K . Percentage of OHC loss in 7 - month old CgndelG mice ( n = 3 - 8 biological replicates from 3 cochleae ) . Data information : Data are presented as mean ± SEM ; two - way ANOVA was used * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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K. Percentage of OHC loss in 7-month old CgndelG mice (n = 3-8 biological replicates from 3 cochleae). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


F . Percentage of OHC loss in noise exposed CgndelG mice ( n = 5 - 9 cochleae ) . Data information : Data are presented as mean ± SEM ; one - way ANOVA was used in ( F ) . * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 .

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F. Percentage of OHC loss in noise exposed CgndelG mice (n = 5-9 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used in (F). ** P < 0.01, *** P < 0.001 and **** P < 0.0001.


Whole mount immunofluorescence with LMO7 antibody on 2 - month old CgndelG mice at 32 kHz cochlear

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Whole mount immunofluorescence with LMO7 antibody on 2-month old CgndelG mice at 32 kHz cochlear region


Whole mount immunofluorescence with Spectrin β II antibody on 2 - month old CgndelG mice at 32 kHz cochlear

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Whole mount immunofluorescence with Spectrin β II antibody on 2-month old CgndelG mice at 32 kHz cochlear region


A . Low - magnification SEM images of hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . B . High - magnification SEM images of IHC hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . C . High - magnification SEM images of OHC hair bundles from 2 - month old Cgn + / + and CgndelG / delG mice . Shown are hair bundles of the second - row OHCs ( OHC2 ) .

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A. Low-magnification SEM images of hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. B. High-magnification SEM images of IHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. C. High-magnification SEM images of OHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. Shown are hair bundles of the second-row OHCs (OHC2).


A WT MDFs were treated ± TSI individually or in combination for 3 hrs . Whole cell lysates ( WCL ) and UBA - pull down ( UBA - PD ) fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments . Samples of UBA - pull down in following figures were analysed in the same way unless otherwise indicated .

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A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.


B WT , Ripk3 - / - , Mlkl - / - , Tnfr1 - / - and Traf2 - / - MDFs were untreated ( UT ) or treated with TSI for 3 hrs . Nec - 1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively .

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B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.


E RIPK3 - gyrase were inducibly expressed in Ripk3 - / - MDFs by doxycycline ( dox ) for 5 hrs , and cells were then treated ± combination of TSI , or coumermycin ( coum ) for 3 hrs .

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E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.


B UBA - pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in ( A ) . Beads eluates were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


C 1 . 4 mL cleared cell lysate from 4 x 106 TSI - treated WT MDFs was split into three parts of the indicated volume , followed by UBA - pull down and DUB incubation . Bead eluates were analysed by Western blot and probed with the indicated antibodies . Representative of three independent experiments .

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C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.


A WT MDFs were treated with TSI for indicated time . Cells were fractionated into cytosol and crude membrane , followed by UBA - pull down . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


B WT USP21 - CaaX and USP21C221R - CaaX ( CR ) were inducibly expressed in WT MDFs by doxycycline for 5 hrs . Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , at the same time cells were untreated ( UT ) or treated with TSQ for 6 hrs . Cells were fractionated into cytosol ( C ) and crude membrane ( M ) . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . B Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above .

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A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


E HT29 cells were stimulated with TSI , ± NSA ( 500 nM ) , or left untreated ( UT ) for 16 hrs , followed by cellular fractionation . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . F Cell lysates from ( E ) were subjected to UBA - pulldown and analysed as described above .

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E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.


A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , cells were untreated or treated with TSQ for 6 hrs ( please note that the same WT control was used in Fig . EV3A ) . Cell death was measured by PI staining based on flow cytometry . Data are plotted as mean ± SEM of three independent experiments . B Cellular fractions from ( A ) were analysed by Western blot from BN - PAGE or SDS - PAGE using antibodies as indicated . The same WT control was used in Fig . 4A . Representative of three independent experiments . C Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above .

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A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


D N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline overnight , and cells were treated with TSI for indicated time , followed by UBA - pull down . Representative of three independent experiments . E N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 16 hrs . Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline . Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 ( BAF ) , PS341 ( PS ) or left untreated ( UT ) . IDN - 6556 was added to all conditions to block apoptosis . Cells were collected 0 , 2 , 4 , 6 hrs after medium replacement , followed by UBA - pull down . Representative of three independent experiments .

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D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.


B WT and 4KR mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 6 hrs and cells were untreated ( UT ) or treated with TSI , followed by UBA - pull down . Representative of three independent experiments .

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B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.


D MLKL - / - HT29 cells stably transfected with constructs encoding human MLKL - USP21 and MLKL - USP21C221R , were treated with doxycycline , NSA ( 1 μM ) , TSI or combinations thereof ( added simultaneously ) . Sytox Green positive cells were quantified in real time by live cell imaging . Data are plotted as mean ± SEM of six independent experiments . ( A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA ) . E Human MLKL - USP21 and MLKL - USP21C221R were inducibly expressed in MLKL - / - HT29 cells by doxycycline ( 10 ng / mL ) for 16 hrs , TSI was added for the indicated times but all samples were collected 25 hrs post - induction , followed by UBA - pulldown . Representative of three independent experiments .

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D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.


B Inhibition zone - based assay for synergy between AMSIN and HNP1 & 2 or lysozyme C ( abbreviated as hLZc ) to the two oral Streptococcus ( SSAN and SSAL ) . Inset ( upper panel , boxed ) , a representative example of the inhibition - zone test showing the synergy . Peptide doses used here were 0 . 05 nmol / well for HNP1 & 2 and hLZc at which no visible inhibition zones were observed for both of them . At the same dose , AMSIN produced a small inhibition zone . For synergetic observation , two peptides were applied to the same well according to the doses indicated . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test ( * * * P < 0 . 001 Note : the nanomolar concentration of HNP1 & 2 was calculated from the average molecular weight of HNP1 and HNP2 .

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B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001 Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2.


C The FIC index assay for synergy between AMSIN and HNP1 & 2 or hLZc to SSAL and SSAN . The dashed line represents the cutoff of the FIC index ( 0 . 5 ) for synergy .

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C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.


A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets . The peptide or the compounds used were 4 × CL . VAN , vancomycin ; RIF , rifampicin ; KAN , kanamycin . OD600 were calculated as measured values subtracting the background value from broth medium ( OD600 = 0 . 356 ) .

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A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL. VAN, vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356).


D Scanning electron microscopic observation of AMSIN - induced BM deformation . The doses used were 10 × CL for AMSIN and vancomycin .

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D Scanning electron microscopic observation of AMSIN-induced BM deformation. The doses used were 10×CL for AMSIN and vancomycin.


E Cytoplasmic accumulation of the soluble cell - wall precursor UMP in vancomycin - treated ( blue line ) , AMSIN - treated ( red dashed line ) or not treated ( green line ) culture of MRSA P1374 . Inset : UMP identified by UPLC - HRMS with singlely and doubly protonated m / z observed .

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E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed.


H ESI - MS detecting the non - covalent complex between AMSIN and UMP . The quadruply protonated complex ( m / z 1304 . 8 ) is observed . Inset : comparison of intensities of the complex between two ratios of UMP : AMSIN Here , AMSIN is abbreviated as AMS due to space limitation .

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H ESI-MS detecting the non-covalent complex between AMSIN and UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of UMP:AMSIN Here, AMSIN is abbreviated as AMS due to space limitation.


J The dose - response unit curve of UMP binding to AMSIN , which is fitted by the BIAevaluation v2 . 0 . 1 . software using 1 : 1 Langmuir binding model .

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J The dose-response unit curve of UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding model.


B Hemolysis by AMSIN . Meucin - 18 was used as a positive control . Mean ± SD of three technical replicates is displayed .

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B Hemolysis by AMSIN. Meucin-18 was used as a positive control. Mean ± SD of three technical replicates is displayed.


C Comparison of cytotoxic effects of AMSIN and LL - 37 on HL - 60 cells . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk ; * P < 0 . 05 , * * * P < 0 . 001 , ns : no

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C Comparison of cytotoxic effects of AMSIN and LL-37 on HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significance


D An inhibition - zone assay showing the inability of AMSIN to bind DNA . The bacterium used was MRSA P1374 and the peptide dose was 1 . 0 nmol / well . Mean ± SD of three biological replicates is displayed . P values were obtained by Mann - Whitney U ( ns : no

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D An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance


E The effect of AMSIN on human ion channels expressed in the CNS and heart . The asterisks mark steady state current traces after administering 100 μM AMSIN .

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E The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady state current traces after administering 100 μM AMSIN.


F Peptide - induced IL - 8 release . A549 cells were treated with AMSIN or LL - 37 for 24 h . IL - 8 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA , as described in Materials and Methods . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significance G Peptide - induced MCP - 1 release . HL - 60 cells were treated with AMSIN or LL - 37 for 24 h . MCP - 1 levels in culture supernatants from the FBS - containing medium ( left ) or the serum - free medium ( right ) were measured with an ELISA . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - test or Mann - Whitney U test ( the latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , ns : no

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F Peptide-induced IL-8 release. A549 cells were treated with AMSIN or LL-37 for 24 h. IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or LL-37 for 24 h. MCP-1 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significance


J Survival after peritoneal infection with MRSA P1374 . The survival fraction of the vehicle - treated control group was zero out of 11 mice and the treatment group contained six mice and all survived .

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J Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle-treated control group was zero out of 11 mice and the treatment group contained six mice and all survived.


A , The indicated cells co - transfected with p5HRE - Luc and either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( empty vector : EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay . B , U2OS cells co - transfected with p5HRE - Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay .

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

A, The indicated cells co-transfected with p5HRE-Luc and either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (empty vector: EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay. B, U2OS cells co-transfected with p5HRE-Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.


C , HCT116 p53 - / - cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 ( at two concentrations : + and + + ) or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies .

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

C, HCT116 p53-/- cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 (at two concentrations: + and ++) or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies.


D , HCT116 p53 + / + cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 R175H , R248W , R273H , or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay .

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

D, HCT116 p53+/+ cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 R175H, R248W, R273H, or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) either pcDNA3 / p53 ( p53 ) or pcDNA3 ( EV ) ( C ) with the indicated siRNA were cultured under < 0 . 1 % O2 conditions for 24 hours and subjected to qRT - PCR for the indicated

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) either pcDNA3/p53 (p53) or pcDNA3 (EV) (C) with the indicated siRNA were cultured under < 0.1% O2 conditions for 24 hours and subjected to qRT-PCR for the indicated mRNA


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) to the transwell invasion

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) to the transwell invasion assay


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) with the indicated siRNA or to the transwell invasion

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) with the indicated siRNA or to the transwell invasion assay


G , The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( left ) and the representative images of lungs ( right ) .

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

G, The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (left) and the representative images of lungs (right).


The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( H ) were cultured under < 0 . 1 % O2 ( H ) for the indicated periods and subjected to the in vitro cell proliferation assay .

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

The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (H) were cultured under < 0.1% O2 (H) for the indicated periods and subjected to the in vitro cell proliferation assay.


The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) transiently transfected with siScr or siZBTB2 ( I ) the indicated oxygen conditions ( I ) for the indicated periods and subjected to the in vitro cell proliferation assay .

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

The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) transiently transfected with siScr or siZBTB2 (I) the indicated oxygen conditions (I) for the indicated periods and subjected to the in vitro cell proliferation assay.


J , K , Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed .

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

J,K, Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed.


A , Tissue microarrays of human lung adenocarcinoma ( 243 samples ) stained with anti - ZBTB2 antibodies . High magnification images are shown in the left lower side of each image . Scale bar , 500 and 100 μm in the low and high magnification images , respectively .

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

A, Tissue microarrays of human lung adenocarcinoma (243 samples) stained with anti-ZBTB2 antibodies. High magnification images are shown in the left lower side of each image. Scale bar, 500 and 100 μm in the low and high magnification images, respectively.


B , Fisher ' s exact test for percentage of CA9 - positive tumors in the four indicated groups . Percentages and absolute numbers of each group are indicated in the graph .

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

B, Fisher's exact test for percentage of CA9-positive tumors in the four indicated groups. Percentages and absolute numbers of each group are indicated in the graph.


C , Kaplan - Meier analysis of the disease - free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status .

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

C, Kaplan-Meier analysis of the disease-free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status.


D - F , TCGA - based Kaplan - Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX ( D ) , ZMAT3 ( E ) , and CEACAM1 ( F ) .

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

D-F, TCGA-based Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX (D), ZMAT3 (E), and CEACAM1 (F).


A , The indicated cells transiently transfected with pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies .

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

A, The indicated cells transiently transfected with pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated ( B , D , E , F ) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated (B,D,E,F) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


H , After transient transfection with the expression vectors for ZBTB2 - V5 , ZBTB2 - myc , or their empty vector ( - ) , ZBTB2 - myc protein was immunoprecipitated using the anti - myc antibody and co - precipitated ZBTB2 - V5 was detected using the anti - V5 antibody ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) .

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

H, After transient transfection with the expression vectors for ZBTB2-V5, ZBTB2-myc, or their empty vector (-), ZBTB2-myc protein was immunoprecipitated using the anti-myc antibody and co-precipitated ZBTB2-V5 was detected using the anti-V5 antibody (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).


I , J , The indicated cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT or pcDNA4 / LgBiT and either pcDNA4 / ZBTB2 - SmBiT or pcDNA4 / SmBiT , as indicated , were cultured under < 0 . 1 % oxygen ( I ) or the indicated oxygen conditions ( J ) and subjected to the split luciferase complementation assay .

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

I,J, The indicated cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT or pcDNA4/LgBiT and either pcDNA4/ZBTB2-SmBiT or pcDNA4/SmBiT, as indicated, were cultured under < 0.1% oxygen (I) or the indicated oxygen conditions (J) and subjected to the split luciferase complementation assay.


K , HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 ( ZF2 or ZF3 - deletion ) were cultured under < 0 . 1 % oxygen conditions , and subjected to immunoprecipitation with anti - myc antibody . Coprecipitated DNA was subjected to the qRT - PCR experiment using primers against HRE regions of the STC1 promoter .

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

K, HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 (ZF2 or ZF3-deletion) were cultured under < 0.1% oxygen conditions, and subjected to immunoprecipitation with anti-myc antibody. Coprecipitated DNA was subjected to the qRT-PCR experiment using primers against HRE regions of the STC1 promoter.


L , The same experiment as I was conducted using the indicated combination of pcDNA4 / ZBTB2 - LgBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - LgBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - LgBiT ( ΔZF3 ) , and pcDNA4 / ZBTB2 - SmBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - SmBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - SmBiT ( ΔZF3 ) .

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

L, The same experiment as I was conducted using the indicated combination of pcDNA4/ZBTB2-LgBiT (WT), pcDNA4/ZBTB2ΔZF2-LgBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-LgBiT (ΔZF3), and pcDNA4/ZBTB2-SmBiT (WT), pcDNA4/ZBTB2ΔZF2-SmBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-SmBiT (ΔZF3).


B , HCT116 p53 - / - cells co - transfected with pG5H1bLuc , pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A , and either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 8 - 11 ] ( ZBTB2 del [ 8 - 11 ] ) , pEF6 / ZBTB2 del [ 51 - 54 ] ( ZBTB2 del [ 51 - 54 ] ) , or pEF6 / myc - His B ( EV ) , as indicated , were cultured under < 0 . 1 % oxygen conditions for 24 hours and subjected to the luciferase assay .

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

B, HCT116 p53-/- cells co-transfected with pG5H1bLuc, pcDNA6/Gal4 DBD-HIF-1α TAD P564A, and either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[8-11] (ZBTB2 del[8-11]), pEF6/ZBTB2 del[51-54] (ZBTB2 del[51-54]), or pEF6/myc-His B (EV), as indicated, were cultured under < 0.1% oxygen conditions for 24 hours and subjected to the luciferase assay.


C , After transient transfection with or without the expression vectors for ZBTB2 - V5 , ZBTB2 4A - V5 , ZBTB2 - myc , ZBTB2 4A - myc , or their empty vector , as indicated , the myc - fused proteins were immunoprecipitated using an anti - myc antibody and co - precipitated ZBTB2 - V5 or ZBTB2 4A - V5 was detected ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) .

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

C, After transient transfection with or without the expression vectors for ZBTB2-V5, ZBTB2 4A-V5, ZBTB2-myc, ZBTB2 4A-myc, or their empty vector, as indicated, the myc-fused proteins were immunoprecipitated using an anti-myc antibody and co-precipitated ZBTB2-V5 or ZBTB2 4A-V5 was detected (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).


D , HCT116 p53 - / - ( left ) cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - LgBiT 4A ( 4A ) , or pcDNA4 / LgBiT ( - ) and either pcDNA4 / ZBTB2 - SmBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - SmBiT ( 4A ) , or pcDNA4 / SmBiT ( - ) , as indicated , were cultured under < 0 . 1 % oxygen and subjected to the split luciferase complementation assay .

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

D, HCT116 p53-/- (left) cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT (ZBTB2), pcDNA4/ZBTB2 4A-LgBiT 4A (4A), or pcDNA4/LgBiT (-) and either pcDNA4/ZBTB2-SmBiT (ZBTB2), pcDNA4/ZBTB2 4A-SmBiT (4A), or pcDNA4/SmBiT (-), as indicated, were cultured under < 0.1% oxygen and subjected to the split luciferase complementation assay.


G , The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 4A ( 4A ) , or pEF6 / myc - His B ( EV ) .

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

G, The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 4A (4A), or pEF6/myc-His B (EV).


H , Growth of the indicated tumor xenografts was analyzed .

H, Growth of the indicated tumor xenografts was analyzed.


( D ) Western blot analysis of a strain expressing TurboID - HA - Emc6 , Bpl1 - AID * - 9myc and OsTIR1 which was grown overnight in SD media with or without auxin . The cells were then diluted into fresh SD media and grown to mid logarithmic phase ( ~ 4hours ) with or without auxin ( respectively ) . When required , biotin was added either 30min before harvesting , or for the entire 4hours . H3 ( histone H3 ) is used as a loading control .

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

(D) Western blot analysis of a strain expressing TurboID-HA-Emc6, Bpl1-AID*-9myc and OsTIR1 which was grown overnight in SD media with or without auxin. The cells were then diluted into fresh SD media and grown to mid logarithmic phase (~4hours) with or without auxin (respectively). When required, biotin was added either 30min before harvesting, or for the entire 4hours. H3 (histone H3) is used as a loading control.


( B ) Volcano plots for each of the four distinct LC - MS / MS experiments highlighted in Figure 2A showing - log ( p - value ) against log2 ( fold - change ) for all proteins identified in the appropriate Emc6 / Sbh1 samples . Proteins enriched in the Emc6 samples are colour / pattern - coded as follows : dotted outline for HA - IP of BioID2 - HA - Emc6 ; blue fill for streptavidin - AP of BioID2 - HA - Emc6 ; black , solid outline for streptavidin - AP of TurboID - HA - Emc6 ; and yellow fill for streptavidin - AP of TurboID - HA - Emc6 / ABOLISH . Grey dots represent proteins that passed the p - value and fold - change criteria , but were only identified by one peptide . Only EMC complex components or proteins with EMC client features are named .

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

(B) Volcano plots for each of the four distinct LC-MS/MS experiments highlighted in Figure 2A showing -log(p-value) against log2(fold-change) for all proteins identified in the appropriate Emc6/Sbh1 samples. Proteins enriched in the Emc6 samples are colour/pattern-coded as follows: dotted outline for HA-IP of BioID2-HA-Emc6; blue fill for streptavidin-AP of BioID2-HA-Emc6; black, solid outline for streptavidin-AP of TurboID-HA-Emc6; and yellow fill for streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Grey dots represent proteins that passed the p-value and fold-change criteria, but were only identified by one peptide. Only EMC complex components or proteins with EMC client features are named.


( A ) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N ' tagged with GFP , Gnp1 C ' tagged with GFP , Pdr12 N ' tagged with GFP or Pho84 C ' tagged with GFP . For the Gnp1 - GFP strain , the quantified signal depletion was ~ 10 % ( p < 0 . 0001 ) . Topological schematics feature beneath each respective image , with predicted TMDs highlighted in blue ( Weill et al , 2019 ) . All images are from two independent biological repeats and are digitally coloured using the ' fire ' lookup table ( LUT ) on Fiji . The same contrast settings were used for each image pair .

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

(A) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N' tagged with GFP, Gnp1 C' tagged with GFP, Pdr12 N' tagged with GFP or Pho84 C' tagged with GFP. For the Gnp1-GFP strain, the quantified signal depletion was ~10% (p < 0.0001). Topological schematics feature beneath each respective image, with predicted TMDs highlighted in blue (Weill et al, 2019). All images are from two independent biological repeats and are digitally coloured using the 'fire' lookup table (LUT) on Fiji. The same contrast settings were used for each image pair.


( C ) Streptavidin blots of diploid strains expressing either AviTag - Fks1 , - Gnp1 , - Pdr5 , - Pdr12 or - Stv1 ( as negative control ) together with BirA - Emc6 , grown in the conditions described in Figure 1E . For each strain , blots showing both the ' lower ' ( L ) and ' higher ' ( H ) contrast settings are shown side - by - side . The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name .

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

(C) Streptavidin blots of diploid strains expressing either AviTag-Fks1, -Gnp1, -Pdr5, -Pdr12 or -Stv1 (as negative control) together with BirA-Emc6, grown in the conditions described in Figure 1E. For each strain, blots showing both the 'lower' (L) and 'higher' (H) contrast settings are shown side-by-side. The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name.


( B ) U2OS stably expressing FLAG - TIP60 or empty vector ( vec ) were subjected to FLAG - immunoprecipitation from nuclear lysates and analysed by Western blotting

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

(B) U2OS stably expressing FLAG-TIP60 or empty vector (vec) were subjected to FLAG- immunoprecipitation from nuclear lysates and analysed by Western blotting


( C ) HEK293T cells were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) . The samples were subjected to FLAG affinity - purification , dephosphorylated with shrimp alkaline phosphatase ( rSAP ) and incubated as indicated with CDK9 / cyclinT1 in presence of ATP . Phosphorylation of TIP60 was analysed by a phosphoS90 - specific TIP60 antibody , and the blot was further probed with antibodies specific for FLAG and CDK9

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

(C) HEK293T cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG- TIP60S90A or empty vector (vec). The samples were subjected to FLAG affinity-purification, dephosphorylated with shrimp alkaline phosphatase (rSAP) and incubated as indicated with CDK9/cyclinT1 in presence of ATP. Phosphorylation of TIP60 was analysed by a phosphoS90-specific TIP60 antibody, and the blot was further probed with antibodies specific for FLAG and CDK9


( D ) p53 - / - hRasG12V MEF were treated with DMSO or 1µM SNS - 032 ( SNS ) for 1h . Nuclear lysates were analysed by Western blotting

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

(D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting


( E ) p53 - / - hRasG12V MEF were treated with DMSO , 1µM SNS - 032 ( SNS ) or 50µM DRB followed by addition of 100nM Calyculin A as indicated . Nuclear lysates were analysed by Western blotting

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

(E) p53-/- hRasG12V MEF were treated with DMSO, 1µM SNS-032 (SNS) or 50µM DRB followed by addition of 100nM Calyculin A as indicated. Nuclear lysates were analysed by Western blotting


( F ) U2OS cells stably expressing FLAG - TIP60WT were transfected with a pool of four different CDK9 - targeting siRNAs or a control siRNA targeting Luciferase . Nuclear lysates were analysed by Western blotting

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

(F) U2OS cells stably expressing FLAG-TIP60WT were transfected with a pool of four different CDK9-targeting siRNAs or a control siRNA targeting Luciferase. Nuclear lysates were analysed by Western blotting


( A ) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or FLAG - TIP60S86A . Nucleoplasm ( nuc . ) and chromatin ( chr . ) fractions were generated and were subjected to Western blot analysis

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

(A) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG- TIP60WT, FLAG-TIP60S90A or FLAG-TIP60S86A. Nucleoplasm (nuc.) and chromatin (chr.) fractions were generated and were subjected to Western blot analysis


( B ) The experiment as described in ( A ) was repeated twice with U2OS cells and twice with Ba / F3 cells and the blots were quantified . Each dot represents the ration of FLAG - TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in ( A )

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

(B) The experiment as described in (A) was repeated twice with U2OS cells and twice with Ba/F3 cells and the blots were quantified. Each dot represents the ration of FLAG-TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in (A)


( C ) Nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT or TIP60S90A , or FLAG - tagged chromodomain mutants TIP60F50A , TIP60Y47A , TIP60Y44F or the empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) and chromatin ( chr . ) fraction . Both fractions were analysed by Western blotting

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

(C) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT or TIP60S90A, or FLAG-tagged chromodomain mutants TIP60F50A, TIP60Y47A, TIP60Y44F or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting


( D ) Nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S90E , TIP60S90D or the empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) and chromatin ( chr . ) fraction . Both fractions were analysed by Western

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

(D) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S90E, TIP60S90D or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting


( E ) U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A or empty vector ( vec ) were treated with 2 µM SNS - 032 ( SNS ) for 3 hours as indicated . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western

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

(E) U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A or empty vector (vec) were treated with 2 µM SNS-032 (SNS) for 3 hours as indicated. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( A ) FLAG - immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S86A or empty vector ( vec ) . Immunoprecipitated material and input lysate were subjected to Western blot analysis ( B ) The experiment as shown in ( A ) was repeated three times . The graphs and error bars represent mean and standard deviation of these three individual experiments ( one - way ANOVA , Fisher " s LSD test , WT vs . S86A p = 0 , 5992 , WT vs . S90A p = 0 , 0429 , S90A vs . S86A p = 0 , 0176 ( * p < 0 , 05 ; n . s . not significant , p > 0 , 05 ) )

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

(A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec). Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) The experiment as shown in (A) was repeated three times. The graphs and error bars represent mean and standard deviation of these three individual experiments (one-way ANOVA, Fisher"s LSD test, WT vs. S86A p=0,5992, WT vs. S90A p=0,0429, S90A vs. S86A p=0,0176 (*p < 0,05; n.s. not significant, p > 0,05))


( C ) HEK293 cells were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector . FLAG - immunoprecipitations were performed from nuclear extracts . Immunoprecipitated material and input lysate were subjected to Western blot analysis

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

(C) HEK293 cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG-TIP60S90A or empty vector. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis


( D ) U2OS cells stably expressing FLAG - TIP60WT or empty vector were treated with 2µM SNS - 032 ( SNS ) for 2 hours as indicated . FLAG - immunoprecipitations were performed from nuclear extracts . This material and input lysate were subjected to Western blot analysis

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

(D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis


( E ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were subjected to ChIP using anti - FLAG antibody . Immunoprecipitated DNA and input were subjected to quantitative real - time PCR using primers annealing in the myc locus about 50bp upstream ( myc + 50 ) or about 500 bp downstream ( myc - 500 ) of the transcription start site . Promoter occupancy of FLAG - TIP60WT or FLAG - TIP60S90A was determined as percent input in relation to FLAG - TIP60WT . The graphs and error bars represent mean and standard deviation of three individual experiments ( one - way ANOVA , Bonferroni " s multiple comparison test , myc + 50 WT vs . S90A p = 0 , 0289 , myc - 500 WT vs . S90A p = 0 , 0155 ( * p < 0 , 05 ) )

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

(E) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were subjected to ChIP using anti-FLAG antibody. Immunoprecipitated DNA and input were subjected to quantitative real-time PCR using primers annealing in the myc locus about 50bp upstream (myc+50) or about 500 bp downstream (myc-500) of the transcription start site. Promoter occupancy of FLAG-TIP60WT or FLAG-TIP60S90A was determined as percent input in relation to FLAG -TIP60WT. The graphs and error bars represent mean and standard deviation of three individual experiments (one-way ANOVA, Bonferroni"s multiple comparison test, myc+50 WT vs. S90A p=0,0289, myc-500 WT vs. S90A p=0,0155 (*p < 0,05))


( A ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were treated with 2µM SNS - 032 ( SNS ) , 2µM JQ1 ( JQ1 ) or the solvent DMSO ( d . ) for 2h as indicated . FLAG - immunoprecipitations were performed from nuclear extracts . Immunoprecipitated material and input lysate were subjected to Western blot

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

(A) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were treated with 2µM SNS-032 (SNS), 2µM JQ1 (JQ1) or the solvent DMSO (d.) for 2h as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis


( B ) U2OS cells stably expressing FLAG - TIP60WT were treated with 2µM JQ1 ( JQ1 ) for 2h . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western blotting

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

(B) U2OS cells stably expressing FLAG-TIP60WT were treated with 2µM JQ1 (JQ1) for 2h. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( C ) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP . The nucleus was fractionated into nucleoplasm and chromatin . Both fractions were analysed by Western blotting

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

(C) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( A ) U2OS cells were transfected with a siRNA pool targeting human TIP60 ( Dharmacon ) or luciferase ( Dharmacon ) . The nuclear lysates were analysed by Western blotting 24h after transfection

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

(A) U2OS cells were transfected with a siRNA pool targeting human TIP60 (Dharmacon) or luciferase (Dharmacon). The nuclear lysates were analysed by Western blotting 24h after transfection


( B ) U2OS cells stably expressing FLAG - TIP60WT , FLAG - TIP60S90A , FLAG - TIP60S86A or empty vector ( vec ) were analysed by Western blotting 60 hours after viral transduction

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

(B) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A, FLAG-TIP60S86A or empty vector (vec) were analysed by Western blotting 60 hours after viral transduction


( A , B ) Isolated epidermal LCs ( A , n = 4 ) or isolated vaginal LCs ( B , n = 5 ) were stimulated O / N with a variety of vaginal microbiota and subsequently exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) . Epidermal LC were stimulated with L . crispatus ( LC ) , M . elsdenii ( ME ) , and P . timonensis ( PT ) whereas vaginal LCs were additionally stimulated with L . iners ( LI ) , G . vaginalis ( GV ) , and A . vaginae ( AV ) . After five days , HIV - 1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(A, B) Isolated epidermal LCs (A, n = 4) or isolated vaginal LCs (B, n = 5) were stimulated O/N with a variety of vaginal microbiota and subsequently exposed to HIV-1 (SF162; MOI 0.5). Epidermal LC were stimulated with L. crispatus (LC), M. elsdenii (ME), and P. timonensis (PT) whereas vaginal LCs were additionally stimulated with L. iners (LI), G. vaginalis (GV), and A. vaginae (AV). After five days, HIV-1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( C ) Isolated epidermal LCs ( n = 3 ) were stimulated O / N with B . fragilis ( BF ) , B . thetaiotaomicron ( BT ) , P . amnii ( PA ) , P . bivia ( PB ) , P . copri ( PC ) , and P . timonensis ( PT ) , followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) infection for 5 days and HIV - 1 p24 determination by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(C) Isolated epidermal LCs (n = 3) were stimulated O/N with B. fragilis (BF), B. thetaiotaomicron (BT), P. amnii (PA), P. bivia (PB), P. copri (PC), and P. timonensis (PT), followed by HIV-1 (SF162; MOI 0.5) infection for 5 days and HIV-1 p24 determination by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( D ) Vaginal epithelium explants ( n = 8 ) were exposed to vaginal microbiota as described for isolated vaginal LCs . HIV - 1 p24 in emigrated LCs was determined by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(D) Vaginal epithelium explants (n = 8) were exposed to vaginal microbiota as described for isolated vaginal LCs. HIV-1 p24 in emigrated LCs was determined by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( A - D ) Isolated epidermal LCs ( A , n = 2 ) , isolated vaginal LCs ( B - C , both n = 3 ) , or vaginal explants ( D , n = 5 ) were stimulated O / N with P . timonensis ( PT ) and exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) for five days in presence or absence of HIV - 1 replication inhibitors ( Inh ) zidovudine ( AZT ) , tenofovir ( TFV ) , iamivudine ( 3TC ) or indinavir ( IDV ) . HIV - 1 levels were determined by intracellular staining for HIV - 1 p24 using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

(A-D) Isolated epidermal LCs (A, n = 2), isolated vaginal LCs (B-C, both n = 3), or vaginal explants (D, n = 5) were stimulated O/N with P. timonensis (PT) and exposed to HIV-1 (SF162; MOI 0.5) for five days in presence or absence of HIV-1 replication inhibitors (Inh) zidovudine (AZT), tenofovir (TFV), iamivudine (3TC) or indinavir (IDV). HIV-1 levels were determined by intracellular staining for HIV-1 p24 using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( G ) Isolated epidermal LCs ( n = 3 ) were treated with T20 , Maraviroc ( MAR ) , neutralizing antibodies against CD4 ( αCD4 ) and isotype control ( Iso ) , followed by O / N stimulation with P . timonensis ( PT ) . Next , LCs were exposed to HIV - 1 for five days ( SF162 ; MOI 0 . 5 ) . Intracellular HIV - 1 p24 levels were determined using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

(G) Isolated epidermal LCs (n = 3) were treated with T20, Maraviroc (MAR), neutralizing antibodies against CD4 (αCD4) and isotype control (Iso), followed by O/N stimulation with P. timonensis (PT). Next, LCs were exposed to HIV-1 for five days (SF162; MOI 0.5). Intracellular HIV-1 p24 levels were determined using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


Immature vaginal ( vLC ) and epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) and activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured and activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . Representative plots of vaginal ( A ) or epidermal ( B ) donors and combined experiments ( MFI expression relative to untreated condition ) of CD80 , CD86 and CCR7 surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). Representative plots of vaginal (A) or epidermal (B) donors and combined experiments (MFI expression relative to untreated condition) of CD80 , CD86 and CCR7 surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Immature vaginal ( vLC ) and epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) and activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured and activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . CD80 ( C , n = 3 ) , CD86 ( D n = 3 ) , and CCR7 ( E , n = 3 ) surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). CD80 (C, n = 3), CD86 (D n = 3), and CCR7 (E, n = 3) surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Ex vivo skin explants were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , P . timonensis ( PT ) or poly ( I : C ) and at day 3 post - inoculation , emigrated LCs were collected , washed and activation phenotype was determined by flow cytometry . Cells were analysed for CD1a expression and the absolute number of CD1a - positive cells , i . e . LCs , migrated from the epidermis was determined using counting beads , the graph shows the migration relative to the untreated condition ( F , n = 5 ) . CD1a - positive cells , i . e . LCs , were analysed using flow cytometry and the graphs show combined experiments ( MFI expression relative to untreated condition ) of CD80 ( G , n = 5 ) and CD86 ( H n = 5 ) surface expression . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), P. timonensis (PT) or poly(I:C) and at day 3 post-inoculation, emigrated LCs were collected, washed and activation phenotype was determined by flow cytometry. Cells were analysed for CD1a expression and the absolute number of CD1a-positive cells, i.e. LCs, migrated from the epidermis was determined using counting beads, the graph shows the migration relative to the untreated condition (F, n = 5). CD1a-positive cells, i.e. LCs, were analysed using flow cytometry and the graphs show combined experiments (MFI expression relative to untreated condition) of CD80 (G, n = 5) and CD86 (H n = 5) surface expression. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure . ( A ) Intracellular HIV - 1 detection was determined by confocal microscopy ( Hoechst in blue , HIV - 1 p24 in green ) , scale bar represents 5 µm ( representative donor ) .

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

Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. (A) Intracellular HIV-1 detection was determined by confocal microscopy (Hoechst in blue, HIV-1 p24 in green), scale bar represents 5 µm (representative donor).


( B ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by a 4 - hour exposure to HIV - 1 . HIV - 1 p24 was determined by ELISA after trypsin treatment and lysis of LCs ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by a 4-hour exposure to HIV-1. HIV-1 p24 was determined by ELISA after trypsin treatment and lysis of LCs (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( C ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure for 3 days . After 3 days , LCs were treated with PBS or trypsin and HIV - 1 p24 was determined by flow cytometry ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(C) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure for 3 days. After 3 days, LCs were treated with PBS or trypsin and HIV-1 p24 was determined by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( D , E ) Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by infection with VSV - g - BlaM - Vpr fusion ( positive control ) and NL4 . 3Bal - BlaM - Vpr fusion . Figure shows representative plots ( D ) and pooled data ( E , n = 3 ) of viral fusion as determined by β - lactamase - Vpr ( BlaM - Vpr ) activity . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(D, E) Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by infection with VSV-g-BlaM-Vpr fusion (positive control) and NL4.3Bal-BlaM-Vpr fusion. Figure shows representative plots (D) and pooled data (E, n = 3) of viral fusion as determined by β-lactamase-Vpr (BlaM-Vpr) activity. Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( F ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 exposure ( SF162 ; MOI 0 . 5 ) in presence or absence of anti - langerin ( 10E2 ) or mannan ( MAN ) . HIV - 1 p24 content was determined by ELISA after trypsin - treatment and subsequent lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(F) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 exposure (SF162; MOI 0.5) in presence or absence of anti-langerin (10E2) or mannan (MAN). HIV-1 p24 content was determined by ELISA after trypsin-treatment and subsequent lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( G ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by treatment with Rapamycin ( µM ) and exposure to HIV - 1 ( SF162 ) . After 3 days HIV - 1 p24 was measured by ELISA after trypsin treatment and lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

(G) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by treatment with Rapamycin (µM) and exposure to HIV-1 (SF162). After 3 days HIV-1 p24 was measured by ELISA after trypsin treatment and lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( A ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure . LCs were subjected to repetitive freeze - thaw cycles and cell lysates were added to U87 . CD4 . CCR5 cell line . HIV - 1 infection of U87 . CD4 . CCR5 was determined by an intracellular staining for p24 by flow cytometry ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

(A) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. LCs were subjected to repetitive freeze-thaw cycles and cell lysates were added to U87.CD4.CCR5 cell line. HIV-1 infection of U87.CD4.CCR5 was determined by an intracellular staining for p24 by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( B , Ex vivo skin explants were stimulated O / N with P . timonensis ( PT ) At day 2 post - inoculation , emigrated LCs were collected , washed and co - cultured with U87 . CD4 . CCR5 cells for 3 days . Cells were analysed for CD1a expression and p24 content by flow cytometry and here presented as representative plots ( B )

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

(B, Ex vivo skin explants were stimulated O/N with P. timonensis (PT) At day 2 post-inoculation, emigrated LCs were collected, washed and co-cultured with U87.CD4.CCR5 cells for 3 days. Cells were analysed for CD1a expression and p24 content by flow cytometry and here presented as representative plots (B)


C ) Ex vivo skin explants were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) and inoculated with HIV - 1 ( JRCSF ; MOI 0 . 5 ) in presence or absence of tenofovir . At day 2 post - inoculation , emigrated LCs were collected , washed and co - cultured with U87 . CD4 . CCR5 cells for 3 days . pooled data of HIV - 1 positive cells ( % ) of the CD1a negative U87 . CD4 . CCR5 cells ( C , n = 4 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

C) Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and inoculated with HIV-1 (JRCSF; MOI 0.5) in presence or absence of tenofovir. At day 2 post-inoculation, emigrated LCs were collected, washed and co-cultured with U87.CD4.CCR5 cells for 3 days. pooled data of HIV-1 positive cells (%) of the CD1a negative U87.CD4.CCR5 cells (C, n = 4). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( D ) Ex vivo skin explants were stimulated with P . timonensis ( PT ) , pre - treated with T20 or maraviroc ( MAR ) and exposed to HIV - 1 ( JRCSF ; MOI 0 . 5 ) for 2 days , after which the migratory fraction was co - cultured with U87 . CD4 . CCR5 cells . HIV - 1 positive cells ( % ) of the CD1a - negative fraction , i . e . U87 . CD4 . CCR5 cells , were determined using flow cytometry ( n = 5 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

(D) Ex vivo skin explants were stimulated with P. timonensis (PT), pre-treated with T20 or maraviroc (MAR) and exposed to HIV-1 (JRCSF; MOI 0.5) for 2 days, after which the migratory fraction was co-cultured with U87.CD4.CCR5 cells. HIV-1 positive cells (%) of the CD1a-negative fraction, i.e. U87.CD4.CCR5 cells, were determined using flow cytometry (n = 5). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( E , F ) Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) and infected with HIV - 1 ( SF162 ; MOI 0 . 5 ) . After 3 days LCs were washed and co - cultured with U87 . CD4 . CCR5 . Infection was analysed by flow cytometry and here shown as representative plots ( E ) and pooled data of HIV - 1 positive cells ( % ) of the CD1a - negative U87 . CD4 . CCR5 cells ( F , n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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

(E, F) Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) and infected with HIV-1 (SF162; MOI 0.5). After 3 days LCs were washed and co-cultured with U87.CD4.CCR5. Infection was analysed by flow cytometry and here shown as representative plots (E) and pooled data of HIV-1 positive cells (%) of the CD1a-negative U87.CD4.CCR5 cells (F, n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( A , B ) Isolated epidermal LCs were stimulated by different Prevotella spp . and Bacteriodus spp . followed by T / F variant ( CH058 ; MOI 0 . 25 ) exposure in presence or absence of AZT ( A , n = 3 ) or tenofovir ( B , n = 2 ) . A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strains . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 .

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

(A, B) Isolated epidermal LCs were stimulated by different Prevotella spp. and Bacteriodus spp. followed by T/F variant (CH058; MOI 0.25) exposure in presence or absence of AZT (A, n = 3) or tenofovir (B, n = 2). A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strains. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.


( C ) HIV - 1 uptake was determined by flow cytometry . Ex vivo skin explants ( n = 3 ) were stimulated O / N with P . timonensis ( PT ) followed by pre - treatment with tenofovir ( TFV ) . After pre - treatment , skin explants were inoculated with lab - adapted strain ( SF162 ; MOI 0 . 25 ) or HIV - 1 T / F strain ( CHO58 ; MOI 0 . 25 ) . After 2 days the migratory fraction was collected and co - cultured with U87 . CD4 . CCR5 cells . Cells were analysed for CD1a expression and p24 content in U87 . CD4 . CCR5 cells by flow cytometry . A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strain SF162 . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 .

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

(C) HIV-1 uptake was determined by flow cytometry. Ex vivo skin explants (n = 3) were stimulated O/N with P. timonensis (PT) followed by pre-treatment with tenofovir (TFV). After pre-treatment, skin explants were inoculated with lab-adapted strain (SF162; MOI 0.25) or HIV-1 T/F strain (CHO58; MOI 0.25). After 2 days the migratory fraction was collected and co-cultured with U87.CD4.CCR5 cells. Cells were analysed for CD1a expression and p24 content in U87.CD4.CCR5 cells by flow cytometry. A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strain SF162. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.


( B , Sequential dissociation of EI24 and BiP from IRE1 elicits a two - phase activation of IRE1 in response to ER stress . Cells were treated with 4 μM tunicamycin ( TM ) for the indicated times . The interactions of EI24 and BiP with IRE1 were analyzed by

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

(B, Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The interactions of EI24 and BiP with IRE1 were analyzed by immunoprecipitation


( D ) EI24 knockout promotes the activation of IRE1 , but not PERK or ATF6 . Control ( sgGFP ) and EI24 knockout ( sgEI24 ) HeLa cells were treated with 4 μM TM for the indicated times . Protein expression was analyzed by immunoblotting .

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

(D) EI24 knockout promotes the activation of IRE1, but not PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM TM for the indicated times. Protein expression was analyzed by immunoblotting.


( G ) Expression of Flag - EI24 in EI24 knockout cells inhibits IRE1 activity . Flag - Metap2 served as the control . Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr . Protein expression was analyzed by immunoblotting .

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

(G) Expression of Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr. Protein expression was analyzed by immunoblotting.


( B ) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress . HeLa cells stably expressing Flag - EI24 were treated with or without 4 μM tunicamycin ( TM ) for 2 hr . Colocalization of EI24 with ER was analyzed . KDEL - mCherry , ER marker . Scale bar , 10 μm .

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

(B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.