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Tatjana P. Harting, Mandy Stubbendorff, Susanne C. Hammer, Patrik Schadzek, Anaclet Ngezahayo, Hugo Murua Escobar, Ingo Nolte
Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines
www.plosone.org
www.plosone.org
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(2%)Tj
1.4513 0 Td
(penicillin-strep-)Tj
-29.7295 -1.2983 Td
(tomycin)Tj
3.5377 0 Td
(\(Biochrom,)Tj
4.7905 0 Td
(Berlin,)Tj
2.88 0 Td
(Germany\))Tj
4.3256 0 Td
(and)Tj
1.7347 0 Td
(incubated)Tj
4.1726 0 Td
(at)Tj
0.9524 0 Td
(37C)Tj
2.228 0 Td
(and)Tj
1.7291 0 Td
(5%)Tj
1.457 0 Td
(CO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 492.1511 648.3968 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 497.9338 650.0976 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(in)Tj
1.0374 0 Td
(humidified)Tj
-30.8293 -1.3039 Td
(atmosphere.)Tj
5.125 0 Td
(At)Tj
1.2076 0 Td
(85%)Tj
1.9275 0 Td
(confluency)Tj
4.6091 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(trypsinized)Tj
4.6715 0 Td
(with)Tj
1.9955 0 Td
(TrypLE)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 466.4125 641.537 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 479.9055 637.0582 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Express)Tj
3.2881 0 Td
(\(Gibco)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 540.3968 641.537 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 551.622 637.0582 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
-35.1607 -1.2982 Td
(Thermofisher)Tj
5.7203 0 Td
(Scientific\))Tj
4.2406 0 Td
(and)Tj
1.7291 0 Td
(were)Tj
2.1203 0 Td
(splitted)Tj
3.1635 0 Td
(1:2.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 597.8834 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(DCA)Tj
2.3102 0 Td
(application)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 580.8755 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cells)Tj
2.1599 0 Td
(used)Tj
2.058 0 Td
(in)Tj
1.0374 0 Td
(experiments)Tj
5.1364 0 Td
(were)Tj
2.126 0 Td
(exposed)Tj
3.4412 0 Td
(to)Tj
1.0261 0 Td
(10)Tj
1.1736 0 Td
(mM)Tj
1.9332 0 Td
(DCA)Tj
2.3017 0 Td
(over)Tj
1.9786 0 Td
(48)Tj
1.1678 0 Td
(hours)Tj
2.5172 0 Td
(and)Tj
1.7291 0 Td
(cultured)Tj
3.566 0 Td
(with)Tj
1.9956 0 Td
(10)Tj
1.1735 0 Td
(ml)Tj
-36.5213 -1.3039 Td
(medium)Tj
3.6056 0 Td
(in)Tj
1.0318 0 Td
(75)Tj
1.1735 0 Td
(cm)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 270.5385 572.3149 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 276.3212 567.8362 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(flasks)Tj
2.4037 0 Td
(\(TPP,)Tj
2.5172 0 Td
(Faust)Tj
2.3697 0 Td
(Lab)Tj
1.6951 0 Td
(Science\).)Tj
3.7757 0 Td
(DCA)Tj
2.3018 0 Td
(was)Tj
1.7007 0 Td
(diluted)Tj
3.0388 0 Td
(in)Tj
1.0318 0 Td
(deionized)Tj
4.1272 0 Td
(water)Tj
2.4321 0 Td
(and)Tj
-35.0246 -1.2982 Td
(filter-sterilized)Tj
6.0605 0 Td
(after)Tj
2.0351 0 Td
(pH)Tj
1.5081 0 Td
(modulation)Tj
4.9039 0 Td
(to)Tj
1.0261 0 Td
(7.4)Tj
1.3946 0 Td
(with)Tj
1.9956 0 Td
(NaOH.)Tj
3.1294 0 Td
(All)Tj
1.4003 0 Td
(cell)Tj
1.5591 0 Td
(lines)Tj
2.0636 0 Td
(were)Tj
2.126 0 Td
(cultured)Tj
3.5659 0 Td
(without)Tj
-32.7682 -1.2983 Td
(DCA)Tj
2.3017 0 Td
(and)Tj
1.7292 0 Td
(used)Tj
2.0636 0 Td
(as)Tj
1.0148 0 Td
(non-treated)Tj
4.9491 0 Td
(control)Tj
3.1238 0 Td
(respectively.)Tj
5.108 0 Td
(The)Tj
1.7858 0 Td
(concentration)Tj
5.8167 0 Td
(of)Tj
1.0148 0 Td
(10)Tj
1.1735 0 Td
(mM)Tj
1.9275 0 Td
(DCA)Tj
2.3074 0 Td
(was)Tj
-34.3159 -1.2982 Td
(chosen)Tj
2.9991 0 Td
(in)Tj
1.0261 0 Td
(order)Tj
2.4094 0 Td
(to)Tj
1.0148 0 Td
(allow)Tj
2.33 0 Td
(comparability)Tj
5.7543 0 Td
(with)Tj
1.9899 0 Td
(previous)Tj
3.6397 0 Td
(human)Tj
3.067 0 Td
([)Tj
0.83 0.64 0.02 0 k
(27)Tj
0 g
(])Tj
1.8425 0 Td
(and)Tj
1.7178 0 Td
(canine)Tj
2.8573 0 Td
([)Tj
0.83 0.64 0.02 0 k
(20)Tj
0 g
(])Tj
/F11 1 Tf
1.8425 0 Td
[()12()]TJ
1.0035 0 Td
[()-13()14()7()20()]TJ
/F5 1 Tf
2.0353 0 Td
(stud-)Tj
-35.5292 -1.304 Td
(ies.)Tj
1.4967 0 Td
(Furthermore,)Tj
5.6126 0 Td
(this)Tj
1.6723 0 Td
(dosage)Tj
2.9424 0 Td
(is)Tj
0.8447 0 Td
(applied)Tj
3.1577 0 Td
(in)Tj
1.0319 0 Td
(an)Tj
1.2018 0 Td
(explorative)Tj
4.6318 0 Td
(study)Tj
2.3868 0 Td
(setting.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 489.6566 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Cell)Tj
1.9134 0 Td
(counting)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 472.6488 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(After)Tj
2.2847 0 Td
(washing)Tj
3.5092 0 Td
(with)Tj
1.9956 0 Td
(PBS)Tj
1.8312 0 Td
(to)Tj
1.0261 0 Td
(remove)Tj
3.2201 0 Td
(dead)Tj
2.1317 0 Td
(cells)Tj
1.9219 0 Td
(and)Tj
1.7291 0 Td
(trypsinization)Tj
5.777 0 Td
(with)Tj
1.9955 0 Td
(TrypLE)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 504.8503 477.0708 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 518.3433 472.6488 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Express)Tj
3.2881 0 Td
(\(Gib-)Tj
-35.1209 -1.2982 Td
(co)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 209.3102 464.0881 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 220.5354 459.6661 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4478 0 Td
(Thermofisher)Tj
5.7203 0 Td
(Scientific\))Tj
4.2406 0 Td
(cell)Tj
1.559 0 Td
(number)Tj
3.4186 0 Td
(was)Tj
1.7008 0 Td
(counted)Tj
3.4752 0 Td
(with)Tj
1.9956 0 Td
(an)Tj
1.2019 0 Td
(automated)Tj
4.4901 0 Td
(Cellometer)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 547.2566 464.0881 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 446.6267 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Auto)Tj
2.2336 0 Td
(T4)Tj
1.3096 0 Td
(\(Nexcelom)Tj
4.6035 0 Td
(Bioscience,)Tj
4.6714 0 Td
(Lawrence,)Tj
4.2917 0 Td
(MA,)Tj
2.0239 0 Td
(USA\))Tj
2.4548 0 Td
(and)Tj
1.7291 0 Td
(compared)Tj
4.2463 0 Td
(with)Tj
1.9955 0 Td
(untreated)Tj
4.0706 0 Td
(control.)Tj
-33.63 -1.2982 Td
(Cells)Tj
2.1599 0 Td
(were)Tj
2.1203 0 Td
(washed)Tj
3.1805 0 Td
(with)Tj
1.9956 0 Td
(PBS,)Tj
2.0579 0 Td
(centrifuged)Tj
4.7849 0 Td
(at)Tj
0.9524 0 Td
(1000)Tj
2.126 0 Td
(rpm)Tj
1.9275 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1735 0 Td
(minutes)Tj
3.464 0 Td
(\(Biochrom\))Tj
4.9039 0 Td
(and)Tj
1.7291 0 Td
(stored)Tj
2.7099 0 Td
(at)Tj
-36.6687 -1.2983 Td
(-80C)Tj
2.5738 0 Td
(for)Tj
1.389 0 Td
(further)Tj
3.0273 0 Td
(examinations)Tj
5.5956 0 Td
(\(quantitative)Tj
5.3178 0 Td
(RT-PCR)Tj
3.6453 0 Td
(and)Tj
1.7291 0 Td
(protein)Tj
3.1578 0 Td
(analysis\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 394.4125 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Lactate)Tj
3.463 0 Td
(levels)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 377.4047 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Lactate)Tj
3.067 0 Td
(release)Tj
2.9027 0 Td
(in)Tj
1.0374 0 Td
(media)Tj
2.6929 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(by)Tj
1.1792 0 Td
(colorimetric)Tj
5.1477 0 Td
(determination)Tj
5.9527 0 Td
(with)Tj
2.0013 0 Td
(Cobas)Tj
/F12 1 Tf
(1)Tj
/F5 1 Tf
3.685 0 Td
(C311)Tj
-33.8341 -1.2982 Td
(\(Hitachi,)Tj
3.77 0 Td
(Tokio,)Tj
2.8346 0 Td
(Japan\).)Tj
3.0501 0 Td
(Therefore)Tj
4.1669 0 Td
(growth)Tj
3.0727 0 Td
(media)Tj
2.6929 0 Td
(from)Tj
2.2054 0 Td
(cell)Tj
1.559 0 Td
(culture)Tj
3.0387 0 Td
(was)Tj
1.6951 0 Td
(centrifuged)Tj
4.7849 0 Td
(at)Tj
0.9524 0 Td
(1000)Tj
-33.8227 -1.2983 Td
(rpm)Tj
1.9275 0 Td
(for)Tj
1.3833 0 Td
(10)Tj
1.1735 0 Td
(minutes)Tj
3.4639 0 Td
(to)Tj
1.0262 0 Td
(remove)Tj
3.2201 0 Td
(floating)Tj
3.2768 0 Td
(cells)Tj
1.9163 0 Td
(and)Tj
1.7347 0 Td
(debris)Tj
2.6759 0 Td
(and)Tj
1.7292 0 Td
(1.3)Tj
1.3946 0 Td
(ml)Tj
1.2869 0 Td
(were)Tj
2.126 0 Td
(removed)Tj
3.7474 0 Td
(to)Tj
1.0261 0 Td
(a)Tj
0.652 0 Td
(sodium)Tj
-33.7604 -1.2982 Td
(fluoride)Tj
3.3788 0 Td
(vessel)Tj
2.4888 0 Td
(\(Sarstedt,)Tj
3.9742 0 Td
(Nu)Tj
0 Tc
0.8107 0 Td
()Tj
-0.004 Tc
0.4535 0 Td
(mbrecht,)Tj
3.8154 0 Td
(Germany\).)Tj
4.5524 0 Td
(To)Tj
1.338 0 Td
(eliminate)Tj
3.9515 0 Td
(influences)Tj
4.2746 0 Td
(of)Tj
1.0148 0 Td
(phenol)Tj
2.9933 0 Td
(red)Tj
1.5421 0 Td
(and)Tj
-34.5881 -1.304 Td
(fetal)Tj
1.9105 0 Td
(calf)Tj
1.6101 0 Td
(serum,)Tj
2.948 0 Td
(medium)Tj
3.6056 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
(as)Tj
1.0148 0 Td
(negative)Tj
3.5376 0 Td
(control)Tj
3.1181 0 Td
(and)Tj
1.7292 0 Td
(deducted)Tj
3.9004 0 Td
(from)Tj
2.2053 0 Td
(measurements.)Tj
6.2589 0 Td
(Fur-)Tj
-35.5972 -1.2982 Td
(ther)Tj
1.8425 0 Td
(the)Tj
1.4683 0 Td
(lactate)Tj
2.7779 0 Td
(content)Tj
3.2655 0 Td
(was)Tj
1.7008 0 Td
(normalized)Tj
4.7792 0 Td
(to)Tj
1.0204 0 Td
(intracellular)Tj
5.0627 0 Td
(protein)Tj
3.1521 0 Td
(concentration)Tj
5.8167 0 Td
(to)Tj
1.0261 0 Td
(prevent)Tj
3.2598 0 Td
(influ-)Tj
-35.172 -1.2983 Td
(ences)Tj
2.398 0 Td
(of)Tj
1.0148 0 Td
(cell)Tj
1.5591 0 Td
(numbers)Tj
3.7814 0 Td
(and)Tj
1.7291 0 Td
(volume.)Tj
3.4242 0 Td
(Protein)Tj
3.1918 0 Td
(concentration)Tj
5.8224 0 Td
(was)Tj
1.7007 0 Td
(assessed)Tj
3.4696 0 Td
(with)Tj
1.9956 0 Td
(Pierce)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 525.6 303.874 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(TM)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 539.0362 299.4519 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(BCA)Tj
-33.9021 -1.3039 Td
(Assay)Tj
2.5115 0 Td
(\(Thermofisher)Tj
6.0604 0 Td
(Scientific\))Tj
4.2406 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(in)Tj
1.0375 0 Td
(manufacturers)Tj
6.4403 0 Td
(instructions.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 260.2204 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Metabolic)Tj
4.4457 0 Td
(activity)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 243.2125 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Depending)Tj
4.6771 0 Td
(on)Tj
1.2756 0 Td
(each)Tj
2.0296 0 Td
(cell)Tj
1.559 0 Td
(line)Tj
1.7065 0 Td
(30006000)Tj
4.541 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(seeded)Tj
2.9083 0 Td
(in)Tj
1.0318 0 Td
(a)Tj
0.6577 0 Td
(96-well-plate)Tj
5.3914 0 Td
(\(Falcon,)Tj
3.4696 0 Td
(Corning,)Tj
-33.2898 -1.2982 Td
(Amsterdam,)Tj
5.1816 0 Td
(The)Tj
1.7859 0 Td
(Netherlands\))Tj
5.4538 0 Td
(with)Tj
2.0012 0 Td
(200)Tj
/F13 1 Tf
1.6441 0 Td
[()]TJ
/F5 1 Tf
(l)Tj
0.9751 0 Td
(medium)Tj
3.6057 0 Td
(199,)Tj
1.8708 0 Td
(10%)Tj
1.9276 0 Td
(fetal)Tj
1.9105 0 Td
(calf)Tj
1.6101 0 Td
(serum,)Tj
2.9537 0 Td
(2%)Tj
1.4513 0 Td
(penicillin-)Tj
-32.3714 -1.3039 Td
(streptomycin,)Tj
5.7372 0 Td
(10)Tj
1.1679 0 Td
(mM)Tj
1.9332 0 Td
(DCA)Tj
2.3017 0 Td
(and)Tj
1.7348 0 Td
(incubated)Tj
4.1726 0 Td
(at)Tj
0.9524 0 Td
(37C)Tj
2.2281 0 Td
(and)Tj
1.7291 0 Td
(5%)Tj
1.457 0 Td
(humidified)Tj
4.6601 0 Td
(CO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 494.8724 215.5464 cm
BT
/F5 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 498.444 217.1905 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(.)Tj
0.4422 0 Td
(Measurements)Tj
-30.2851 -1.2982 Td
(were)Tj
2.1203 0 Td
(performed)Tj
4.473 0 Td
(every)Tj
2.3471 0 Td
(24)Tj
1.1679 0 Td
(hours)Tj
2.5171 0 Td
(for)Tj
1.3833 0 Td
(four)Tj
1.9162 0 Td
(days.)Tj
2.2224 0 Td
(Therefore)Tj
4.1669 0 Td
(medium)Tj
3.6056 0 Td
(was)Tj
1.7008 0 Td
(replaced)Tj
3.5829 0 Td
(and)Tj
1.7348 0 Td
(20)Tj
/F13 1 Tf
1.1678 0 Td
[()]TJ
/F5 1 Tf
(l)Tj
0.9751 0 Td
(MTT)Tj
-35.0812 -1.2983 Td
(\(CellTiter96)Tj
/F12 1 Tf
(1)Tj
/F5 1 Tf
6.0604 0 Td
(Aqueous)Tj
3.7531 0 Td
(One)Tj
1.9389 0 Td
(Solution)Tj
3.5999 0 Td
(Assay,)Tj
2.7383 0 Td
(Promega,)Tj
4.0252 0 Td
(Mannheim,)Tj
4.9095 0 Td
(Germany\))Tj
4.3256 0 Td
(was)Tj
1.7007 0 Td
(added)Tj
2.6589 0 Td
(and)Tj
-35.7105 -1.2982 Td
(incubated)Tj
4.1782 0 Td
(for)Tj
1.3833 0 Td
(two)Tj
1.7121 0 Td
(hours)Tj
2.5115 0 Td
(at)Tj
0.9525 0 Td
(37C)Tj
2.228 0 Td
(in)Tj
1.0374 0 Td
(the)Tj
1.4684 0 Td
(dark.)Tj
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(ImageJ)Tj
3.033 0 Td
(and)Tj
-35.6312 -1.2983 Td
(normalized)Tj
4.7791 0 Td
(to)Tj
1.0205 0 Td
(cell)Tj
1.5647 0 Td
(counts.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 587.7921 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Mitochondrial)Tj
6.085 0 Td
(ATP-synthesis)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 570.7842 cm
BT
/F5 1 Tf
1 TL
-0.02 Tc
0 0 Td
(Mitochondrial)Tj
5.8279 0 Td
(respiration)Tj
4.4107 0 Td
(was)Tj
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(directly)Tj
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(by)Tj
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(measuring)Tj
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(cellular)Tj
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(ATP)Tj
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(synthesis.)Tj
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(cell)Tj
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(lines)Tj
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(were)Tj
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(seeded)Tj
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(\(5000)Tj
2.3811 0 Td
(cells/well\))Tj
4.0139 0 Td
(as)Tj
0.9807 0 Td
(triplicate)Tj
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1.0034 0 Td
(a)Tj
0.635 0 Td
(96-opaque-well)Tj
6.1681 0 Td
(plate)Tj
2.058 0 Td
(\(Greiner)Tj
3.5546 0 Td
(Bio-One\))Tj
3.8551 0 Td
(with)Tj
1.9332 0 Td
(100)Tj
/F13 1 Tf
1.5987 0 Td
[()]TJ
/F5 1 Tf
(l)Tj
-36.663 -1.2983 Td
(medium)Tj
3.5092 0 Td
(199,)Tj
1.8085 0 Td
(10%)Tj
1.8765 0 Td
(fetal)Tj
1.8312 0 Td
(calf)Tj
1.5477 0 Td
(serum,)Tj
2.8516 0 Td
(2%)Tj
1.423 0 Td
(streptomycin-penicillin)Tj
9.0312 0 Td
(,)Tj
0.4251 0 Td
(10)Tj
1.1396 0 Td
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1.8992 0 Td
(DCA)Tj
2.2563 0 Td
(and)Tj
1.6838 0 Td
(incubated)Tj
4.0365 0 Td
(at)Tj
-35.3194 -1.3039 Td
(37C)Tj
2.1599 0 Td
(humidified)Tj
4.5071 0 Td
(CO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 280.4598 530.1354 cm
BT
/F5 1 Tf
1 TL
-0.0266 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 283.8614 531.7795 cm
BT
/F5 1 Tf
1 TL
-0.02 Tc
0 0 Td
(.)Tj
0.4251 0 Td
(After)Tj
2.2054 0 Td
(48)Tj
1.1395 0 Td
(hours)Tj
2.4378 0 Td
(incubation)Tj
4.388 0 Td
(100)Tj
/F13 1 Tf
1.5987 0 Td
[()]TJ
/F5 1 Tf
(l)Tj
0.9411 0 Td
(ATP-solution)Tj
5.4935 0 Td
(\(CellTiter-Glo)Tj
/F12 1 Tf
(1)Tj
/F5 1 Tf
6.6897 0 Td
(2.0)Tj
1.3493 0 Td
(Assay,)Tj
-35.0529 -1.2982 Td
(Promega\))Tj
4.0082 0 Td
(was)Tj
1.6554 0 Td
(added.)Tj
2.7892 0 Td
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4.0762 0 Td
(10)Tj
1.1395 0 Td
(minutes)Tj
3.3505 0 Td
(incubation)Tj
4.388 0 Td
(at)Tj
0.9241 0 Td
(room)Tj
2.3584 0 Td
(temperature,)Tj
5.1647 0 Td
(luminescence)Tj
5.4652 0 Td
(mea-)Tj
-35.3194 -1.2983 Td
(surement)Tj
3.8665 0 Td
(was)Tj
1.6497 0 Td
(performed)Tj
4.3255 0 Td
(with)Tj
1.9333 0 Td
(Synergy2)Tj
3.7587 0 Td
(Plate)Tj
2.1033 0 Td
(Reader)Tj
2.9253 0 Td
(\(BioTek\).)Tj
3.8664 0 Td
(In)Tj
1.0772 0 Td
(order)Tj
2.3414 0 Td
(to)Tj
0.9921 0 Td
(normalize)Tj
4.1046 0 Td
(the)Tj
1.4229 0 Td
(data)Tj
-34.3669 -1.3039 Td
(with)Tj
1.9332 0 Td
(the)Tj
1.423 0 Td
(respective)Tj
4.0195 0 Td
(cell)Tj
1.4966 0 Td
(numbers,)Tj
3.8778 0 Td
(cells)Tj
1.8425 0 Td
(were)Tj
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(counted)Tj
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1.0035 0 Td
(a)Tj
0.6406 0 Td
(simultaneously)Tj
6.0151 0 Td
(performed)Tj
4.3256 0 Td
(experiment)Tj
4.6204 0 Td
(by)Tj
-36.6176 -1.2983 Td
(flow)Tj
1.8822 0 Td
(cytometry)Tj
4.1272 0 Td
(\(MACSQuant)Tj
5.6692 0 Td
(Analyzer)Tj
3.668 0 Td
(10,)Tj
1.3493 0 Td
(Miltenyi)Tj
3.4866 0 Td
(Biotek,)Tj
2.9083 0 Td
(Bergisch)Tj
3.5093 0 Td
(Gladbach,)Tj
4.1215 0 Td
(Germany\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 451.2188 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Immunofluorescenc)Tj
8.6315 0 Td
(e)Tj
0.7701 0 Td
(staining)Tj
3.6189 0 Td
(of)Tj
1.0441 0 Td
(Ki67)Tj
2.1922 0 Td
(and)Tj
1.8661 0 Td
(TUNEL)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 434.211 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cultivation,)Tj
4.9549 0 Td
(fixation)Tj
3.2938 0 Td
(and)Tj
1.7348 0 Td
(washing)Tj
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(were)Tj
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(performed)Tj
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(as)Tj
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(described)Tj
4.0535 0 Td
(above.)Tj
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(permeabilization)Tj
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(using)Tj
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(0.2%)Tj
2.1543 0 Td
(Triton)Tj
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(X-100)Tj
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(for)Tj
1.3833 0 Td
(20)Tj
1.1736 0 Td
(minutes,)Tj
3.6906 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(exposed)Tj
3.4469 0 Td
(overnight)Tj
4.0819 0 Td
(to)Tj
1.0261 0 Td
(a)Tj
0.652 0 Td
(canine)Tj
2.8686 0 Td
(specific)Tj
3.1862 0 Td
(rab-)Tj
-35.5689 -1.3039 Td
(bit-polyclonal)Tj
5.7713 0 Td
(Ki67)Tj
2.1146 0 Td
(antibody)Tj
3.7644 0 Td
(\(dilution)Tj
3.7473 0 Td
(of)Tj
1.0148 0 Td
(1:150;)Tj
2.5739 0 Td
(Life)Tj
1.7348 0 Td
(Technologies,)Tj
5.7599 0 Td
(Thermofisher)Tj
5.7203 0 Td
(Scientific\).)Tj
-32.2013 -1.2983 Td
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1.6157 0 Td
(labeling,)Tj
3.5773 0 Td
(a)Tj
0.6463 0 Td
(monoclonal)Tj
5.0003 0 Td
(anti-rabbit)Tj
4.4957 0 Td
(Alexa)Tj
2.4661 0 Td
(Fluor)Tj
/F12 1 Tf
(1)Tj
/F5 1 Tf
3.3902 0 Td
(555)Tj
1.6441 0 Td
(antibody)Tj
3.7531 0 Td
(\(Cell)Tj
2.1373 0 Td
(Signaling)Tj
3.9288 0 Td
(Technology,)Tj
-32.6549 -1.2983 Td
(Leiden,)Tj
3.1747 0 Td
(The)Tj
1.7802 0 Td
(Netherlands\))Tj
5.4595 0 Td
(was)Tj
1.7007 0 Td
(incubated)Tj
4.1726 0 Td
(for)Tj
1.389 0 Td
(1)Tj
0.6973 0 Td
(hour)Tj
2.1487 0 Td
(\(1:250\))Tj
3.033 0 Td
(and)Tj
1.7291 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
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(counterstained)Tj
6.1795 0 Td
(with)Tj
-35.5065 -1.3039 Td
(DAPI)Tj
2.5398 0 Td
(\(1:1000\))Tj
3.5036 0 Td
(for)Tj
1.3833 0 Td
(5)Tj
0.6973 0 Td
(minutes.)Tj
3.6907 0 Td
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5.4255 0 Td
(imaging)Tj
3.4865 0 Td
(protocol)Tj
3.6 0 Td
(was)Tj
1.7008 0 Td
(the)Tj
1.4683 0 Td
(same)Tj
2.2564 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(above.)Tj
-34.8205 -1.2982 Td
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2.313 0 Td
(cell)Tj
1.5591 0 Td
(fluorescence)Tj
5.1873 0 Td
(was)Tj
1.7008 0 Td
(established)Tj
4.5978 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(above.)Tj
2.7722 0 Td
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1.6215 0 Td
(TUNEL)Tj
3.4128 0 Td
(staining)Tj
3.4129 0 Td
(Apoptag)Tj
3.6397 0 Td
(Fluo-)Tj
-35.2854 -1.2983 Td
(rescein)Tj
3.0387 0 Td
(Direct)Tj
2.7382 0 Td
(Kit)Tj
1.457 0 Td
(\(Merck)Tj
3.1691 0 Td
(Millipore\))Tj
4.2973 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(as)Tj
1.0148 0 Td
(manufacturers)Tj
6.4403 0 Td
(instructions.)Tj
5.227 0 Td
(The)Tj
1.7858 0 Td
(exci-)Tj
-35.3364 -1.3039 Td
(tation)Tj
2.5738 0 Td
(occurred)Tj
3.7927 0 Td
(with)Tj
1.9956 0 Td
(an)Tj
1.2076 0 Td
(argonlaser)Tj
4.3823 0 Td
(at)Tj
0.9524 0 Td
(488)Tj
1.6441 0 Td
(nm)Tj
1.5874 0 Td
(and)Tj
1.7291 0 Td
(imaging)Tj
3.4866 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(above.)Tj
-34.5881 -1.2983 Td
(The)Tj
1.7801 0 Td
(amount)Tj
3.3562 0 Td
(of)Tj
1.0148 0 Td
(TUNEL)Tj
3.4129 0 Td
(positive)Tj
3.3165 0 Td
(cells)Tj
1.9162 0 Td
(was)Tj
1.7008 0 Td
(evaluated.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 288.6236 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Western)Tj
3.9118 0 Td
(blot)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 271.6157 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Protein)Tj
3.1917 0 Td
(expression)Tj
4.4674 0 Td
(analysis)Tj
3.3279 0 Td
(was)Tj
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(performed)Tj
4.473 0 Td
(with)Tj
1.9956 0 Td
(Immunoblotting)Tj
6.9222 0 Td
(\(Western)Tj
3.9514 0 Td
(Blot\))Tj
2.1884 0 Td
(as)Tj
1.0148 0 Td
(descri-)Tj
-33.2275 -1.3039 Td
(bed)Tj
1.678 0 Td
(in)Tj
1.0319 0 Td
(detail)Tj
2.4207 0 Td
(by)Tj
1.1792 0 Td
(Wagner)Tj
3.4299 0 Td
(et)Tj
0.9411 0 Td
(al.)Tj
1.1282 0 Td
([)Tj
0.83 0.64 0.02 0 k
(28)Tj
0 g
(].)Tj
2.075 0 Td
(The)Tj
1.7801 0 Td
(protein)Tj
3.1521 0 Td
(concentration)Tj
5.8223 0 Td
(was)Tj
1.7008 0 Td
(determined)Tj
4.8472 0 Td
(using)Tj
2.3925 0 Td
(Pierce)Tj
-33.579 -1.2982 Td
(BCA)Tj
2.1486 0 Td
(Assay)Tj
2.5115 0 Td
(\(Thermofisher)Tj
6.0604 0 Td
(Scientific\))Tj
4.2406 0 Td
(and)Tj
1.7291 0 Td
(for)Tj
1.389 0 Td
(electrophoresis)Tj
6.2475 0 Td
(60)Tj
/F13 1 Tf
1.1736 0 Td
[()]TJ
/F5 1 Tf
(g)Tj
1.1905 0 Td
(protein)Tj
3.1521 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
2.0296 0 Td
(sample)Tj
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(was)Tj
1.6951 0 Td
(used.)Tj
2.2903 0 Td
(The)Tj
1.7802 0 Td
(PVDF)Tj
2.7382 0 Td
(membrane)Tj
4.5638 0 Td
(was)Tj
1.7008 0 Td
(incubated)Tj
4.1782 0 Td
(overnight)Tj
4.0819 0 Td
(at)Tj
0.9524 0 Td
(4C)Tj
1.7518 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6576 0 Td
(canine)Tj
2.863 0 Td
(specific)Tj
3.1918 0 Td
(poly-)Tj
-34.4407 -1.2983 Td
(clonal)Tj
2.6248 0 Td
(primary)Tj
3.4526 0 Td
(antibody)Tj
3.7644 0 Td
(anti-rabbit)Tj
4.507 0 Td
(anti-PDK-1)Tj
4.9096 0 Td
(\(1:1000,)Tj
3.3902 0 Td
(ABIN2499649,)Tj
6.1172 0 Td
(antibodies-online,)Tj
-28.7658 -1.3039 Td
(Aachen,)Tj
3.4865 0 Td
(Germany\))Tj
4.3257 0 Td
(or)Tj
1.0998 0 Td
(a)Tj
0.652 0 Td
(canine)Tj
2.8686 0 Td
(specific)Tj
3.1861 0 Td
(polyclonal)Tj
4.354 0 Td
(anti-rabbit)Tj
4.5014 0 Td
(anti-PDK-3)Tj
4.9153 0 Td
(antibody)Tj
3.7644 0 Td
(\(1:500,)Tj
-33.1538 -1.2982 Td
(ABIN278250,)Tj
5.6409 0 Td
(antibodies-online\).)Tj
7.8179 0 Td
(Afterwards,)Tj
4.8869 0 Td
(the)Tj
1.4683 0 Td
(PVDF)Tj
2.7382 0 Td
(membrane)Tj
4.5695 0 Td
(was)Tj
1.6951 0 Td
(incubated)Tj
4.1782 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6576 0 Td
(sec-)Tj
-35.6482 -1.2983 Td
(ondary)Tj
3.0613 0 Td
(antibody)Tj
3.7644 0 Td
(anti-rabbit)Tj
4.5014 0 Td
(IgG,)Tj
1.9673 0 Td
(AP)Tj
1.4626 0 Td
(Conjugate)Tj
4.3483 0 Td
(for)Tj
1.3833 0 Td
(2)Tj
0.6974 0 Td
(hours)Tj
2.5114 0 Td
(\(Promega,)Tj
4.3654 0 Td
(Mannheim,)Tj
4.9095 0 Td
(Germany\).)Tj
-32.9723 -1.3039 Td
(A)Tj
0.9014 0 Td
(canine)Tj
2.8686 0 Td
(specific)Tj
3.1861 0 Td
(monoclonal)Tj
5.0116 0 Td
(anti-mouse)Tj
4.7566 0 Td
(anti-GAPDH)Tj
5.5785 0 Td
(antibody)Tj
3.7644 0 Td
(\(Abcam,)Tj
3.6567 0 Td
(Cambridge,)Tj
4.9209 0 Td
(United)Tj
-34.6448 -1.2983 Td
(Kingdom\))Tj
4.3653 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
(as)Tj
1.0148 0 Td
(loading)Tj
3.2201 0 Td
(control.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 126.0283 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Statistical)Tj
4.4504 0 Td
(analysis)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 109.0204 cm
BT
/F5 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Statistical)Tj
4.0195 0 Td
(analysis)Tj
3.3278 0 Td
(of)Tj
1.0148 0 Td
(data)Tj
1.9162 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(with)Tj
1.9955 0 Td
(SAS)Tj
1.8539 0 Td
(software)Tj
3.5886 0 Td
(7.1)Tj
1.3947 0 Td
(\(SAS)Tj
2.1996 0 Td
(Institute)Tj
3.583 0 Td
(Inc.,)Tj
1.9842 0 Td
(Cary,)Tj
2.3641 0 Td
(NC,)Tj
-35.4101 -1.3039 Td
(USA\).)Tj
2.6815 0 Td
(For)Tj
1.6271 0 Td
(comparison)Tj
4.9719 0 Td
(of)Tj
1.0148 0 Td
(two)Tj
1.7121 0 Td
(means,)Tj
3.0331 0 Td
(two-tailed)Tj
4.2576 0 Td
(t-test)Tj
2.245 0 Td
(was)Tj
1.7008 0 Td
(used.)Tj
2.2847 0 Td
(The)Tj
1.7858 0 Td
(confidence)Tj
4.5978 0 Td
(value)Tj
2.3074 0 Td
(was)Tj
1.7008 0 Td
(set)Tj
-35.9204 -1.2982 Td
(to)Tj
1.0261 0 Td
(5%)Tj
1.4513 0 Td
(\(p)Tj
/F14 1 Tf
0 Tc
(<)Tj
/F5 1 Tf
-0.004 Tc
(0.05\))Tj
3.8381 0 Td
(and)Tj
1.7348 0 Td
(was)Tj
1.6951 0 Td
(considered)Tj
4.6034 0 Td
(statistically)Tj
4.6091 0 Td
(significant.)Tj
ET
Q
q
1 j
1 J
0 w
576 737.1 m
36 737.1 l
36 737.6 l
576 737.6 l
f*
36 741.2598 107.1496 23.6976 re
W* n
q
107.0929 0 0 23.6409 36 741.3165 cm
q
/I0 Do
Q
Q
Q
q
0 0 612 792 re
W* n
1 j
1 J
0 w
7.9999 0 0 7.9999 391.5779 745.7952 cm
BT
/F0 1 Tf
1 TL
-0.005 Tc
0 0 Td
(Effect)Tj
2.7425 0 Td
(of)Tj
1.0417 0 Td
(dichloroac)Tj
4.4646 0 Td
(etate)Tj
2.4095 0 Td
(in)Tj
0.978 0 Td
(canine)Tj
3.111 0 Td
(mamma)Tj
3.5646 0 Td
(ry)Tj
1.0417 0 Td
(cell)Tj
1.6867 0 Td
(lines)Tj
ET
Q
q
1 j
1 J
0 w
36 48.0002 m
576 48.0002 l
576 47.5002 l
36 47.5002 l
f*
0.83 0.64 0.02 0 k
81.5811 34.9228 m
237.8835 34.9228 l
h
f*
0 g
7.9999 0 0 7.9999 36 36 cm
BT
/F0 1 Tf
1 TL
-0.005 Tc
0 0 Td
(PLOS)Tj
2.8559 0 Td
(ONE)Tj
2.3669 0 Td
(|)Tj
0.83 0.64 0.02 0 k
0.4748 0 Td
(https://doi.or)Tj
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