Time - lapse imaging of an embryo with transiently halted cardiac contraction ( using Tricaine ) . Despite absent blood flow kugeln still changed shape ( grey arrowhead ) , retained shape ( white arrowhead ) or protruded and retracted ( black arrowhead ; time post cessation of flow is indicated on micrographs ; 3dpf ; grey inverted LUT ) . Time - lapse of an embryo with transiently halted cardiac contraction as in ( C ) showed that kugel diameter still oscillates ( time post cessation of flow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).


Dextran microangiography filled perfused vessels with dextran ( arrowhead ) , while dextran was not observed to enter kugeln ( unfilled arrowhead ) .

O O O O O O O O O O O O O O O O O O O O O O O O

Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).


Inhibition of cardiac contraction by tnnt2a morpholino ( MO ) knockdown statistically significantly reduced kugel number per embryo ( * * * * p < 0 . 0001 ; control n = 20 embryos 5 . 10 ± 1 . 47 ( mean ± s . e . m . ) , tnnt2a MO = 18 embryos 0 . 06 ± 0 . 06 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugeln were studied in the double - transgenic Tg ( fli1a : LifeAct - mClover ) sh467 , Tg ( kdrl : HRAS - mCherry ) s916 . BLECs were mClover positive and mCherry - negative ( BLECs - black arrowheads ; kugeln - white arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).


No direct physical interaction between kugeln ( BLECs - black arrowheads ; kugeln - white arrowheads ) and BLECs was observed ( n = 21 4dpf embryos ; 3 experimental repeats ) . Depth - coded MIPs showed that BLECs and kugeln are present on different anatomical planes ( depths ; purple ventral ( v ) , white dorsal ( d ) ; BLECs - black arrowheads ; kugeln - white arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).


Ccbe1 morpholino ( MO ) injection led to a loss of lymphatics ( white arrowhead ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).


Kugel number was not statistically significantly altered by ccbe1 morpholino ( MO ) knockdown ( p = 0 . 3496 ; control MO n = 22 embryos 0 . 95 ± 0 . 28 ( mean ± s . e . m . ) , ccbe1 MO n = 23 embryos 0 . 57 ± 0 . 14 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown ( p = 0 . 8783 ; control MO n = 21 kugeln from 22 embryos 7 . 75 ± 1 . 29 ( mean ± s . e . m . ) , ccbe1 MO n = 13 kugeln from 23 embryos 8 . 93 ± 2 . 35 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Injection of IgG - conjugated Alexa 647 into the tectum showed no uptake ( blue ; unfilled arrowhead ) by kugeln ( white arrowhead ; 140 kugeln from 17 4dpf embryos ; 2 experimental repeats ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).


The possible interaction of macrophages with kugeln was studied in the transgenic Tg ( kdrl : HRAS - mCherry ) s916 , Tg ( fms : GAL4 . VP16 ) i186 , Tg ( UAS - E1b : nfsB . mCherry ) il149 , visualizing macrophages and the vasculature simultaneously . Coloured depth - coding indicates the different anatomical depths of kugeln and macrophages ( ventral purple , dorsal white ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).


Maximum intensity projections of z - stacks acquired every 10 minutes during a time - lapse showed no interaction of macrophages ( unfilled arrowhead ) with kugeln ( filled arrowhead ; n = 21 kugeln in 8 3dpf embryos ) . Macrophages are all white , indicating they are on a different z - plane to the vessels seen .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.


VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number ( * * * * p < 0 . 0001 ; DMSO control n = 30 embryos 8 . 53 ± 2 . 62 ( mean ± s . e . m . ) ; AV951 n = 31 embryos 21 . 10 ± 2 . 71 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Mann - Whitney U test ) .

B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly different after AV951 treatment ( p = 0 . 7890 ; DMSO control n = 243 kugeln from 30 embryos 9 . 94 ± 0 . 76 ( mean ± s . e . m . ) ; AV951 n = 652 kugeln from 31 embryos 9 . 73 ± 0 . 32 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Student ' s t - test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).


DAF - FM staining , a vital dye for nitric oxide ( NO ) , showed that 57 . 56 % of kugeln were positive for nitric oxide reactivity ( 2 . 5µM incubation from 96 - 102hpf ; n = 22 4dpf embryos ; 118 of 205 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( DAF - FM positive ) and " unfilled " ( DAF - FM negative ) kugeln .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.


Time - lapse acquisition with DAF - FM revealed that kugeln contained NO early in their biogenesis ( grey LUT ; inverted ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).


Application of LysoTracker , a vital dye that stains lysosomes or acidic compartments , showed that 17 . 08 % of kugeln contained acidic contents ( 8 . 33µM ; 96 - 101hpf ; n = 22 4dpf embryos ; 62 of 363 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( Lysotracker positive ) and " unfilled " ( Lysotracker negative ) kugeln .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.


The nitric oxide synthase ( NOS ) inhibitor L - NAME had no statistically significant effect on kugel number per embryo ( 0 . 5mM L - NAME 18h ; p = 0 . 4870 ; control n = 22 embryos 14 . 27 ± 3 . 35 ( mean ± s . e . m . ) , L - NAME n = 24 embryos 7 . 46 ± 1 . 61 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Diameter of kugeln was not affected by L - NAME ( p = 0 . 4161 ; control n = 315 kugeln from 22 embryos 8 . 77 ± 0 . 49 ( mean ± s . e . m . ) , L - NAME n = 179 kugeln from 24 8 . 66 ± 0 . 71 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT ( * * * * p < 0 . 0001 ; control n = 24 embryos 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 embryos 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly altered by DAPT treatment ( p = 0 . 0832 ; control n = 225 kugeln from 24 embryos 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 embryos 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln ( white arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf embryos ; 2 experimental repeats ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).


Studying Notch signalling in the Notch reporter line Tg ( TP1glob : venusPest ) s940 , showed high Notch levels in the mid - brain , but , again , no difference in expression at the sites of kugeln ( n = 45 kugeln from 19 4dpf embryos ; 2 experimental repeats ) .

O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).


Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV - 939 ( * * * p = 0 . 0003 ; control n = 22 embryos 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 embryos 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by XAV - 939 ( p = 0 . 4098 ; control n = 29 kugeln from 22 embryos 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 embryos 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - test )

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)


Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV ( p 0 . 0359 ; control n = 22 embryos 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 embryos 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


The diameter of kugel was not statistically significantly altered by GSK3 inhibition ( p 0 . 5555 ; control n = 23 kugeln from 22 embryos 7 . 26 ± 0 . 89 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 89 kugeln from 21 embryos 8 . 09 ± 0 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97


( C ) Co - immunoprecipitation between VP30 and the indicated PPxPxY containing host factors . Empty expression plasmid ( Vector ) or FLAG - tagged host protein expression plasmids were co - transfected with an HA - VP30 expression plasmid in HEK293T cells . An anti - FLAG immunoprecipitation ( IP : Anti - FLAG ) was performed . Western blots of IP and whole cell lysates ( WCL ) are shown . Anti - β - tubulin was used as a loading control for the WCLs .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Co-immunoprecipitation between VP30 and the indicated PPxPxY containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs.


( D ) Cells were transfected with empty vector or HA - VP30 expression plasmid and IPs were performed with anti - HA antibody ( IP : Anti - HA ) . Western blotting with anti - HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates ( WCL ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cells were transfected with empty vector or HA-VP30 expression plasmid and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates (WCL).


( B ) Co - immunoprecipitation between HA - VP30 and either GFP or GFP - fused to peptides derived from the host proteins was performed . Western blots of the co - immunoprecipitation and whole - cell lysate ( WCL ) are shown .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown.


( C ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 in the presence of increasing concentrations ( 0 . 13 - 500 μM ) of the PPxPxY containing peptides . Fluorescence polarization was determined with constant concentrations of FITC - RBBP6 and eVP30130 - 272 , at 0 . 50 μM and 3 . 8 μM respectively . Experiments were performed in two independent replicates . Error bars represent standard deviation .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.


( A ) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown . VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve . The error bars indicate mean ± S . D . ( N = 3 ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3).


( A ) Co - immunoprecipitation experiment demonstrating that NP interferes with VP30 - PPxPxY binding . VP30 was co - expressed with the indicated HA - tagged PPxPxY proteins and either empty vector ( - ) or increasing amounts of FLAG - tagged NP expression plasmid . IPs were performed with anti - HA antibodies followed by Western blots of IPs or WCL by using anti - HA , anti - FLAG and anti - VP30 antibodies .

O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.


( C ) Co - immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6 . VP30 was co - expressed with FLAG - tagged PPxPxY containing host proteins and either a 5 - fold or 10 - fold excess of RBBP6 plasmid . IP was performed using anti - FLAG beads followed by Western blotting using the indicated antibodies .

O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies.


( B ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 as it is outcompeted by increasing concentrations ( 0 . 13 - 500 μM ) of RBBP6 alanine mutants . Fluorescence polarization was determined with constant concentrations of FITC - RBBP6 and eVP30130 - 272 at 0 . 50 µM and 3 . 8 µM respectively . Experiments were performed in two independent replicates . Error bars represent the standard deviation .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 as it is outcompeted by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation.


( D ) Minigenome ( MG ) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant - RBBP6 peptides . GFP alone or GFP - RBBP6 peptide plasmids were transfected at 12 . 5 and 25 ng amounts along with the plasmids of the MG system . The data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments ( see Appendix Figure S2 ) . Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Minigenome (MG) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant-RBBP6 peptides. GFP alone or GFP-RBBP6 peptide plasmids were transfected at 12.5 and 25 ng amounts along with the plasmids of the MG system. The data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figure S2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005


( C ) MG activity upon knockdown of host genes . HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6 , hnRNP L , hnRNPUL1 or PEG10 . Twenty - four hours post - transfection , cells were transfected with MG assay plasmids including VP30 at 12 . 5 and 25ng doses . Data information : , the data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.


( A ) HeLa cells were transfected with scrambled siRNA ( SCR si ) or siRNAs targeting hnRNP L , hnRNPUL1 or PEG10 , 48 h post - transfection , cells were challenged with EBOV - EGFP at an MOI of 0 . 5 . 24 h post - infection , cells were fixed , stained for nuclei with Hoechst dye and imaged . Scale bar is 200 µm . The graph ( right ) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA . The data shows a representative result of two independent experiments with the mean ± S . D . calculated from three independently treated wells . Statistical significance was calculated relative to the values obtained in SCR siRNA - treated cells using ANOVA with Dunnett ' s multiple comparisons test . * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01


( B ) Levels of transcription and replication in the MG assay upon over - expression of the indicated host factors . MG assays were performed with 50 and 500 ng of host protein expression plasmid . RNA was isolated and analyzed by qRT - PCR to measure the levels of viral mRNA / cRNA and vRNA . The sample with vector control was set to 100 % and mean ± S . D . values from two independent experiments are shown ( each experiment was performed in duplicate , n = 2 ) . * * p < 0 . 005 , * p < 0 . 05 . Statistical significance was calculated relative to vector control using ANOVA with Tukey ' s multiple comparisons test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Levels of transcription and replication in the MG assay upon over-expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT-PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100% and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n=2). **p<0.005, *p<0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey's multiple comparisons test.


( C ) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6 , hnRNP L or hnRNPUL1 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O

(C) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L or hnRNPUL1.


( D ) Immunoblots to assess the levels of VP30 , pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6 . HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6 . 48 h post - transfection cells were infected with EBOV at MOI = 0 . 1 . At 24 h post - infection , cells were lysed in TRIzol . Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30 , pSer29 VP30 and NP .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O

(D) Immunoblots to assess the levels of VP30, pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with EBOV at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30 and NP.


( E ) Western blots detecting pVP30 , VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L , hnRNPUL1 or PEG10 knockdown .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.


All liposome permeabilization assays were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( a - d ) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes . ( a ) Pore formation of 50 nM BakΔTM ( red ) is activated by 20 nM cBid ( blue ) . 20 nM cBid alone ( orange ) shows no liposome permeabilization . Addition of 25 nM BclxLΔTM ( green ) inhibits pore - formation . ( b ) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence ( blue ) and presence ( green ) of 25 nM BclxLΔTM . ( c , d ) BakΔTM becomes autoactive at increasing concentrations . The concentrations are color coded as indicated in ( d ) . ( c ) The averaged kinetics for increasing BakΔTM concentrations . Bar diagrams in ( b ) and ( d ) represent data from the kinetic experiments averaged between 9000 - 10000 s . The standard deviation was calculated from three measurements . (

O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

All liposome permeabilization assays were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. (


All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( e ) Far - UV - CD spectra of BakΔTM + / - liposomes and the activators cBid or Puma - BH3 peptide as indicated .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated.


All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( f ) CD - detected thermal melting experiments of BakΔTM ( black ) with liposomes ( red ) and Puma - BH3 ( blue ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O

All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (f) CD-detected thermal melting experiments of BakΔTM (black) with liposomes (red) and Puma-BH3 (blue).


( c , d ) Liposome permeabilization assay comparing the variants in ( a , green ) and ( b , blue ) to wild - type BakΔTM ( black ) . ( c ) Comparison of 50 nM of wild - type BakΔTM ( black ) to the two variants after activation by 25 nM cBid . ( d ) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM . The data derived from the liposome permeabilization kinetics shown in panel ( c ) is averaged for the time interval of 9000 - 10000 s and the standard deviations were derived from three technical replicates . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c, d) Liposome permeabilization assay comparing the variants in (a, green) and (b, blue) to wild-type BakΔTM (black). (c) Comparison of 50 nM of wild-type BakΔTM (black) to the two variants after activation by 25 nM cBid. (d) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM. The data derived from the liposome permeabilization kinetics shown in panel (c) is averaged for the time interval of 9000-10000 s and the standard deviations were derived from three technical replicates. (


A . HeLa , H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , cell lysates were subjected to Western blot analysis with anti - c - Myc antibody .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody.


C . Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA - MIF . Actin was also included as a positive control .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control.


D . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with anti - c - Myc and anti - GAPDH antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies.


E . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


F . HeLa cells expressing either control RNA or lncRNA - MIF were treated with or without MG132 ( 20 μM ) for 6 h . Cell lysates were analyzed by Western blot with the indicated antibodies .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.


G . HeLa cells expressing either control shRNA or lncRNA - MIF shRNA were treated with MG132 for 6 h . Cell lysates were analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.


H . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were treated with cycloheximide ( CHX , 50 μg / ml ) for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the c - Myc protein half - life . Protein band intensity were analyzed by ImageJ . I . Band intensity of c - Myc in Fig 1H was analyzed by ImageJ .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.


J . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 as indicated . Forty - eight hours after infection , cells were treated with CHX for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the half - life of c - Myc protein . K . Band intensity of c - Myc in Fig 1J was analyzed by ImageJ .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.


A . P493 - 6 cells ( human B cell lymphoma cell line ) carrying a c - Myc tet - off system were treated with Doxycycline ( 1 μg / ml ) for the indicated periods of time . Total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.


B . HeLa , HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c - Myc shRNA . Forty - eight hours after infection , total RNA and cell lysates were analyzed by real - time RT - PCR and Western blotting , respectively . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa, HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c-Myc shRNA. Forty-eight hours after infection, total RNA and cell lysates were analyzed by real-time RT-PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


C . HeLa , HCT116 and MCF10A cells were transfected with control vector or Flag - c - Myc . Twenty - four hours after transfection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa, HCT116 and MCF10A cells were transfected with control vector or Flag-c-Myc. Twenty-four hours after transfection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting.


E . HeLa cells were co - transfected with either Flag - c - Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells were co-transfected with either Flag-c-Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


F . HeLa cells expressing control shRNA or c - Myc shRNA were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. HeLa cells expressing control shRNA or c-Myc shRNA were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


G . P493 - 6 cells were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after trasfection , cells were treated with doxycycline for 0 hour and 12 hours respectively . Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


H . Lysates from HeLa cells were subjected to ChIP assay . ChIP products were amplified by qPCR reaction with the indicated pairs of primers . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Lysates from HeLa cells were subjected to ChIP assay. ChIP products were amplified by qPCR reaction with the indicated pairs of primers. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


A . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF or biotin - labeled antisense RNA for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF or biotin-labeled antisense RNA for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies.


B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.


C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


E . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


F . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF and antisense RNA for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 and miR - 34a levels . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF and antisense RNA for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 and miR-34a levels. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


H . miR - 586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. miR-586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


I . miR - 586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

I. miR-586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


J . HeLa cells were subjected to fractionation into cytoplasmic ( C ) and nuclear ( N ) extracts . Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . Actin and PARP were used as markers for cytoplasmic and nuclear fractions , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. HeLa cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). Actin and PARP were used as markers for cytoplasmic and nuclear fractions, respectively.


K . HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes of lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes of lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


B . miR - 586 mimics or negative control mimics ( NC mimics ) were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


C . miR - 586 inhibitor or negative control inhibitor ( NC inhibitor ) was transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

C. miR-586 inhibitor or negative control inhibitor (NC inhibitor)was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


D . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.


E . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , total RNA was extracted and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


F . The copy numbers of miR - 586 and lncRNA - MIF transcript per cell in the indicated cells were quantified using a quantitative real - time RT - PCR assay . Data shown are mean ± SD ( n = 3 ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3).


G . HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . HeLa cells were infected with lentiviruses expressing lncRNA - MIF or control RNA . Forty - eight hours after infection , total RNA was analyzed by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


H . HeLa cells expressing lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.


I . HeLa cells expressing lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting .

I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.


K . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , lncRNA - MIF harboring nonsense mutation or miR - 586 binding defective lncRNA - MIF . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR-586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.


A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By Forty - eight hours infection , cells were treated with MG132 or DMSO ( vehicle ) for 6 h . Cell lysates were then analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies.


B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . Forty - eight hours after infection , total RNA was subjected to analyzing the levels of lncRNA - MIF and lncRNA - MIF - AS by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By 48 h infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By 48 h infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . By 48 h infection , cells were treated with MG132 or DMSO for 6 h . Cell lysates were then analyzed by Western blot analysis with the indicated antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.


E . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , acidification of the culture medium was evaluated by visually inspecting the color of the medium . Yellow medium indicates the presence of more lactate .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.


B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.


C . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


D . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


E . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


F . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


B . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


E . The percentage numbers of cells ( Fig 7C ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


F . The percentage numbers of cells ( Fig 7D ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


A . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics as indicated . Colonies were stained with crystal violet and counted after 14 days incubation .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


B . HeLa cells expressing either lncRNA - MIF shRNA or control shRNA were transfected with miR - 586 inhibitors or NC inhibitors as indicated . Colonies were stained with crystal violet and counted after 14 days incubation .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

B. HeLa cells expressing either lncRNA-MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


C . 2 × 106 HeLa cells expressing either lncRNA - MIF , lncRNA - MIF control RNA , lncRNA - MIF - shRNA or lncRNA - MIF control shRNA were individually injected subcutaneously into flanks of nude mice ( n = 7 for each group ) . Representative photographs of xenografttumors in situ were taken 3 weeks after injection . D . Three weeks post graft , mice were euthanized , tumors were excised and weighed , and the mean tumor weight was plotted on the graph . Data shown are mean ± SD ( n = 7 ; * * P < 0 . 01 , two - tailed t - test ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 2×106 HeLa cells expressing either lncRNA-MIF, lncRNA-MIF control RNA, lncRNA-MIF-shRNA or lncRNA-MIF control shRNA were individually injected subcutaneously into flanks of nude mice (n = 7 for each group). Representative photographs of xenografttumors in situ were taken 3 weeks after injection.D. Three weeks post graft, mice were euthanized, tumors were excised and weighed, and the mean tumor weight was plotted on the graph. Data shown are mean ± SD (n = 7; **P < 0.01, two-tailed t-test).


A Peroxisomes were induced by growing the WT strain expressing Pot1 ‐ GFP in oleate medium to mid ‐ log ‐ phase , then transferred to SD ‐ N starvation medium with or without GABA to trigger pexophagy for 6 h . GFP cleavage was analyzed at the indicated time points by immunoblotting .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


B Mitochondria were induced by growing the WT strain expressing OM45 ‐ GFP in YPL medium to mid ‐ log ‐ phase and subsequently transferring cells to either SD ‐ N with or without GABA to trigger mitophagy for 12 h . GFP cleavage was analyzed at the indicated time points by immunoblotting .

B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45 ‐ GFP grown in YPL medium for 12 h to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA for 24 h . Bar , 5 μm .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.


D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA , grown to mid ‐ log ‐ phase , after which samples were analyzed for Ape1 maturation .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O

D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.


E Ribophagy was monitored by growing the WT strain expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 24 h .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.


F Autophagy was monitored by growing the WT strain expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 6 h .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.


APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A .

APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A.


B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA and rapamycin for 6 h . Bar , 5 μm .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.


C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .


A WT cells expressing OM45 ‐ GFP , along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing OM45 ‐ GFP were grown in YPL medium to mid ‐ log ‐ phase . To monitor mitophagy , strains were transferred to SD ‐ N starvation medium ( with or without rapamycin ) .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).


B WT strain along with the uga2 ∆ strain over ‐ expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin . Samples were taken at the indicated time points , and Pot1 degradation was analyzed by immunoblotting ( 45 kD ) .

O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).


C To monitor autophagy , WT cells expressing GFP ‐ Atg8 along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing GFP ‐ Atg8 were grown in SD medium and transferred to SD ‐ N .

O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O

C To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.


A , B WT cells were cultured under pexophagy ( A ) or mitophagy ( B ) conditions with or without GABA and rapamycin . S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.


C Samples were analyzed for Pot1 degradation by immunoblotting ( 45 kD ) .

O O O O O B-MEASURED_VAR O O O O O O O O

C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD).


D GFP production during mitophagy was analyzed by immunoblotting .

D GFP production during mitophagy was analyzed by immunoblotting.


A S6 phosphorylation after 6 h in SD ‐ N was analyzed by immunoblotting with a loading control .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control.


B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting .

B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.


A , B WT , WT with GABA , WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under ( A ) pexophagy and ( B ) mitophagy conditions . After 24 h incubation , cells were stained with DHR − 123 and propidium iodide for 1 h . Living cells were analyzed for DHR − 123 fluorescence by flow cytometry . Data represent mean + s . d . ( n = 3 ) . * P < 0 . 005 , * * P < 0 .

O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR−123 and propidium iodide for 1 h. Living cells were analyzed for DHR−123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01


C , D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under ( C ) pexophagy or ( D ) mitophagy conditions . Significant differences between the treatments and strains were determined using an unpaired two ‐ tailed t ‐ test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C,D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two‐tailed t‐test. **P < 0.01.


E Pexophagy assay was monitored by the degradation of Pot1 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

E Pexophagy assay was monitored by the degradation of Pot1‐GFP and analyzed for GFP cleavage by immunoblotting.


F Mitophagy assay was monitored by the degradation of Om45 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

F Mitophagy assay was monitored by the degradation of Om45‐GFP and analyzed for GFP cleavage by immunoblotting.


A , B Example images of Parkin ‐ expressing HeLa cells analyzed using a tandem fluorochrome protein ( mito ‐ RFP ‐ GFP ) mitophagy assay under ( A ) control conditions or ( B ) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes . Bar , 10 μm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm.


C Percentage of cells displaying mitophagy + s . d . , * * P < 0 . 01 using an unpaired two ‐ tailed t ‐ test , n > 80 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.


A Electron microscopy images of mitochondria from WT ( n = 44 ) and SSADH ‐ deficient mice ( Aldh5a1 − / − ) ( n = 80 ) were calculated for area size .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

A Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size.


B Electron microscopy images showing typical sizes of WT and Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm.


C Quantification of mitochondrial numbers from electron microscopy images of liver from WT ( n = 31 ) and Aldh5a1 − / − mice treated with vehicle ( n = 39 ) or rapamycin ( n = 34 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


D Quantification of mitochondrial numbers from electron microscopy images of brain from WT ( n = 23 ) and Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


E Aldh5a1 − / − mice were treated with vehicle or rapamycin ( 10 mg / kg body weight per day ) via intraperitoneal injections for 10 successive days starting at day 10 of life . WT mice served as non ‐ disease controls ( set to 1 ) . After sacrifice , liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay .

O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.


F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis ( WT set to 1 ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).


G Immunofluorescence images showing typical nuclear staining ( DAPI , blue ) and SOD2 staining ( red ) from WT , Aldh5a1 − / − mice treated with vehicle and Aldh5a1 − / − mice treated with rapamycin . Bar , 10 μm .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O

G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.


A Quantification of S6 phosphorylation of liver lysates from WT ( n = 5 ) and Aldh5a1 − / − mice treated with vehicle ( n = 4 ) or rapamycin ( n = 5 ) after normalization ( WT set to 1 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

A Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).


B S6 phosphorylation of liver lysates analyzed by immunoblotting .

B S6 phosphorylation of liver lysates analyzed by immunoblotting.


C Quantification of S6 phosphorylation of brain lysates from WT ( n = 2 ) and Aldh5a1 − / − mice treated with vehicle ( n = 3 ) or rapamycin ( n = 3 ) after normalization ( WT set to 1 ) .

C Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1−/− mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).


D S6 phosphorylation of brain lysates analyzed by immunoblotting .

D S6 phosphorylation of brain lysates analyzed by immunoblotting.


( A ) Raji cells stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) and analyzed by flow cytometry . The numbers indicate the % of positive cells . One representative experiment is shown .

O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.


( B ) Binding of the 24 indicated antibodies to the surface of Raji - Env cells . Results are expressed as the median fluorescence intensity ( MFI ) of staining . n = 6 independent experiments . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.