DAF - FM , 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 ; 118 of 205 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( DAF - FM positive ) " unfilled " ( DAF - FM negative ) kugeln .

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

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 ; 62 of 363 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( Lysotracker positive ) " 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

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.


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

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 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 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

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 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 ) .

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 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

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 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

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 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - ) .

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 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 showed no indication of different dll4 expression at sites of kugeln ( arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf ; 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

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 Notch reporter Tg ( TP1glob : venusPest ) s940 , showed high Notch in mid - brain , but , again , no difference in expression at sites of kugeln ( n = 45 kugeln from 19 4dpf ; 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 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 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

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 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - )

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 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

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 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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).


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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 either GFP or GFP - fused to peptides derived from host was performed . Western blots of co - immunoprecipitation whole - lysate ( WCL ) are shown .

O O O O O O O O O O O O O O O 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 presence of increasing concentrations ( 0 . 13 - 500 μM ) of PPxPxY containing peptides . polarization was determined with constant concentrations of FITC - RBBP6 eVP30130 - 272 , at 0 . 50 μM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for indicated host peptides incubated with VP30 are shown . VP30 peptide acid residue positions sequences are indicated at top of each curve . error bars indicate mean ± S . D . ( N = 3 ) .

(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 indicated HA - tagged PPxPxY 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 anti - VP30 antibodies .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 . polarization was determined with constant concentrations of FITC - RBBP6 eVP30130 - 272 at 0 . 50 µM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 25 ng amounts along with plasmids of MG system . data represent mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . experiment was reproduced in two additional , independent experiments ( see Appendix Figure S2 ) . Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons . * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transfected with scrambled siRNA or siRNA targeting RBBP6 , hnRNP L , hnRNPUL1 or PEG10 . Twenty - four hours post - transfection , were transfected with MG plasmids including VP30 at 12 . 5 25ng doses . Data information : , data represent mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . experiment was reproduced in two additional , independent experiments Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons . * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transfected with scrambled siRNA ( SCR si ) or siRNAs targeting hnRNP L , hnRNPUL1 or PEG10 , 48 h post - transfection , were challenged with EBOV - EGFP at an MOI of 0 . 5 . 24 h post - infection , were fixed , stained for nuclei with Hoechst dye imaged . Scale bar is 200 µm . graph ( right ) depicts relative number of infected as determined by calculating of number of infected to nuclei shown relative to SCR siRNA . data shows a representative result of two independent experiments with mean ± S . D . calculated from three independently treated wells . Statistical significance was calculated relative to values obtained in SCR siRNA - treated using ANOVA with Dunnett ' s multiple comparisons . * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) of transcription replication in MG upon over - expression of indicated host factors . MG assays were performed with 50 500 ng of host expression plasmid . was isolated analyzed by qRT - PCR to measure of viral mRNA / cRNA vRNA . sample with vector control was set to 100 % 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 pVP30 in lysates from MG upon knockdown of RBBP6 , hnRNP L or hnRNPUL1 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 NP in EBOV infected HeLa upon hnRNP L , hnRNPUL1 or PEG10 knockdown .

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

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


liposome permeabilization assays were performed with C - terminally His - tagged BakΔTM BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( a - d ) Liposome permeabilization assays detecting release of a 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 absence ( blue ) presence ( green ) of 25 nM BclxLΔTM . ( c , d ) BakΔTM becomes autoactive at increasing concentrations . concentrations are color coded as indicated in ( d ) . ( c ) averaged kinetics for increasing BakΔTM concentrations . Bar diagrams in ( b ) ( d ) represent data from kinetic experiments averaged between 9000 - 10000 s . standard deviation was calculated from three measurements . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (


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

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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.


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

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

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(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 A549 were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , lysates were subjected to Western blot with anti - c - Myc antibody .

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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 from indicated lines was subjected to Northern blot to determine molecular of lncRNA - MIF . Actin was also included as a positive control .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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 together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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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 together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

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

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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 together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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 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 were measured normalized on . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

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

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

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

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

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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 . numbers of ( 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 expressing either lncRNA - MIF or control were transfected with miR - 586 mimics or NC mimics as indicated . Colonies were stained with crystal violet counted after 14 days incubation .

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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 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 counted after 14 days incubation .

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

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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 WT 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 indicated time points by immunoblotting .

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

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

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

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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 WT expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase transferring to SD ‐ N either with or without GABA for 24 h .

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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 WT expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase transferring to SD ‐ N either with or without GABA for 6 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.


APeroxisomes were induced in oleate medium 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 microscopy using a WT expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in presence of FM4 ‐ 64 , transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA rapamycin for 6 h . Bar , 5 μm .

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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 mitophagy was assessed as described for Fig .

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


A WT expressing OM45 ‐ GFP , along with uga2 ∆ over ‐ expressing GAD1 gene 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 ) .

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

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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 expressing GFP ‐ Atg8 along with uga2 ∆ over ‐ expressing GAD1 gene expressing GFP ‐ Atg8 were grown in SD medium transferred to SD ‐ N .

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

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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 ) .

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 .

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


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

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

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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 was monitored by degradation of Pot1 ‐ GFP analyzed for GFP cleavage by immunoblotting .

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


F Mitophagy was monitored by degradation of Om45 ‐ GFP 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 of Parkin ‐ expressing HeLa analyzed using a tandem fluorochrome ( mito ‐ RFP ‐ GFP ) mitophagy under ( A ) control conditions or ( B ) displaying mitophagy depicted by red mitochondrial structures localized to lysosomes . Bar , 10 μm .

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

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


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

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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 showing typical sizes of WT Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm .

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


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

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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 of mitochondrial numbers from electron microscopy of brain from WT ( n = 23 ) Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg 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 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 .

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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 were quantified from liver microsections using immunofluorescence microscopy automated image ( WT set to 1 ) .

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


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

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

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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 of S6 phosphorylation of brain lysates from WT ( n = 2 ) 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 stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) analyzed by flow cytometry . numbers indicate % of positive . One representative experiment is shown .

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

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(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.


( C ) Raji - Env were incubated with indicated antibodies either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 1h , surface of C3 were analyzed by flow cytometry . One representative experiment is shown .

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(C) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 1h, surface levels of C3 were analyzed by flow cytometry. One representative experiment is shown.


( D ) C3 deposition by 24 indicated antibodies . For each antibody , a normalized MFI is calculated by subtracting MFI of " no antibody " condition . ( nd . not done ) . n = 3 donors of serum . Data information : Error bars indicate . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney .

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(D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. 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.


( E ) Raji - Env were incubated with indicated antibodies either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , was determined by flow cytometry . numbers indicate of dead . One representative experiment is shown .

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(E) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, cell death was determined by flow cytometry. The numbers indicate the percentage of dead cells. One representative experiment is shown.


( F ) CDC induction by 24 indicated antibodies . % of CDC was calculated as relative of dead compared to " no antibody " condition . n = 3 donors of serum . Data information : Error bars indicate . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. 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.


( G ) Raji - Env were incubated for 24h with NHS indicated concentrations of WT or E430G mutant bNAbs ( 10 - 1074 , 3BNC117 or PGDM1400 ) or isotype control ( mGO53 ) . was measured by flow cytometry % of CDC was calculated as relative % of dead compared to " no antibody " condition . n = 3 donors of serum .

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 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

(G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( A ) Activated CD4 T were infected by HIV - 1 ( NLAD8 ) subjected to antibody binding . numbers indicate % of Gag + that are decorated by indicated antibodies . One representative experiment is shown .

O O O O O O O O O O O O O O O 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) Activated primary CD4 T cells were infected by HIV-1 (strain NLAD8) and subjected to antibody binding assay. The numbers indicate the % of Gag+ cells that are decorated by the indicated antibodies. One representative experiment is shown.


( B ) Antibody binding at surface of HIV - 1 - infected CD4 T . Results are expressed as median ( MFI ) of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 9 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Antibody binding at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as median fluorescence (MFI) of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=9 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( C ) HIV - 1 - infected ( NLAD8 ) CD4 T were cultured with indicated antibody NHS . After 24h , surface of C3 frequency of infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O 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) HIV-1-infected (strain NLAD8) CD4 T cells were cultured with the indicated antibody and NHS. After 24h, the surface levels of C3 and the frequency of infected cells (Gag+) were determined by flow cytometry. One representative experiment is shown.


( D ) C3 surface at surface of HIV - 1 - infected CD4 T . Results are expressed as MFI of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 7 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

(D) C3 surface levels at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( E ) HIV - 1 - infected CD4 T ( NLAD8 ) were cultured with indicated antibody normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , surface of C5b - 9 ( MAC ) on infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, the surface levels of C5b-9 (MAC) on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.


( F ) C5b - 9 ( MAC ) surface on HIV - 1 - infected ( NLAD8 ) . Results are expressed as MFI of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C5b-9 (MAC) surface levels on HIV-1-infected cells (strain NLAD8). Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( G ) HIV - 1 - infected CD4 T ( NLAD8 ) were cultured with indicated antibody NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , of infected ( Gag + ) in each condition was determined by flow cytometry . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. One representative experiment is shown.


( H ) Complement - mediated cytotoxicity of HIV - 1 - infected CD4 T ( NLAD8 ) . For each antibody , % of CDC was calculated at 24h as % of disappearance of Gag + in NHS compared to HIHS . Each dot represents a single donor of CD4 T . n = 7 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complement-mediated cytotoxicity of HIV-1-infected CD4 T cells (strain NLAD8). For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( A ) Raji - Env were subcloned to obtain lines expressing increasing amounts of Env . Env surface were determined by binding of 10 - 1074 measured by flow cytometry . One representative experiment is shown .

O O O O O O O O O O O O O O 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-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown.


( B ) Parental Raji different Raji - Env clones were cultured with 10 - 1074 or an isotype control ( mGO53 ) NHS for 24h . % of CDC was calculated as relative of dead compared to " no antibody " condition . n = 3 donors of serum .

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

(B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( C ) Non - infected ( NI ) HIV - 1 - infected CD4 T ( NLAD8 ) as well as three Raji - Env clones ( D1 , D2 D4 ) were incubated with 10 - 1074 antibody surface were analyzed by flow cytometry . Results are expressed as median ( MFI ) of in total ( Raji , n = 3 independent experiments ) or in infected ( Gag + ) ( CD4 T , n = 5 donors ) .

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

(C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors).


( D ) HIV - or HIV ∆ Vpu - infected CD4 T ( NLAD8 ) were subjected to antibody binding with indicated antibodies surface were determined by flow cytometry ( left panel ) . obtained with mGO53 was similar to background signal observed on unstained . Infected were cultured with indicated antibodies NHS for 24h C3 surface on infected ( Gag + ) were determined by flow cytometry ( right panel ) . One representative experiment is shown .

(D) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown.


( E ) HIV - or HIV ∆ Vpu - infected CD4 T ( NLAD8 ) were incubated with NHS either 10 - 1074 ( left panel ) or 3BNC117 ( right panel ) . Both WT or E430G - mutated antibodies were used . After 24h , C3 surface on infected were analyzed by flow cytometry . Results are expressed as MFI of on infected ( Gag + ) . MFI of C3 on Gag - was included as a control . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( A ) CD4 T were nucleofected with a control ( CRISPR Control ) or anti - CD59 ( CRISPR CD59 ) guide / Cas9 . CD59 surface were analyzed by flow cytometry 7 days after nucleofection . Isotype control ( grey ) CD59 stainings ( blue ) . One representative experiment is shown on left . Numbers indicate % of CD59 + ( middle ) or MFI of in CD59 - ( Right ) . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary CD4 T cells were nucleofected with a control (CRISPR Control) or anti-CD59 (CRISPR CD59) guide RNA/Cas9 complex. CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection. Isotype control (grey) and CD59 stainings (blue). One representative experiment is shown on the left. Numbers indicate the % of CD59+ cells (middle) or the MFI of staining in CD59- cells (Right). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( B ) NLAD8 ∆ Vpu - infected CD59 KO CD4 T were cultured with indicated antibodies NHS or HIHS for 24h . Numbers indicate of infected ( Gag + ) . One representative experiment is shown .

(B) NLAD8∆Vpu-infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) CD59 - KO ( CRISPR CD59 ) or control ( CRISPR Control ) CD4 T infected with HIV WT ( left ) or HIVΔVpu ( right ) ( NLAD8 ) were cultured with indicated antibody , NHS or HIHS for 24h . % of CDC was calculated as % of disappearance of Gag + after culture with NHS compared to HIHS . Each dot represents a single donor of CD4 T . n = at least 6 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD59-KO (CRISPR CD59) or control (CRISPR Control) CD4 T cells infected with HIV WT (left) or HIVΔVpu (right) (strain NLAD8) were cultured with the indicated antibody, and NHS or HIHS for 24h. The % of CDC was calculated as the % of disappearance of Gag+ cells after culture with NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n= at least 6 donors of CD4 T cells.


( D ) CD59 surface on non - infected ( NI ) or HIV - 1 - infected ( NLAD8 ) CD4 T were analyzed by flow cytometry . Results are expressed as MFI of with an isotype control or anti - CD59 antibody . n = 4 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD59 surface levels on non-infected (NI) or HIV-1-infected (strain NLAD8) primary CD4 T cells were analyzed by flow cytometry. Results are expressed as the MFI of staining with an isotype control or the anti-CD59 antibody. n=4 donors of CD4 T cells.


( B ) HIV - 1 - infected CD4 T were cultured with indicated antibodies ( E430G ) with NHS or HIHS . frequency of infected was assessed by flow cytometry in live at days 1 , 2 , 3 6 . Numbers indicate of infected ( Gag + ) . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIV-1-infected primary CD4 T cells were cultured with the indicated antibodies (E430G) and with NHS or HIHS. The frequency of infected cells was assessed by flow cytometry in live cells at days 1, 2, 3 and 6. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) Frequency of live HIV - 1 - infected ( NLAD8 WT or ΔVpu ) 6 days after incubation with indicated antibodies , with NHS or HIHS . Each donor of CD4 T is represented by a different symbol . n = 8 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Frequency of live HIV-1-infected cells (NLAD8 WT or ΔVpu strain) 6 days after incubation with the indicated antibodies, and with NHS or HIHS. Each donor of CD4 T cells is represented by a different symbol. n=8 donors of CD4 T cells.


( D ) Elimination of HIV - 1 - infected ( NLAD8 WT or ΔVpu ) over culture with indicated antibody ( E430G ) , NHS or HIHS . frequency of % infected ( Gag + ) is determinined by flow cytometry . mean relative of infected compared to HIHS condition is depicted . n = 6 - 8 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Elimination of HIV-1-infected cells (strain NLAD8 WT or ΔVpu) over culture with the indicated antibody (E430G), and NHS or HIHS. The frequency of % infected (Gag+) cells is determinined by flow cytometry. The mean relative percentage of infected cells compared to the HIHS condition is depicted. n=6-8 donors of CD4 T cells.


( A ) CD4 T infected with HIV - 1 ( Transmitted / Founder strains CH058 CH077 , vKB18 ) were incubated with indicated bNAbs surface were analyzed by flow cytometry . numbers indicate % of bNAb + among infected ( Gag + ) . percentages of infected were 27 % , 30 % 6 % for CH058 , CH077 vKB18 , respectively . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary CD4 T cells infected with HIV-1 (Transmitted/Founder strains CH058 and CH077, and the primary strain vKB18) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. The percentages of infected cells were 27%, 30% and 6% for CH058, CH077 and vKB18, respectively. One representative experiment is shown.


( B ) CH058 - infected CD4 T were cultured with indicated WT or E430G mutant antibody NHS . After 24h , C3 surface on infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O 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) CH058-infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS. After 24h, the C3 surface levels on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.