Supporting Information. Cytosolic Irradiation of Femtosecond Laser Induces Mitochondria-dependent Apoptosis-like
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1 Supporting Informtion Cytosolic Irrdition of Femtosecond Lser Induces Mitochondri-dependent Apoptosis-like Cell Deth vi Intrinsic Rective Oxygen Cscdes Jonghee Yoon 1,2, Seung-wook Ryu 1,3, Seunghee Lee 1 & Chulhee Choi 1,2,3* 1 Deprtment of Bio nd Brin Engineering, KAIST, Dejeon, Kore 2 KAIST Institute for Opticl Science nd Technology, KAIST, Dejeon, Kore 3 KAIST Institute for the BioCentury, KAIST, Dejeon, Kore
2 Supplementry Figure 1. Lser-induced mitochondril frgmenttion. () Miniml overlp etween punctuted C 2+ fluorescence nd mitochondril frgmenttion. Green fluorescence indictes intrcellulr C 2+ nd red fluorescence indictes mitochondri. The squre with white lines t the ottom right is higher mgnifiction view of the squre with white dshed lines. The white spot indictes the region of lser irrdition. Scle r, 2 μm. () Temporl dynmics of lser-induced mitochondril frg menttion. White rrows indicte the region of mitochondril frgmenttion. The white spot indictes the region of lser irrdition. Scle r, 2 μm. (c) Quntifiction of mitochondril potentil through vrition in MitoTrcker in oth reversile nd irreversile responses. Red fluorescence. The red rrow indictes l ser irrdition time. c Bseline Activtion 5 min MitoTrcker( F/F, %) Irreversile Reversile Time (s) Bseline 11 s 126 s 157 s
3 Supplementry Figure 2. Long-term oservtion of lser-induced specific poptosis-like cell deth y phse microscopy. (, ) Arrows indicte lser-irrdited cells. The yellow rrow indictes the reversile response, nd the red rrow indictes the irreversile response. The numers ove ech imge indicte the time fter l ser stimultion. h 5 h h 1 h 5 h
4 Supplementry Figure 3. Lser-induced cell deth in vrious cell types. The white rrows indicte lser-irrdited cells. Red fluorescence indictes mitochondri s tined with MitoTrcker Red nd green fluorescence indictes intrcellulr C 2+ ions stined with Fluo4-m. Scle r, 5 μm. HBdSMC HBSMC HutSMC Huh-7 Cell deth Control
5 Supplementry Figure 4. C 2+ -independent lser-induced cell deth. () Temporl lser-induced C 2+ dynmics with rynodine receptor inhiition using the nt gonist, rynodine. The white circle indictes the region of lser irrdition. Scle r, 2 μm. () Quntifiction of lser-induced C 2+ dynmics in the irrdited cell i n () nd rynodine-free cells. The red rrow indictes lser irrdition time. (n = 5) ***P.1 (unpired t-test). (c) Mximum C 2+ indictor signls of irrdited cellw which occure cell deth nd djcent cells which re live nd increse intrcellulr C 2+ level. (n = 6) Bseline Activtion 1 s 5 s Clcium (ΔF/F ; %) *** Control RyR inhiitor Time (s) c Mximum Clcium Vlue (%) Irrdited cells N.S Adjcent cells
6 Supplementry Figure 5. Propgtion of lser-induced ROS from the vicinity of the focl region to the nucleus. () Genertion of intrinsic ROS vi lser stimultion. Red fluorescence indictes endogenous ROS stined with DHE. Green fluorescence indictes mitochondri. The red spot indictes the region of lser irrd ition. Scle r, 2 μm. () Line scn imge of the white rrow in () demonstrting lser-induced ROS propgtion from the vicinity of the focl region to the nucleu s. Lser-induced intrinsic ROS strted to propgte to the lterl re. The white spot indictes the region of lser irrdition nd time. Bseline 4 min 2 μm 25 s
7 Supplementry Figure 6. Cellulr signling involved in lser-induced cell deth. () Antioxidnt effect ginst lser irrdition t μj. () Effects of 3AB (P ARPinhiitor) nd BocD-fmk (cspse inhiitor) on lser irrdition t μj. (n 35 cells). (c) Synergistic effects of 3AB nd BocD-fmk on lser-induced cell de th. The lser irrdition energy ws 6.39 μj. (n 1 cells). (d) Effect of cyclosporine A ginst lser-induced cell deth. HeL cells were treted with CysA (mptp in hiitor, 5 mg/l). (n 1 cells) *P.5, **P.1, ***P.1 (Chi-squre test). Cell deth(%) Control NAC Cell Deth(%) 8 Control 3AB 6 BocD-fmk Energy(µJ) Energy(µJ) c d Cell Deth(%) ** ** Control BocD-fmk 3AB BocD-fmk + 3AB *** Cell Deth(%) * Control CysA 6.36 Energy(µJ) 6.36 Energy(µJ)
8 Supplementry Figure 7. Geneticlly modified mitochondril morphology. () Rel-time PCR evluted levels of Drp1 nd Mfn1 mrna. Dt re the men ± S D of two experiments. *** p<.1. () Altered mitochondril morphology throughout sicontrol, sidrp1, nd simfn1 tretment. Scle r, 2 μm. (c, d) Effects of sirnas ginst lser-induced cell deth (n 2 cells). c Expression level of mrnas (fold) *** *** Ctl Drp1 Mfn1 Ctl Drp1 Mfn1 d Cell deth (%) Cell deth (%) sicontrol sidrp1 sicontrol simfn1 sidrp1 simfn1 sicontrol sidrp1 simfn-1
9 Supplementry Movie 1. Lser-induced mitochondril frgmenttion. Green fluorescence indictes intrcellulr C 2+ stined y Fluo4-AM. Red fluorescence i ndictes mitochondri stined y MitoTrcker Red. Scle r, 2 μm.
10 Supplementry Movie 2. Rpid plsm memrne retrction in the irrdited cells with the irreversile response. Chnges in plsm memrne stining with CellMsk. Green fluorescence indictes sl plsm memrne oundry nd red fluorescence indictes chnged plsm memrne oundry. Scle r, 2 μm.
11 Supplementry Movie 3. Femtosecond lser pulses produce intrinsic ROS in the irrdited cells with irreversile response. Green fluorescence indictes mitochondri stined with MitoTrcker Green. Red fluorescence indictes ROS genertion stined with DHE. Scle r, 5 μm.
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doi:1.138/nture11444 CMKIIN Gold prticles/ mitochondril re ( m ) 4 3 1 CMKIIN mtcmkiin mtcmkiin SERCA ATP synthse HA mitoplsts mtcmkiin CMKIIN cytosolic mtcmkiin CMKIIN 97 kdl 64 kdl 51 kdl 14 kdl 6 kdl
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doi:.38/nture8499 doi:.38/nture8499 5 6 5 4.5 Firing rte (Hz) -67-65 -66-6 -58 V m (mv) -7-67 -68-66 -64 c Thet power (mv ) -73-69 -7-7 -7.5.8 3....9.9.4.6.6. 9 8 9 8 9 8 9 8 9 8 Supplementry Figure Firing
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