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 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 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

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

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

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

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

(B) 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_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 O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 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 B-CONTROLLED_VAR 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

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

(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.


( D ) The C - terminus of EI24 is required for IRE1 binding . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) , after which protein - protein interactions were analyzed by immunoprecipitation ( IP ) . The star represents IgG light chains . ( E ) The C - terminus of EI24 binds to IRE1 in a persistent fashion . HeLa cells transfected with the indicated EI24 domains were treated as in ( B ) . Protein - protein interactions were analyzed by IP . The star represents IgG light chains . (

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

(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. (


( I ) EI24 overexpression cannot prevent the activation of the phospho - mimicking ( 3SE ) IRE1 mutant . Wild - type and IRE1 3SE knock - in HeLa cells were transfected with empty vector or Flag - EI24 . Transfected cells were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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 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

(I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting.


( L ) Expression of full - length EI24 or the △ TM34 or △ C mutant in EI24 knockout HeLa cells for comparison of their functions . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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 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

(L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting.


( B , C ) EI24 knockout causes resting - state ER calcium reduction ( B ) and cytosolic calcium elevation ( C ) under ER stress . Control ( sgGFP ) and EI24 knockout ( sgEI24 ) HeLa cells were first perfused in TM - free buffer , and switched to the buffer containing 4 μM TM to induce ER stress where indicated . Resting state calcium levels in the ER ( B ) and cytosol ( C ) with or without TM are displayed in columns . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

O O O O O B-CONTROLLED_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 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 B-CONTROLLED_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 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

(B, C) EI24 knockout causes resting-state ER calcium reduction (B) and cytosolic calcium elevation (C) under ER stress. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were first perfused in TM-free buffer, and switched to the buffer containing 4 μM TM to induce ER stress where indicated. Resting state calcium levels in the ER (B) and cytosol (C) with or without TM are displayed in columns. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( G ) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( I ) Wild - type EI24 , but not the TM34 - or C - terminus - deleted mutant , preserves ER calcium stores in EI24 knockout cells . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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 B-MEASURED_VAR 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

(I) Wild-type EI24, but not the TM34- or C-terminus-deleted mutant, preserves ER calcium stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( B ) Knockdown of IP3R1 prevents ER stress - induced apoptosis in EI24 knockout cells . Cells were cultured with or without 4 μM TM for 8 hr . Percentages of early apoptotic cells were measured by flow cytometry . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t -

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 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

(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test


( D ) Knockdown of IP3R1 prevents ER stress - induced apoptosis . Cells were treated with 4 μM TM for the indicated times . Protein expressions were analyzed by immunoblotting .

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

(D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.


( F - H ) Reintroduction of full - length EI24 , but not the TM34 - or C - terminus - deleted mutant ( F , G ) or the C - terminus alone ( H ) , inhibited ER stress - induced apoptosis in EI24 knockout cells . Cells treated with or without TM were analyzed for apoptosis by flow cytometry ( F , H ) or by immunoblotting ( G ) . Arrows in ( H ) denote decreased or increased apoptotic levels . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test for F ) , one - tailed Student ' s t - test for ( H ) .

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 B-CONTROLLED_VAR 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 O O 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-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).


B t - SNE plots showing integrated analysis of Prrx1 - Cre - ( 8 - week - old mice , n = 3 males ) and Lepr - Cre - traced ( 8 - week - old mice , n = 4 males ) cells . Cells were colored by clusters ( left ) or samples ( right ) . Top 20 PCs were chosen for the clustering .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE plots showing integrated analysis of Prrx1-Cre- (8-week-old mice, n=3 males) and Lepr-Cre-traced (8-week-old mice, n=4 males) cells. Cells were colored by clusters (left) or samples (right). Top 20 PCs were chosen for the clustering.


D Violin plots showing the expression of feature genes for each cluster .

D Violin plots showing the expression of feature genes for each cluster.


E In situ hybridization of Col3a1 on femur sections . Col3a1 was expressed in the perichondral ( a ) and periosteal ( b ) regions , but not in the bone marrow . B : Bone ; BM : Bone marrow ; Peri : Periosteum ; Endo : Endosteum . Scale bars were 100 µm . Arrowheads indicated the periosteum .

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

E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.


E Violin plots showing the expression of feature genes for each cluster .

E Violin plots showing the expression of feature genes for each cluster.


F Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Arrowheads indicated tdTomato + periosteal cells . Two representative periosteal regions were magnified ( a and b ) . GP : Growth plate . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm . G Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice 7 days after fracture . Aggrecan ( green ) , tdTomato ( red ) , DAPI ( blue ) . Arrowheads indicated tdTomato + periosteal cells . Two representative regions were magnified ( a and b ) . Scale bars were 100 µm .

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

F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.


B Dot plots showing the gene expression patterns of selected genes in each cluster .

B Dot plots showing the gene expression patterns of selected genes in each cluster.


D Flow cytometry analysis of BrdU incorporation in uncultured Lepr - Cre + Notch3 + / - BMSCs ( n = 4 independent experiments ) . Eight - week - old Lepr - Cre ; tdTomato mice were given a single intraperitoneal injection of BrdU ( 100 mg / kg body mass ) and maintained on 0 . 5 mg / ml of BrdU in the drinking water for 14 days .

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

D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.


E Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Notch3 ( green ) , tdTomato ( red ) , CD31 ( gray ) , DAPI ( blue ) . Sinusoid ( a ) , arteriole ( b ) , artery ( c ) and avascular bone marrow ( BM , d ) regions were shown . Arrowheads indicated Notch3 + tdTomato + adventitial cells closely associated with ECs . Arrows indicated Notch3 - tdTomato + reticular cells in the bone marrow that were not associated with ECs . Scale bars were 10 µm . F Localization quantification of Lepr - Cre + Notch3 + cells relative to bone marrow ECs ( n = 5 biological replicates from three independent experiments ) . Cells within 5 µm diameter of ECs were considered adjacent .

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

E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm. F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent. G


H Volcano plot showing DEGs in the bulk RNA - seq analysis ( n = 3 biological replicates ) . Green dots represent down - regulated genes . Red dots represent up - regulated genes .

O O O O O O O O O O O O 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 Volcano plot showing DEGs in the bulk RNA-seq analysis (n=3 biological replicates). Green dots represent down-regulated genes. Red dots represent up-regulated genes.


O Heatmap showing the average expression levels ( column - wide Z score ) of secreted factors with clustering .

O Heatmap showing the average expression levels (column-wide Z score) of secreted factors with clustering.


C Heatmap showing pseudotime - dependent gene expression . Bars on the top were colored as in ( A ) . Representative genes were highlighted on both sides .

O O O O O 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 Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides.


F Representative images of adipogenic , osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr - Cre + BMSCs . Scale bars were 100 µm .

O O 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 Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm.


G qPCR analysis of the expression levels of Pparg , Bglap and Acan in control or knockdown group ( n = 3 independent experiments ) . The statistical significance of differences was analyzed by one - way ANOVAs with Tukey ' s multiple comparison tests . Data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


B Violin plots showing the expression of feature genes for each cluster .

B Violin plots showing the expression of feature genes for each cluster.


F Immunostaining of Sca - 1 on femur sections of 8 - week - old Lepr - Cre ; tdTomato mice . Arrows indicated Sca - 1 + tdTomato + cells inside cortical bones of the femur diaphysis . Arrowheads indicated Sca - 1 + tdTomato + cells in the femur periosteum . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm .

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

F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.


G Representative flow cytometry plots of long bone periosteum in 8 - week - old Lepr - Cre ; tdTomato mice . The percentage of DAPI - CD45 - Ter119 - Tie2 - tdTomato + Sca - 1 + / - cells were shown .

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

G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.


I , J The average number of CFU - F colonies formed ( I ) and the average cell number per colony ( J ) were shown ( n = 4 independent experiments ) . The statistical significance of differences was assessed using one - way ANOVAs with Tukey ' s multiple comparison tests . All data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * 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

I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


A , B Representative oil red staining after 7 days of adipogenic differentiation in Lepr - Cre + subsets ( A ) and qPCR analysis of Adipoq and Pparg expression ( B ) ( n = 3 independent experiments ) . Scale bars were 100 µm .

O O 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 O O O

A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.


C , D In vitro chondrogenic differentiation of sorted periosteal cells . Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection ( C ) . Scale bars were 100 µm . Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression ( D ) ( n = 3 independent experiments ) .

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

C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).


E , F In vitro osteogenic differentiation of sorted periosteal cells . Representative alizarin red staining after osteogenic differentiation for 14 days . Scale bars were 200 µm . Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression ( F ) ( n = 3 independent experiments ) .

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

E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).


D ) SDS - PAGE analysis of purified tubulin from brains of all mouse models used in the current study , stained with Coomassie Brilliant - Blue . Alpha - and β - tubulin are separated using a specific SDS - PAGE

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

D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocol


E ) Immunoblot analysis of all purified tubulin variants . The molecular weight in ( D ) and ( E ) is indicated in kDa .

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

E) Immunoblot analysis of all purified tubulin variants. The molecular weight in (D) and (E) is indicated in kDa.


B ) Representative IRM and TIRF microscopy images showing microtubule positions and Tau - meGFP intensities for different PTM variants ( grey : wild type ; blue : Ttll1 - / - ; yellow : Ttll7 - / - ; green : Ttll1 - / - Ttll7 - / - ; pink : Atat1 ‑ / ‑ ) . In the last column , false - coloured TIRF images highlight differences of Tau - meGFP intensities on different types of microtubules ( Movie EV1 ) . C ) Quantification of integrated intensity of Tau - meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples . Single assays ( Fig EV2A ) of one experiment were combined after normalisation to the wild - type microtubule values ( A . U . = arbitrary units ) . Each data point represents the normalised fluorescence intensity value of one microtubule . D ) Summary quantification the three independent experiments shown in ( C ) . Each point is a normalised mean of the triplicates , error bars represent standard deviation .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation.


E ) Saturation curve of increasing concentration Tau - mCherry binding to GMPCPP wild - type and Ttll1 - / - Ttll7 - / - microtubules . Each data point is shown as mean ± standard deviation of the raw integrated intensity values ( A . U . × 104 ) of three experiments with independently purified tubulin samples ( Fig EV2D ) . Dissociation constants Kd and R2 coefficient from non - linear fits of each microtubule type are indicated below the plot .

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 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

E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.


C ) Decay curves of normalised microtubule length plotted against time upon incubation with katanin ( A . U . = arbitrary units ) for single microtubules highlighted by arrow heads with yellow borders in ( B ) . D ) Half - life times ( seconds ) of all microtubules from the assays of which representative images are displayed in ( B ) and representative decay curves are shown in ( C ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown . Mann - Whitney test , p - values displayed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Decay curves of normalised microtubule length plotted against time upon incubation with katanin (A.U. = arbitrary units) for single microtubules highlighted by arrow heads with yellow borders in (B). D) Half-life times (seconds) of all microtubules from the assays of which representative images are displayed in (B) and representative decay curves are shown in (C). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown. Mann-Whitney test, p-values displayed.


E ) Analysis of normalised microtubule half - lifetimes from 14 independent experiments ( Fig EV3 ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown , Mann - Whitney test , p - values displayed .

O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of normalised microtubule half-lifetimes from 14 independent experiments (Fig EV3). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


B ) Kymographs showing single Kif5B - EGFP motor molecules moving on one single microtubule of known PTM state ( Movie EV4 ) . Horizontal scale bars 2 µm ; vertical scale bars 5 C ) Scatter plots showing the average velocities ( µm / s ) exhibited by single Kif5b - EGFP molecules on the different microtubule types ( each data point represents one Kif5b - EGFP molecule ) . Data pooled from 29 independent experiments ( Appendix Fig S2 ) . Medians with interquartile ranges shown , Mann - Whitney test , p - values displayed .

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

B) Kymographs showing single Kif5B-EGFP motor molecules moving on one single microtubule of known PTM state (Movie EV4). Horizontal scale bars 2 µm; vertical scale bars 5 C) Scatter plots showing the average velocities (µm/s) exhibited by single Kif5b-EGFP molecules on the different microtubule types (each data point represents one Kif5b-EGFP molecule). Data pooled from 29 independent experiments (Appendix Fig S2). Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


D , E ) Survival probability ( Kaplan - Meier plots , taking varying microtubule lengths into account ; see Methods ) of the interaction time ( D ) and run length ( E ) of Kif5b - EGFP molecules on different microtubule types . Data in ( C , D , E ) were pooled from individual experiments ( Appendix Fig S2 ) without normalisation . The 50 % probability values are show in the plots . Statistical analysis displayed in the tables below : hazard ratios with 95 % confidence intervals pairing all data from different microtubule types ( * pairs differing with significance level 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 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

D,E) Survival probability (Kaplan-Meier plots, taking varying microtubule lengths into account; see Methods) of the interaction time (D) and run length (E) of Kif5b-EGFP molecules on different microtubule types. Data in (C,D,E) were pooled from individual experiments (Appendix Fig S2) without normalisation. The 50% probability values are show in the plots. Statistical analysis displayed in the tables below: hazard ratios with 95% confidence intervals pairing all data from different microtubule types (*pairs differing with significance level p<0.05).


( B ) Phenotyping of the same mice , presented in the same order , inoculated i . n . with 0 . 3 × 105 TCID50 of SARS - CoV - 2 at the age of 5 - 7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E - specific qRT - PCR . Red lines indicate the qRT - PCR limits of detection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phenotyping of the same mice, presented in the same order, inoculated i.n. with 0.3 × 105 TCID50 of SARS-CoV-2 at the age of 5-7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection.


( C , D ) Quantification ( C ) and images ( D ) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B , presented in the same order .

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

(C, D) Quantification (C) and images (D) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B, presented in the same order.