (Carl Zeiss, Munich, Germany), 10X (NA= 0.50). Images were taken before ATP addition, directly after addition, and then every 30 seconds for a period of 4 minutes. We used PBS for a control. BTS was chosen as the myosin ATPase inhibitor due to the photo-inactivation of BBS in the presence of the blue light, necessary to image the QDs 2-4. Additionally, similar to the other fluorimetric experiments, we also measured the spectro- 1

2 fluorimetric intensity changes of BC QD myosin complexes in the presence of BTS at the same concentration mentioned above. For these measurements a Hitachi F-2000 Fluorescence spectrophotometer was used. CdTe QDs incorporation into cancer cells MCF10A (non malignant) breast epithelial cells, MCF10AneoT and TGF3B breast cancer cells (pre-malignant) and MCF10CA1h (malignant) breast cancer cell lines were grown separately on 35mm glass bottom Petri dishes by dividing the dish into 4 different quadrants. Cell plates were rinsed with PBS (1x) and incubated with μl of 10mg/ ml CdTe QDs in 2-3 ml of fresh cell culture media for minutes at 37 o C. Post incubation, cells were again rinsed twice with PBS (1x) to wash off excess QDs and fresh 1 ml PBS (1x) was added. Cells were then imaged under a blue filter using Axiovert 200 (Carl Zeiss, Munich, Germany), 10X (NA= 0.5). Additionally, cancer cell lines, WSU12 and UP154, were cultured on 35mm glass bottom petri dishes and treated in a similar way to the breast cancer cell lines. Once the CdTe QDs were incorporated into the cells, they were imaged using Axiovert 200 (Carl Zeiss, Munich, Germany), 40X (NA= 1.3). REFERENCES (1) Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. J. Biol. Chem. 1991, 266, (26), (2) Bond, L. M.; Tumbarello, D. A.; Kendrick-Jones, J.; Buss, F. Future Med. Chem. 2013, 5, (1),

3 (3) Shaw, M. A.; Ostap, E. M.; Goldman, Y. E. Biochemistry 2003, 42, (20), (4) Cheung, A.; Dantzig, J. A.; Hollingworth, S.; Baylor, S. M.; Goldman, Y. E.; Mitchison, T. J.; Straight, A. F. Nat. Cell Biol. 2002, 4, (1),

4 SUPPORTING FIGURES Figure S1. Estimation of thermal resolution from fluorescent intensity of CdTe QDs. (A) Relative fluorescent (FL) intensity of CdTe QDs dispersed in PBS ph 7.4 at different temperatures for four representative experiments. The black line is a linear fit of four trials (R 2 = 0.98). (B), (C) and (D) Relative FL intensity drop corresponding to 0.1 0, and C rise in temperature respectively. FL value at a = , FL value at b = , Difference in 4

5 FL value = Therefore, ~0.002% drop in fluorescence corresponds to 1mK rise in temperature. Figure S2. Inorganic phosphate standard curve. Standard curve showing absorbance at 650 nm of various concentrations of inorganic phosphate (ip) release from ATP in nmoles. 5

6 Figure S3. ATP dose-dependent decrease in fluorescence intensity of CdTe QD-RS myosin complex. Figure shows the percent decrease in fluorescent intensity with exposure of rabbit skeletal (RS) myosin to increasing concentrations of ATP. The drop in fluorescence is maximal at 3 mm ATP, and saturates beyond this point. 6

7 Figure S4. Rate of loss in fluorescence intensity of CdTe QDs associated with Drosophila skeletal muscles preparations. Muscles from y 1 w 1 ; UAS-srl (control, Green) and mef2> UASsrl (experimental, Red) fly, in the initial 30 sec following exposure to ATP. Note the rate of heat loss is twice for the control compared to the spargel-expressing experimental muscles. 7

8 Figure S5. ATP hydrolysis by fly muscle is primarily governed by myosin. Inhibition of fly muscle myosin using N-benzyl-p-toluenesulfonamide (BTS) results in abrogation of ATP hydrolysis resulting in negligible drop of fluorescence, reflecting little or no generation of heat. (A) Relative fluorescent (FL) intensity decay of CdTe QDs over 4 minutes after addition of 5 mm ATP in the presence of 250 µm BTS (green), 1 nm bafilomycin (brown) and vehicle, PBS 1x (red). (B) Fluorescent microscopy images obtained at 30 second intervals of D. melanogaster skeletal muscle over 4 minutes in the presence of 250 µm BTS, 1 nm bafilomycin, and vehicle (PBS). Scale bar = 500 µm. (n= 3). Note little loss of florescence in the BTS-treated muscle compared to the bafilomycin and vehicle control. 8

9 Figure S6. N-benzyl-p-toluenesulfonamide (BTS) inhibits myosin ATPase. Real-time spectrofluorimetry of CdTe QDs associated with BC myosin following exposure to ATP in presence and absence of BTS. Note no loss of fluorescence in presence of BTS. One of three similar experiments. 9

10 Figure S7. Cancer cells exhibit reduced CdTe QD fluorescence. (A) Fluorescent microscopy images of MCF10A (non malignant), MCF10AneoT and TGF3B (pre-malignant) and MCF10CA1h (malignant) mammary cells. A quantification of the mean fluorescence intensities of each of the cell types is depicted in the bar graph. Non-malignant MCF10A cells exhibit significantly higher (p<0.05) fluorescent intensity compared to the premalignant and malignant. Scale bar= 100µm (B) Human head-neck cancer cell line WSU12 with a primarily glycolytic metabolism is warmer than oxidative UP154 cells, reflected in low fluorescence of the CdTe QDs associated with WSU12 cells. In contrast, QDs associated with the UP154 cells, which have primarily an oxidative metabolism, fluoresce much brighter. Scale bar= 20µm. 10