Adaptations in heart structure and function likely enabled endurance and survival in preindustrial humans.
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Contributed by Stefan W. Hell, November 1, 2019 (sent for review August 6, 2019; reviewed by Peter Macheroux and Alice Y. Ting)
Article Figures & SI
Figures
- Fig. 1.
Bioluminescence of wild-type Lux and co Lux. HEK 293 cells were transfected with (A) wild-type lux and frp genes without codon optimization or (B) the co lux genes in a ratio of luxA:luxB:luxC:luxD:luxE:frp = 1:1:3:3:3:1. Cells were imaged in a 24-well plate with an Amersham Imager 600. (Left) White-light images of the respective well with a monolayer of cells, and (Right) bioluminescence signal after the indicated exposure time. The color maps were scaled to the minimum and maximum camera counts per pixel of the bioluminescence images.
- Fig. 2.
Bioluminescence of HEK 293 cells expressing different combinations of co lux genes or FLuc from pcDNA3.1(+). Cells were grown in 24-well plates, transfected with the same total amount of DNA, and imaged with an Amersham Imager 600. Asterisk (*) indicates transfection of all plasmids in equal amounts. Otherwise, plasmids encoding LuxA, LuxB, LuxC, LuxD, LuxE, and Frp were transfected in a ratio of 1:1:3:3:3:1. Plasmids encoding 2 or 3 genes were transfected in 2- and 3-fold amounts, respectively. The following abbreviations are used: luxAB, luxA-P2A-luxB; luxBA, luxB-P2A-luxA; luxCD, luxC-P2A-luxD; luxDE, luxD-P2A-luxE; luxABfrp, luxA-P2A-luxB-T2A-frp; luxDEC, luxD-P2A-luxE-T2A-luxC. Plus (+) signs indicate expression from separate plasmids. For imaging of FLuc, 150 µg/mL d-luciferin was added to the medium. Error bars represent SD of the signal of 5 separate wells.
- Fig. 3.
Co lux toxicity control measurements. HeLa cells were cotransfected with co lux genes in pcDNA3.1(+) or the empty vector, and a fluorescent sensor where indicated. (A) Absorption of cell lysates from MTT assay with untransfected cells (−) for comparison. Error bars represent SD of 3 separate coverslips. (B–D) Comparison of (B) ATP, (C) NADPH, and (D) H2O2 levels by ratiometric fluorescence imaging of the sensors (B) PercevalHR, (C) iNap1, and (D) HyPer-3 using the indicated excitation wavelengths. Error bars represent SD of 50 cells. ** represents a P value of <0.01 as calculated by a 2-tailed Student’s t test.
- Fig. 4.
Bioluminescence images of different cell lines expressing the co lux genes. Images were taken with the indicated exposure times using a (A) 60× or (B) 100× objective lens. The color map was scaled to the minimum and maximum pixel values of each image. For the (A) 3-min and (B) 5-min images, the color bars represent the number of detected photons per pixel. Blue pixels represent saturation of the camera. Fluorescence images (fluo) of cotransfected lifeact-EYFP excited at 491 nm are shown in gray. (Scale bars, 20 µm.)
- Fig. 5.
Long-term bioluminescence measurements of HeLa cells expressing co lux. Cells were imaged in cell culture medium with (A) no additive, (B) gramicidin (25 µg/mL), or (C) DNP (25 µg/mL) or (D) in EBSS without glucose. In C, cells were washed 5 times with EBSS after 8 h, and imaging was continued in cell culture medium without additives. In D, cells were washed 10 times with EBSS before starting the measurement, and EBSS was replaced with cell culture medium after 12 h. Bioluminescence images were taken with a 60× objective lens and exposure times of (A, C, and D) 3 min or (B) 2 min. The color map was scaled to the minimum and maximum pixel values of each image series. Blue pixels represent saturation. Arrows indicate dividing cells. Fluorescence images (fluo) of cotransfected lifeact-EYFP excited at 491 nm are shown in gray. Complete time series are shown in Movies S1–S4. (Scale bars, 20 µm.)
Data supplements
Supporting Information
- Download Appendix (PDF)
- Download Movie_S01 (MOV) - Cell division of HeLa cells expressing co lux. Cells were imaged in cell culture medium without additives. Single images were taken with 3-min exposure time. (Scale bar: 20 μm.)
- Download Movie_S02 (MOV) - HeLa cells expressing co lux treated with gramicidin. Cells were imaged in cell culture medium with gramicidin (25 μg/mL). Single images were taken with 2-min exposure time. (Scale bar: 20 μm.)
- Download Movie_S03 (MOV) - HeLa cells expressing co lux treated with DNP. Cells were imaged in cell culture medium with DNP (25 μg/mL). After 8 h, cells were washed and the medium was replaced by fresh cell culture medium without additives. Single images were taken with 3-min exposure time. (Scale bar: 20 μm.)
- Download Movie_S04 (MOV) - HeLa cells expressing co lux imaged in EBSS without glucose. After 12 h, EBSS was replaced by fresh cell culture medium without additives. Single images were taken with 3-min exposure time. (Scale bar: 20 μm.)
Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system
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