Figure 4 ( A ) Co - immunoprecipitation experiment demonstrating that NP interferes with VP30 - PPxPxY binding . VP30 was co - expressed with the indicated HA - tagged PPxPxY proteins and either empty vector ( - ) or increasing amounts of FLAG - tagged NP expression plasmid . IPs were performed with anti - HA antibodies followed by Western blots of IPs or WCL by using anti - HA , anti - FLAG and anti - VP30 antibodies . ( C ) Co - immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6 . VP30 was co - expressed with FLAG - tagged PPxPxY containing host proteins and either a 5 - fold or 10 - fold excess of RBBP6 plasmid . IP was performed using anti - FLAG beads followed by Western blotting using the indicated antibodies .

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Figure 4(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.(C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies.


Figure 5 ( B ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 as it is outcompeted by increasing concentrations ( 0 . 13 - 500 μM ) of RBBP6 alanine mutants . Fluorescence polarization was determined with constant concentrations of FITC - RBBP6 and eVP30130 - 272 at 0 . 50 µM and 3 . 8 µM respectively . Experiments were performed in two independent replicates . Error bars represent the standard deviation . ( D ) Minigenome ( MG ) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant - RBBP6 peptides . GFP alone or GFP - RBBP6 peptide plasmids were transfected at 12 . 5 and 25 ng amounts along with the plasmids of the MG system . The data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments ( see Appendix Figure S2 ) . Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 as it is outcompeted by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation. (D) Minigenome (MG) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant-RBBP6 peptides. GFP alone or GFP-RBBP6 peptide plasmids were transfected at 12.5 and 25 ng amounts along with the plasmids of the MG system. The data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figure S2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005


Figure 6 ( C ) MG activity upon knockdown of host genes . HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6 , hnRNP L , hnRNPUL1 or PEG10 . Twenty - four hours post - transfection , cells were transfected with MG assay plasmids including VP30 at 12 . 5 and 25ng doses . Data information : , the data represent the mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . The experiment was reproduced in two additional , independent experiments Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons test . * * * * p < 0 . 00005 ; * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7 ( A ) HeLa cells were transfected with scrambled siRNA ( SCR si ) or siRNAs targeting hnRNP L , hnRNPUL1 or PEG10 , 48 h post - transfection , cells were challenged with EBOV - EGFP at an MOI of 0 . 5 . 24 h post - infection , cells were fixed , stained for nuclei with Hoechst dye and imaged . Scale bar is 200 µm . The graph ( right ) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA . The data shows a representative result of two independent experiments with the mean ± S . D . calculated from three independently treated wells . Statistical significance was calculated relative to the values obtained in SCR siRNA - treated cells using ANOVA with Dunnett ' s multiple comparisons test . * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 ( B ) Levels of transcription and replication in the MG assay upon over - expression of the indicated host factors . MG assays were performed with 50 and 500 ng of host protein expression plasmid . RNA was isolated and analyzed by qRT - PCR to measure the levels of viral mRNA / cRNA and vRNA . The sample with vector control was set to 100 % and mean ± S . D . values from two independent experiments are shown ( each experiment was performed in duplicate , n = 2 ) . * * p < 0 . 005 , * p < 0 . 05 . Statistical significance was calculated relative to vector control using ANOVA with Tukey ' s multiple comparisons test . ( C ) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6 , hnRNP L or hnRNPUL1 . ( D ) Immunoblots to assess the levels of VP30 , pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6 . HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6 . 48 h post - transfection cells were infected with EBOV at MOI = 0 . 1 . At 24 h post - infection , cells were lysed in TRIzol . Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30 , pSer29 VP30 and NP . ( E ) Western blots detecting pVP30 , VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L , hnRNPUL1 or PEG10 knockdown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01(B) Levels of transcription and replication in the MG assay upon over-expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT-PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100% and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n=2). **p<0.005, *p<0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey's multiple comparisons test.(C) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L or hnRNPUL1.(D) Immunoblots to assess the levels of VP30, pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with EBOV at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30 and NP.(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.


Figure 1All liposome permeabilization assays were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( a - d ) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes . ( a ) Pore formation of 50 nM BakΔTM ( red ) is activated by 20 nM cBid ( blue ) . 20 nM cBid alone ( orange ) shows no liposome permeabilization . Addition of 25 nM BclxLΔTM ( green ) inhibits pore - formation . ( b ) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence ( blue ) and presence ( green ) of 25 nM BclxLΔTM . ( c , d ) BakΔTM becomes autoactive at increasing concentrations . The concentrations are color coded as indicated in ( d ) . ( c ) The averaged kinetics for increasing BakΔTM concentrations . Bar diagrams in ( b ) and ( d ) represent data from the kinetic experiments averaged between 9000 - 10000 s . The standard deviation was calculated from three measurements . ( All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( e ) Far - UV - CD spectra of BakΔTM + / - liposomes and the activators cBid or Puma - BH3 peptide as indicated . All CD measurements were performed with C - terminally His - tagged BakΔTM and BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( f ) CD - detected thermal melting experiments of BakΔTM ( black ) with liposomes ( red ) and Puma - BH3 ( blue ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1All liposome permeabilization assays were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. ( All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated.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).


Figure 6 ( c , d ) Liposome permeabilization assay comparing the variants in ( a , green ) and ( b , blue ) to wild - type BakΔTM ( black ) . ( c ) Comparison of 50 nM of wild - type BakΔTM ( black ) to the two variants after activation by 25 nM cBid . ( d ) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM . The data derived from the liposome permeabilization kinetics shown in panel ( c ) is averaged for the time interval of 9000 - 10000 s and the standard deviations were derived from three technical replicates . (

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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. (


Figure 1A . HeLa , H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , cell lysates were subjected to Western blot analysis with anti - c - Myc antibody . C . Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA - MIF . Actin was also included as a positive control . D . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with anti - c - Myc and anti - GAPDH antibodies . E . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . F . HeLa cells expressing either control RNA or lncRNA - MIF were treated with or without MG132 ( 20 μM ) for 6 h . Cell lysates were analyzed by Western blot with the indicated antibodies . G . HeLa cells expressing either control shRNA or lncRNA - MIF shRNA were treated with MG132 for 6 h . Cell lysates were analyzed by Western blotting with the indicated antibodies . H . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were treated with cycloheximide ( CHX , 50 μg / ml ) for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the c - Myc protein half - life . Protein band intensity were analyzed by ImageJ . I . Band intensity of c - Myc in Fig 1H was analyzed by ImageJ . J . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 as indicated . Forty - eight hours after infection , cells were treated with CHX for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the half - life of c - Myc protein . K . Band intensity of c - Myc in Fig 1J was analyzed by ImageJ .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody.C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control.D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies.E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.


Figure 2A . P493 - 6 cells ( human B cell lymphoma cell line ) carrying a c - Myc tet - off system were treated with Doxycycline ( 1 μg / ml ) for the indicated periods of time . Total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting with the indicated antibodies . B . HeLa , HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c - Myc shRNA . Forty - eight hours after infection , total RNA and cell lysates were analyzed by real - time RT - PCR and Western blotting , respectively . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . C . HeLa , HCT116 and MCF10A cells were transfected with control vector or Flag - c - Myc . Twenty - four hours after transfection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting . E . HeLa cells were co - transfected with either Flag - c - Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . F . HeLa cells expressing control shRNA or c - Myc shRNA were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . G . P493 - 6 cells were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after trasfection , cells were treated with doxycycline for 0 hour and 12 hours respectively . Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . H . Lysates from HeLa cells were subjected to ChIP assay . ChIP products were amplified by qPCR reaction with the indicated pairs of primers . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.B. HeLa, HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c-Myc shRNA. Forty-eight hours after infection, total RNA and cell lysates were analyzed by real-time RT-PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).C. HeLa, HCT116 and MCF10A cells were transfected with control vector or Flag-c-Myc. Twenty-four hours after transfection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting.E. HeLa cells were co-transfected with either Flag-c-Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).F. HeLa cells expressing control shRNA or c-Myc shRNA were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).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).


Figure 3A . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF or biotin - labeled antisense RNA for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . E . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . F . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF and antisense RNA for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 and miR - 34a levels . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . H . miR - 586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . I . miR - 586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . J . HeLa cells were subjected to fractionation into cytoplasmic ( C ) and nuclear ( N ) extracts . Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . Actin and PARP were used as markers for cytoplasmic and nuclear fractions , respectively . K . HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes of lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF or biotin-labeled antisense RNA for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).E. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).F. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF and antisense RNA for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 and miR-34a levels. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).H. miR-586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).I. miR-586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).J. HeLa cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). Actin and PARP were used as markers for cytoplasmic and nuclear fractions, respectively.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).


Figure 4B . miR - 586 mimics or negative control mimics ( NC mimics ) were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . C . miR - 586 inhibitor or negative control inhibitor ( NC inhibitor ) was transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . D . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting . E . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , total RNA was extracted and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . F . The copy numbers of miR - 586 and lncRNA - MIF transcript per cell in the indicated cells were quantified using a quantitative real - time RT - PCR assay . Data shown are mean ± SD ( n = 3 ) . G . HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . HeLa cells were infected with lentiviruses expressing lncRNA - MIF or control RNA . Forty - eight hours after infection , total RNA was analyzed by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . H . HeLa cells expressing lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting . I . HeLa cells expressing lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting . K . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , lncRNA - MIF harboring nonsense mutation or miR - 586 binding defective lncRNA - MIF . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).C. miR-586 inhibitor or negative control inhibitor (NC inhibitor)was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3).G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.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.


Figure 5A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By Forty - eight hours infection , cells were treated with MG132 or DMSO ( vehicle ) for 6 h . Cell lysates were then analyzed by Western blotting with the indicated antibodies . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . Forty - eight hours after infection , total RNA was subjected to analyzing the levels of lncRNA - MIF and lncRNA - MIF - AS by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By 48 h infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . By 48 h infection , cells were treated with MG132 or DMSO for 6 h . Cell lysates were then analyzed by Western blot analysis with the indicated antibodies . E . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By 48 h infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.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).


Figure 6A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , acidification of the culture medium was evaluated by visually inspecting the color of the medium . Yellow medium indicates the presence of more lactate . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies . C . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . D . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . E . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . F . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


Figure 7A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) . B . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle . D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle . E . The percentage numbers of cells ( Fig 7C ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) . F . The percentage numbers of cells ( Fig 7D ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

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Figure 7A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


Figure 8A . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics as indicated . Colonies were stained with crystal violet and counted after 14 days incubation . B . HeLa cells expressing either lncRNA - MIF shRNA or control shRNA were transfected with miR - 586 inhibitors or NC inhibitors as indicated . Colonies were stained with crystal violet and counted after 14 days incubation . 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 ) .

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Figure 8A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.B. HeLa cells expressing either lncRNA-MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.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).


Figure 1A Peroxisomes were induced by growing the WT strain expressing Pot1 ‐ GFP in oleate medium to mid ‐ log ‐ phase , then transferred to SD ‐ N starvation medium with or without GABA to trigger pexophagy for 6 h . GFP cleavage was analyzed at the indicated time points by immunoblotting . B Mitochondria were induced by growing the WT strain expressing OM45 ‐ GFP in YPL medium to mid ‐ log ‐ phase and subsequently transferring cells to either SD ‐ N with or without GABA to trigger mitophagy for 12 h . GFP cleavage was analyzed at the indicated time points by immunoblotting . C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45 ‐ GFP grown in YPL medium for 12 h to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA for 24 h . Bar , 5 μm . D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA , grown to mid ‐ log ‐ phase , after which samples were analyzed for Ape1 maturation . E Ribophagy was monitored by growing the WT strain expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 24 h . F Autophagy was monitored by growing the WT strain expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 6 h .

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Figure 1A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.


Figure 2APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A . B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA and rapamycin for 6 h . Bar , 5 μm . C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .

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Figure 2APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A.B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .


Figure 3A WT cells expressing OM45 ‐ GFP , along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing OM45 ‐ GFP were grown in YPL medium to mid ‐ log ‐ phase . To monitor mitophagy , strains were transferred to SD ‐ N starvation medium ( with or without rapamycin ) . B WT strain along with the uga2 ∆ strain over ‐ expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin . Samples were taken at the indicated time points , and Pot1 degradation was analyzed by immunoblotting ( 45 kD ) . 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 .

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Figure 3A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).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.


Figure 4A , 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 ) . D GFP production during mitophagy was analyzed by immunoblotting .

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Figure 4A,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).D GFP production during mitophagy was analyzed by immunoblotting.


Figure 5A S6 phosphorylation after 6 h in SD ‐ N was analyzed by immunoblotting with a loading control . B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting .

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Figure 5A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control.B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.


Figure 6A , 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 . 01C , D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under ( C ) pexophagy or ( D ) mitophagy conditions . Significant differences between the treatments and strains were determined using an unpaired two ‐ tailed t ‐ test . * * P < 0 . 01 . E Pexophagy assay was monitored by the degradation of Pot1 ‐ GFP and analyzed for GFP cleavage by immunoblotting . F Mitophagy assay was monitored by the degradation of Om45 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

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Figure 6A,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 < 0.01C,D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two‐tailed t‐test. **P < 0.01.E Pexophagy assay was monitored by the degradation of Pot1‐GFP and analyzed for GFP cleavage by immunoblotting.F Mitophagy assay was monitored by the degradation of Om45‐GFP and analyzed for GFP cleavage by immunoblotting.


Figure 7A , B Example images of Parkin ‐ expressing HeLa cells analyzed using a tandem fluorochrome protein ( mito ‐ RFP ‐ GFP ) mitophagy assay under ( A ) control conditions or ( B ) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes . Bar , 10 μm . C Percentage of cells displaying mitophagy + s . d . , * * P < 0 . 01 using an unpaired two ‐ tailed t ‐ test , n > 80 .

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Figure 7A,B Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm.C Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.


Figure 8A Electron microscopy images of mitochondria from WT ( n = 44 ) and SSADH ‐ deficient mice ( Aldh5a1 − / − ) ( n = 80 ) were calculated for area size . B Electron microscopy images showing typical sizes of WT and Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm . C Quantification of mitochondrial numbers from electron microscopy images of liver from WT ( n = 31 ) and Aldh5a1 − / − mice treated with vehicle ( n = 39 ) or rapamycin ( n = 34 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life . D Quantification of mitochondrial numbers from electron microscopy images of brain from WT ( n = 23 ) and Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life . E Aldh5a1 − / − mice were treated with vehicle or rapamycin ( 10 mg / kg body weight per day ) via intraperitoneal injections for 10 successive days starting at day 10 of life . WT mice served as non ‐ disease controls ( set to 1 ) . After sacrifice , liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay . F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis ( WT set to 1 ) . 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 .

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Figure 8A Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size.B Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm.C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).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.


Figure 9A 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 . 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 .

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Figure 9A 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.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.


Figure 1 ( A ) Raji cells stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) and analyzed by flow cytometry . The numbers indicate the % of positive cells . One representative experiment is shown . ( B ) Binding of the 24 indicated antibodies to the surface of Raji - Env cells . Results are expressed as the median fluorescence intensity ( MFI ) of staining . n = 6 independent experiments . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test . ( 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.(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 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 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 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 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.


Figure 2 ( 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 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 ( 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 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 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 (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 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 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 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 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 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.


Figure 3 ( 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 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 ) 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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) 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 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 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.


Figure 4 ( 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 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 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 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 .

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Figure 4(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 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 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 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.


Figure 5 ( 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 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 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 .

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Figure 5(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 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 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.


Figure 6 ( 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 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 . ( C ) CH058 - , CH077 - and vKB18 - infected primary CD4 T cells were incubated with the indicated antibodies and NHS . After 24h , C3 surface levels on infected cells were analyzed by flow cytometry . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a donor of CD4 T cells . n = 6 donors of CD4 T cells . ( D ) Primary CD4 T cells infected with the indicated HIV - 1 strains ( CH058 , CH077 or vKB18 ) were cultured with the indicated WT or E430G mutant antibodies 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 . 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 = 6 donors of CD4 T cells .

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Figure 6(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 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.(C) CH058-, CH077- and vKB18-infected primary CD4 T cells were incubated with the indicated antibodies and NHS. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a donor of CD4 T cells. n=6 donors of CD4 T cells.(D) Primary CD4 T cells infected with the indicated HIV-1 strains (CH058, CH077 or vKB18) were cultured with the indicated WT or E430G mutant antibodies 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. 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=6 donors of CD4 T cells.