( H ) GFP - MST2 was expressed with or without FLAG - TRIP6 in HEK293 cells and the levels of MST2 , MST2 activating phosphorylation ( pMST2 - T180 ) , and FLAG - TRIP6 were measured by western blotting with the indicated antibodies . ( Mean ± SD ; n = 3 ; n . s . ≥ 0 . 05 , T - test ) .

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

(H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test).


( I ) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6 , and YAP target gene expression was analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , T - test ) .

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

(I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test).


( J ) The levels of YAP target gene expression was analyzed using RT - qPCR in control ( WT ) and TRIP6 - KO HEK293A cells . ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , T - test )

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

(J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test)


( K ) Control ( WT ) and TRIP6 - KO HEK293A cells were stained for YAP and TRIP6 . Merged image shows YAP ( green ) , TRIP6 ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * * P ≤ 0 . 0001 , Fisher ' s test ) . Scale bar = 20µm .

O O O O O O O O O O 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

(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.


( A ) LATS2 - GFP and Myc - MOB1A were overexpressed in HEK293 cells with or without co - overexpression of full length FLAG - TRIP6 and FLAG - TRIP6 1 - 277 . Myc - MOB1A was immunoprecipitated using anti - Myc antibodies and immune complexes were assayed for Myc - MOB1A and LATS2 - GFP levels . Levels of FLAG - TRIP6 , FLAG - TRIP6 1 - 277 , Myc - MOB1A , and LATS2 - GFP in the lysate are also shown . The levels of LATS2 - GFP in immune complexes relative to the level of Myc - MOB1A are shown in the graph ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LATS2-GFP and Myc-MOB1A were overexpressed in HEK293 cells with or without co-overexpression of full length FLAG-TRIP6 and FLAG-TRIP6 1-277. Myc-MOB1A was immunoprecipitated using anti-Myc antibodies and immune complexes were assayed for Myc-MOB1A and LATS2-GFP levels. Levels of FLAG-TRIP6, FLAG-TRIP6 1-277, Myc-MOB1A, and LATS2-GFP in the lysate are also shown. The levels of LATS2-GFP in immune complexes relative to the level of Myc-MOB1A are shown in the graph (Mean ± SD; n=3; **P≤0.01, T-test)


( B ) Competitive binding experiments were done using purified recombinant MBP - LATS2 , GST - TRIP6 , and 6His - MOB1A . MBP - LATS2 bound to maltose beads was incubated with GST - TRIP6 with or without increasing amounts of 6His - MOB1A , and the levels of each protein bound to MBP - LATS2 on the beads at the end of the experiment was determined by western blotting . The levels of input proteins are shown ( lysate ) . The binding of 6His - MOB1A and GST - TRIP6 to MBP alone , and the use BSA as a competitor instead of 6His - MOB1A are shown as controls . The numbers at the bottom are referred to in the text .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Competitive binding experiments were done using purified recombinant MBP-LATS2, GST-TRIP6, and 6His-MOB1A. MBP-LATS2 bound to maltose beads was incubated with GST-TRIP6 with or without increasing amounts of 6His-MOB1A, and the levels of each protein bound to MBP-LATS2 on the beads at the end of the experiment was determined by western blotting. The levels of input proteins are shown (lysate). The binding of 6His-MOB1A and GST-TRIP6 to MBP alone, and the use BSA as a competitor instead of 6His-MOB1A are shown as controls. The numbers at the bottom are referred to in the text.


( A ) MCF10A cells were infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) ( quantification of LATS1 localization at cell - cell junctions is shown in Figure EV3D ) . Scale bar = 20µm .

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

(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.


( B ) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells . MCF10A control cells were treated with control siRNA ( siControl ) against fire fly luciferase . Cells were stained for TRIP6 and LATS1 as in ( A ) ( quantification of TRIP6 localization at cell - cell junctions is shown in Figure EV3E ) . Scale bar = 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.


( C ) MCF10A cells were grown to high density , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

O O O O O 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) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( D ) Cells were grown as in ( C ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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

(D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( E ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and lysed while under tension . Anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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

(E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( F ) Cells were treated as in ( E ) , fixed while under tension , and stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

O O O O O O O O O O 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) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( G ) MCF10A cells infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) , were grown at high density on PDMS membranes and were stretched or not ( only stretched cells shown ) at 17 % elongation for 2 hours , fixed while under tension , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( H ) Cells were treated as in ( G ) and YAP target gene ( CTGF and Cyr61 ) and TRIP6 expression were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , T - test ) .

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

(H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test).


( I ) TRIP6 expression levels in ( H ) in control ( shEGFP ) and TRIP6 ( shTRIP6 ) knockdown cells just prior to stretch were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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

(I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).


( A ) MCF10A cells were either not treated or treated separately with Latrunculin B , Blebbistatin , EGTA , serum starvation , and Y27632 , and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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

(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) Cells were treated as in ( A ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 .

O O O O O O O O O O O O O O O O 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) Cells were treated as in (A), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6.


( A ) MCF10A cells were lysed and anti - TRIP6 or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin and TRIP6 .

O O O O O O O O O O O O O O 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) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( B ) MCF10A cells were either not treated ( control ) or treated separately by growth to high density , serum starvation , Latrunculin B , Blebbistatin , EGTA , or Y27632 treatment , and stained using anti - vinculin antibodies by immunofluorescence . Scale bar = 20µm .

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

(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.


( C ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and fixed while under tension and stained for vinculin . Scale bar = 20µm .

O O O O O O O O O O O O O O O O O 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) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm.


( D ) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho - LATS1 ( T1079 and S909 ) , phospho - YAP ( S127 ) , LATS1 , YAP , vinculin , and tubulin antibodies and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) .

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

(D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( E ) Vinculin was knocked down as described in ( D ) and cells were stained for YAP and vinculin . Merged image shows YAP ( green ) , vinculin ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , Fisher ' s test ) . Scale bar = 20µm .

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

(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.


( F ) Vinculin was knocked down as described in ( D ) and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) .

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

(F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( G ) Vinculin was knocked down in MCF10A cells as described in ( D ) , grown at high density on PDMS membranes and cells were stretched ( or not ) at 17 % elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) .

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

(G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( A ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells and cells were stained for LATS1 and TRIP6 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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

(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) Vinculin was knocked down as described ( Figure 5D ) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1 , TRIP6 , and vinculin and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) .

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

(B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( C ) MCF10A cells were subjected to different treatments as described ( Figure 4A ) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6 .

O O O O O O O O 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) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( D ) Quantification of TRIP6 - vinculin binding from part ( C ) is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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

(D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( E , F ) MCF10A cells were treated separately ( or not ) with Latrunculin B ( E ) and by serum starvation ( F ) and LATS1 immune complexes and lysates were probed by western blotting for vinculin , LATS1 , and TRIP6 . Quantification of LATS1 - TRIP6 - vinculin binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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

(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( G ) Wild type and T12 mutant of vinculin were co - expressed with LATS2 and FLAG - TRIP6 and anti - FLAG antibody was used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin , LATS2 and FLAG - TRIP6 . Quantification of LATS2 - TRIP6 - vinculin ( WT or T12 ) binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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

(G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( H ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells grown at high density on PDMS membranes . The membranes were stretched at 17 % elongation for 2 hours and fixed while under tension and stained for TRIP6 . Merged images show TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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

(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.


( A ) RNAscope fluorescence in - situ hybridization of Ch25h transcripts ( red ) in the spinal cord of non - immunized mice ( NI ) ( left panels ) and mice 17 days after EAE immunization ( peak disease ) ( 2 right panels ) shown in endothelial cells ( Isolectin B4 ( IsoB4 ) green top panels ) and activated macrophages / microglia ( Ionized calcium biding adaptor molecule 1 ( IBA1 ) green lower panels ) . Nuclei are shown in blue . ( B ) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages / microglia ( MG ) and endothelial cells ( EC ) , n = 6 biological replicates / group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. (B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.


( E ) Percentage of eGFP + CNS ECs in the same condition as in ( D ) . Symbols indicate individual mice and bars indicate mean ± SD . Ch25hfl / fl NI : n = 4 biological replicates , Ch25hfl / fl EAE : n = 5 biological replicates , Ch25hECKO NI : n = 4 biological replicates , Ch25hECKO EAE : n = 4 biological replicates . Representative results of 1 experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.


( A ) EAE disease course in Ch25hECKO and Cre negative littermates ( Ch25hfl / fl ) . Top panel : EAE clinical score . Bars indicate mean ± SEM . Bottom panel : Survival analysis of EAE , depicting disease incidence . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( B ) As in ( A ) except that Ch25h fl / fl - Pdgfb - iCreERT2 mice that express Cre recombinase in blood endothelial cells ( Ch25hBECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph . ( C ) As in ( A ) except that Ch25hfl / fl - Prox1 - CreERT2 mice that express Cre recombinase in lymphatic endothelial cells ( Ch25hLECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on the graph .

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(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.


( D , E ) Histopathological quantifications ( D ) and representatives staining ( E ) of spinal cord sections of immunized Ch25hfl / fl mice ( n = 7 biological replicates ) and Ch25hECKO mice ( n = 10 biological replicates ) at day 21 post - immunization for cellular infiltration ( HE ) . Five sections per mouse were quantified . Scale bars 200 µm ( top panels ) , 100 µm ( bottom panels ) .

(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).


( A ) RT - qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells ( pMBMECs ) isolated from Ch25hfl / fl and Ch25hECKO mice . Cells were left unstimulated ( NS ) or stimulated with IL - 1β . n = 5 biological replicates / group . The experiment was performed 3 times .

(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.


( F ) RT - qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl / fl mice and stimulated with IL - 1β . n = 4 biological replicates / group . The experiment was realized once .

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(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.


( G ) RT - qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl / fl mice during EAE ( day 10 after immunization ) . Ch25hECKO n = 3 biological replicates , Ch25hfl / fl n = 4 biological replicates . The experiment was performed once .

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(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.


( C ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC concentration in the same conditions as in ( B ) . Bars represent mean ± SD . n = 6 biological replicates / group except for Ch25fl / fl IL - 1β were n = 5 biological replicates . The experiment was performed once . ( D ) 25 - OHC , 7 - keto - 25 - OHC , and 24 ( S ) - OHC levels in spinal cord tissue extracted from Ch25hfl / fl ( n = 8 ) , Ch25hECKO ( n = 3 ) and Ch25hBBBKO ( n = 6 ) at the peak of EAE .

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(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.


( G ) Prostaglandin concentration ( nM ) in the supernatant of pMBMECs comparing conditions mentioned in ( A ) . n = 5 biological replicates / group and bars represent mean ± SD .

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(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.


( C ) Impact of CNS PMN - MDSC and BM - PMN on CD4 + T cell proliferation assessed by CFSE dilution using flow cytometry . PMN - MDSC were FACS - sorted from the CNS ( right panel ) and BM ( left panel ) of WT mice at the peak of EAE . NS = non stimulated . n = 4 biological replicates / group . Bars indicate mean ± SD . CNS PMN - MDSC / CD4 + T cells : representative results of 2 independent experiments ; BM - PMN : 1 experiment .

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(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.


( H ) Percentage ( upper left panel ) and absolute number ( lower left panel ) of CNS live cells CD45 + CD11b + Ly6CintLy6G + PMN - MDSC ; Percentage of live CD4 + CD44 + Ki67 + cells ( upper right panel ) and ratio ( lower right panel ) of absolute number of PMN - MDSC and proliferating CD4 + T cells in the CNS at the peak of the disease of Ch25hfl / fl ( n = 9 ) and Ch25hECKO mice ( n = 8 ) . Symbols depict individual mice and bars indicate mean ± SD . Combined results of 2 independent experiments .

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(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.


( I ) EAE disease course in Ch25hBBBKO mice ( n = 11 ) where the Cre - recombinase is expressed in endothelial cells of the CNS and Cre negative littermates ( Ch25hfl / fl : n = 7 and Ch25hfl / wt : n = 2 ) . Bars indicate mean ± SEM . Representative results of 2 independent experiments .

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(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.


( B ) Survival analysis in the same conditions as in ( A ) . Ch25hfl / fl isotype : n = 7 , Ch25hECKO isotype : n = 8 , Ch25hfl / fl Combo : n = 8 , Ch25hECKO Combo : n = 9 .

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(B) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.


( C ) Representative contour plots of PMN - MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in ( A ) . ( D ) As in ( C ) except that statistical analysis and percentage of PMN - MDSC in live cells are shown . Symbols depict individual mice and bars mean ± SD . Ch25hfl / fl Isotype n = 4 , Ch25hECKO isotype n = 4 , Ch25hfl / fl Combo , n = 3 , Ch25hECKO Combo n = 4 . (

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(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A). (D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4. (


( E ) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry . Ch25hECKO and Ch25hfl / fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE ( day 14 after immunization ) . ( F ) As in ( E ) except that statistical analysis is shown . Symbols depict individual mice , bars mean ± SD . Ch25hfl / lfl isotype n = 7 , Ch25hECKO isotype n = 5 , Ch25hfl / lfl Combo n = 6 , Ch25hECKO Combo n = 5 . The experiment was performed once . (

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(E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization). (F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once. (


Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by the MACS peak caller and ordered on the y - axis according to HIF - 1β signal intensity . Heatmaps show HIF ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity , averaged across two independent ChIP - seq experiments ) at HIF binding sites and across the flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal intensity and ( B ) HIF - 1α signal intensity at HIF - 1α binding sites and ( C ) HIF - 1β signal intensity and ( D ) HIF - 2α signal intensity at HIF - 2α binding sites . HIF - 1β signal intensity above local background levels is observed at all HIF - 1α and HIF - 2α binding sites . ( E & F ) Line plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal intensity ( solid blue lines ) within the MACS defined ( E ) HIF - 1α and ( F ) HIF - 2α binding sites compared to the average background HIF - 1β binding signal ( dashed black line ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on the x - axis according to HIF - 1β signal . HIF - 1β signal intensity at both HIF - 1α and HIF - 2α binding sites was consistently above genome - wide background levels . ( G ) HIF - 1β signal intensity was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal intensity for all sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α and HIF - 1β signal intensities . ( H ) The ratio of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site and plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) The non - reproducibility of the total HIF - α / HIF - 1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate

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

Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


For each cell line , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in RNA - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) and both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment Analysis ( ES = enrichment score , NES = normalized enrichment score )

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For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Immunoblots show progressive induction of HIF - 1α and HIF - 2α protein levels with increasing severity of

Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia


HIF - 1α ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

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HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

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(C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


Frequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . The ratio of total HIF - α signal was unimodally distributed ( purple line ) . Upper tertile HIF - α sites ( solid red line ) had a significantly ( p = 10 - 7 , Wilcox rank sum test ) higher ratio of HIF - 1β signal ( dotted red line ) and vice versa ( blue lines )

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Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the two oxygen

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Immunoblots show induction of HIF - 1α and HIF - 2α protein level , which is highest at 6 hours and falls by 48

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Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours


HIF - 1α , ( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for all canonical HIF - binding sites that bound at any of the three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between the two timepoints and no novel sites were observed at either timepoint . ( E - F ) The same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α and ( G ) HIF - 1β ChIP - seq signals at 48 hours and 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either

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HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint


Frequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . The ratio of total HIF - α signal had a unimodal distribution ( purple line ) . However , upper tertile HIF - α sites ( solid red line ) had a comparable ratio of HIF - 1β signal ( dotted red line ) to the ratio of HIF - 1β signal in the lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at ( I ) 16 hours hypoxia and at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the various

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


Cells were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α and HIF - 1β protein levels in the wild type and single clones of CRISPR - Cas9 engineered cell

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Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines


HIF - 1α ChIP - seq signal in the HIF - 2α KO cells was plotted against that in the wild - type cells for all canonical HIF - 1 binding sites in either cell

HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type


HIF - 2α ChIP - seq signal in the HIF - 1α KO cells was plotted against that in the wild - type cells for all canonical HIF - 2 binding sites in either cell type . No significant increase ( Wilcoxon rank sum test ) in binding of either isoform was observed following deletion of the

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HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other


Box - and - whisker plots showing : ( D ) The effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type cells showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank test ) decrease rather than increase ( compare red boxes ) consistent with the reduction in HIF - 1α protein observed in panel

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

Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A


The effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type cells showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank test )

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

The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)


The effect , at sites that bind both isoforms in wild - type cells , of ( F ) HIF - 2α inactivation on HIF - 1α binding intensity showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank test ) as

The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abov


The effect , at sites that bind both isoforms in wild - type cells , o HIF - 1α inactivation on HIF - 2α binding intensity showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank test )

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

The effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Canonical HIF - 1 and HIF - 2 binding sites were determined by overlap of HIF - α and HIF - 1β binding sites identified by the MACS peak caller as for Figure 4 . The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for ( A ) HIF - 1 and ( B ) HIF - 2 sites in HKC - 8 cells , ( C ) HIF - 1 and ( D ) HIF - 2 sites in RCC4 cells , ( E ) HIF - 1 and ( F ) HIF - 2 sites in HepG2 cells . The distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite the sites themselves differing between cell

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

Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types


The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) and HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared test )

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

The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Immunoblots showing HIF - 1α , HIF - 2α and HIF - 1β protein levels in HKC - 8 , HepG2 , RCC4 and MCF - 7 cell lines in normoxia and following 16 hours incubation at 0 . 5 %

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

Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen


Sites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α and both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α and both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 and HKC - 8 cells following 16 hours incubation in 0 . 5 % oxygen and from normoxic RCC4 cells and compared with previously published data from MCF - 7 cells . ( D ) The proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 cell lines is plotted . For sites that are shared between ( E ) HepG2 and HKC - 8 cells , ( F ) HKC - 8 and RCC4 cells , or ( G ) RCC4 and HepG2 cells , the ratio of HIF - 1α / HIF - 2α signal for one cell line was plotted against the other . The ratio of HIF - 1α / HIF - 2α in one cell line correlates well with that in the other . ChIP - seq signal is averaged across two independent

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

Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity ) at canonical HIF - 1 and HIF - 2 binding sites and across the flanking ± 5kb regions ( x - axis ) in HepG2 cells incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on the y - axis according to HIF signal derived from HepG2 cells incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in

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

Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia


Bar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 and HIF - 2 sites in HepG2 cells ( identified as in Figure 4 ) . TFBSs for 61 additional DNA - binding proteins were determined from ENCODE ChIP - seq data in normoxic HepG2 cells . The significance of the overlap between each set of binding sites and canonical HIF - 1 and HIF - 2 sites , was determined using a hypergeometric test . Accessible sites determined from ENCODE DNAse - seq data in HepG2 cells was used as a negative

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

Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Time lapse images of T6SS activity in tagged parental strain ( VipA - mCherry2 ) and ∆ tssA mutant background . The parental strain forms multiple dynamic structures per cell . ∆ tssA mutant predominantly forms dynamic sheath spots ( white arrow ) and few WT - like sheath structures ( blue arrow ) . Scale bars : 2 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

Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.


H2 - T6SS dynamics in tagged parental strain ( ∆ retS TssB2 - mCherry2 , referred to as WT H2 ) and ΔtssA2PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . The WT H2 strain harbors multiple dynamic sheath structures per cell , while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures . Scale bars : 2 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

H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.


Time lapse images of T6SS activity in tagged strain ( VipA - mCherry2 TssAVC - mNeonGreen ) . White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics . Scale bar : 1 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

Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.


Kymographs of dynamics observed for TssAVC . Frequency of specific dynamics is indicated in the upper right corner of the merge image . ( B ) Sheath and TssAVC dynamics from A . TssAVC localizes to T6SS initiation site and to distal end of growing sheath ( white arrow ) . ( C ) Once sheath reaches cell periphery , TssAVC dissociates from distal end ( white arrow ) and sheath stays extended a prolonged period of time . ( D ) TssAVC dissociates from growing sheath structure and re - associates at a later time point ( white arrows ) to continue catalysis of polymerization . ( E ) TssAVC stays attached to sheath distal end after contraction ( white arrow ) .

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

Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).


Fluorescence microscopy of T6SS dynamics in a double tagged strain ( ∆ retS TssB2 - mCherry2 TssA2PA - mNeonGreen ) . White arrow indicates full cycle of extending and contracting T6SS sheath . Scale bar : 1 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

Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.


Kymographs of TssB2 and TssA2PA dynamics . TssA2PA localizes at sheath initiation site and on distal end during polymerization ( G ) , dissociates before contraction ( H ) , dissociates and re - associated during sheath extension ( I ) or stays attached to distal end after contraction ( J ) .

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

Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).


Top view ( XY - plane ) and side view ( XZ - plane ) of the TssAVC Nt2 domain cryo - EM reconstruction ( pink , non transparent ) shown with fitted Nt2 - dimer model ( left ) . Partial Nt2 - CTD and Nt1 - Nt2 linker densities are highlighted with black arrows . Top and side views of ribbon diagram of Nt2 - dimer ( middle ) . Representative 2D class averages of TssAVC particles with visible connections between Nt2 - dimer and CTD ( top right ) , representative 2D class averages of Nt2 - dimer particles , Nt2 - CTD linker and Nt1 - Nt2 linker densities are highlighted with blue and yellow arrows ( bottom right ) .

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

Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).


The ∆ tagA mutant displays long and bent but still dynamic structures . Scale bar : 2 µm . Frames from time lapse movies showing sheath dynamics in the ∆ tagA mutant . Sheath polymerization does not stop when the distal end reaches opposing membrane ( white arrow ) . Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole , ultimately effecting bending of the whole structure . C . Sheath detaches from its membrane anchor ( orange arrow ) , keeps extending to maximum cell length and then contracts . Scale bars : 1 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 O O O O O 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 ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.


Time lapse series of T6SS activity in tagged strain ( VipA - mCherry2 TagAVC - mNeonGreen ) . Orange arrow indicates sheath distal end , white arrow indicates initiation site of another T6SS sheath . Scale bar : 1 µm .

Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.


Kymographs of dynamics observed for TagAVC . E . TagAVC localizes at the distal end when sheath reaches the cell periphery . Signal of TagAVC - mNeonGreen disappeared and reappeared on this site repeatedly . F . TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction . G . TagAVC briefly localizes at T6SS initiation site . H . TagAVC briefly localizes at T6SS initiation site and at distal end , where it stays attached after contraction .

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

Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.


H1 - T6SS dynamics in tagged parental strain ( ∆ retS TssB1 - mCherry2 , referred to as WT H1 ) and ΔtssA1PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . Scale bars : 2 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

H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.


Fluorescence microscopy of T6SS dynamics in ∆ retS TssB1 - mCherry2 strain harboring pPSV35 plasmid with TssA1PA - mNeonGreen fusion . Scale bar : 2 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

Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.


Kymographs of TssB1 and TssA1PA dynamics . TssA1PA localizes exclusively at sheath initiation site ( B - E ) , disappears after contraction ( C , D ) or remains in place for longer timeframe ( C , E ) . TssA1PA signal appears in neighboring cell shortly after T6SS attack ( B , C ) .

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

Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).


Representative confocal images of cell - type - specific promoter : : GFP : AGO1 translational fusions .

Representative confocal images of cell-type-specific promoter::GFP:AGO1 translational fusions.


Immunoblot analysis of FLAG : AGO1 IPed in each root - tip layer . Scale bars : 50μM , NT : Non - transformed . CB : Coomassie blue .

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

Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.


Left : input and IPed AGO1 following post - lysis addition of a radiolabeled siRNA duplex ( siGFP ) . Right : northern analysis of AGO1 - bound sRNAs .

Left: input and IPed AGO1 following post-lysis addition of a radiolabeled siRNA duplex (siGFP). Right: northern analysis of AGO1-bound sRNAs.


Layer - specific distribution of all AGO1 - loaded miRNAs .

Layer-specific distribution of all AGO1-loaded miRNAs.


Northern analysis of layer - specific AGO1 - loaded miRNAs using sRNAs IPed independently from that used for deep - sequencing . Bar - plots underneath each panel display the deep - sequencing results as normalized reads counts .

Northern analysis of layer-specific AGO1-loaded miRNAs using sRNAs IPed independently from that used for deep-sequencing. Bar-plots underneath each panel display the deep-sequencing results as normalized reads counts.


Representative confocal images of cell - type - specific promoter : : GFP transcriptional fusions in hyl1 vs WT roots .

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

Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.


Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots .

Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.


qRT - PCR validation of three distinct classes of direct miRNA target transcripts from total root - , total root polysome - or epidermis - specific polysome - associated RNA . The AT2G37050 kinase is positively regulated by LAC17 . Scale bars : 50μM . All qPCR values were normalized to the internal control ACTIN2 .

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

qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.


miRoot snapshots of whole - root ( left ) and layer - specific ( right ) LAC17 mRNA accumulation in WT versus hyl1 , as assessed by polysome profiling .

miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.


miRoot snapshot of cell - type specific miRNAs showing , in this case , a sum group of the miR156 family . The right panel shows the gradient of expression revealed upon removal of the stele ( blue box ) .

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

miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).


Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds . Removal of the stele ( blue box ) reveals subtleties of miR156 - mediated gene regulation in the other layers , resulting in a near - mirror image of ( 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 O O O O O O O

Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).


Confocal image of pmiR396a : : H2B : GFP . Normalized read counts from AGO1 - IP - Seq and independent northern analysis of AGO1 - loaded miR396 .

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

Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.


Representative confocal image of the pGRF2 : : H2B : GFP translational fusion .

Representative confocal image of the pGRF2::H2B:GFP translational fusion.


Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella . Graph : GRF2 normalized read counts .

Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.


Representative confocal images of the pGRF2 : : GRF2 : GFP translational fusion in WT or hyl1 backgrounds .

Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.


Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella .

Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.


Layer - specific normalized read counts of miR169 paralogs ( separated into 4 different groups based on sequence identity : a , b - c , d - g , h - n ) from AGO1 - IP - deep - Seq and independent northern analysis , as in Fig . 1F .

Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.


Cell - type specific AGO1 - IP signals for each miR169 sequence - group with ( upper ) or without ( lower panel ) the miRoot scale normalization option .

Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.


Predicted base - pairing of indicated miR169 sequence - group to four NF - YA target transcripts . Pairing with expectation value greater than 3 was not considered . The color gradient ( pale : low , dark : strong ) qualitatively approximates the pairing contribution of each miR169 sequence - group according to the upper panel of ( B ) . miRoot snapshots of layer - specific translatome signals of four out - of - seven miR169 NF - YA targets in WT versus hyl1 backgrounds .

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

Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B). miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.


Browser representation and independent northern analysis and AGO1 - loaded miR165 / 166 .

Browser representation and independent northern analysis and AGO1-loaded miR165/166.


miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds .

miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.


Representative confocal images of pmiR395 ( a / c / e ) : : H2B : GFP transcriptional fusions under SO4 - - deficient or SO4 - - proficient growth conditions . Northern analysis of AGO1 - loaded miR395 under the conditions in ( C ) .

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Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).


Representative confocal images of the pAPS4 : : APS4 : GFP translational fusion under the conditions in ( C ) .

Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).


Representative confocal image of the pSHR : : GFP : P19 translational fusion and ( B ) principle of P19 - based miRNA duplex sequestration .

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Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.


Binding of miR395 by SHR : : HA : P19 under SO4 - - sufficient ( + ) and - deficient ( - ) growth conditions .

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Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.


qRT - PCR analysis of polysome - associated APS4 in the cortex or epidermis in the presence ( + ) or absence ( - ) of SHR : : HA : P19 . Signals are relative to SHR : : HA : P19 ( - ) lines given a value of 1 .

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qRT-PCR analysis of polysome-associated APS4 in the cortex or epidermis in the presence (+) or absence (-) of SHR::HA:P19. Signals are relative to SHR::HA:P19 (-) lines given a value of 1.


Representative confocal image of the pmiR397b : : H2B : GFP transcriptional fusion ( left ) and the browser AGO1 - loaded signal for miR397b ( right ) .

Representative confocal image of the pmiR397b::H2B:GFP transcriptional fusion (left) and the browser AGO1-loaded signal for miR397b (right).


Cross section of Arabidopsis leaf showing cell - types isolated via Meselect .

Cross section of Arabidopsis leaf showing cell-types isolated via Meselect.


miR160 northern analysis in whole - leaf , vasculature and epidermis . U6 : loading control .

miR160 northern analysis in whole-leaf, vasculature and epidermis. U6: loading control.


Western analysis of the promoter : : H2B : GFP translational fusions shown in ( 5C ) in the cell types isolated in ( G ) .

Western analysis of the promoter::H2B:GFP translational fusions shown in (5C) in the cell types isolated in (G).


qRT - PCR analysis of ARF17 in whole - leaves or in separated cell types in WT versus hyl1 backgrounds .

qRT-PCR analysis of ARF17 in whole-leaves or in separated cell types in WT versus hyl1 backgrounds.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . a , Samples were processed for western blot using anti - Rad53 , Srs2 and Ddc2 antibodies .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and − VPA . b , A schematic diagram showing probe locations with respect to the HO cut site . The resection rate was calculated as the rate of HO cut band disappearance .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance.


a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . c , Fold enrichment of the 0 . 2 - kb fragment was calculated after ChIP of Rfa1 - Flag , Ddc2 - Myc .

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a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc.


d , VPA effect on ectopic recombination ( rec . ) in wild - type ( WT ) cells . Error bars represent standard deviation ( s . d . ) calculated from four independent experiments .

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d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.


a , MRE11 : : MYC cells were treated as in Fig . 1a . Cell samples were processed for ChIP analysis and the fold enrichment of the 0 . 2 - kb fragment after ChIP of Mre11 - Myc without ( - VPA ) or with ( + VPA ) VPA was calculated .

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a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated.


b , EXO1 : : FLAG SAE2 : : PK MRE11 : : MYC cells were grown as in a . Cell samples were taken and processed for western blot analysis using anti - Flag , PK and Myc antibodies .

b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.


a , Cherry : : APE1GFP : : ATG8 cells were grown and shifted to YPD , nitrogen starvation ( SD - N ) or YPD + VPA medium for 3 h . Samples were processed for microscopy . The table shows numbers corresponding to the experiment . Percentage of fluorescence signals is presented and error bars represent the s . d . obtained from three independent experiments . DIC , differential interference contrast . Scale bars , 3 µm .

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a, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm.


b , pho8Δ60 and pho8Δ60 atg1Δ ; cells were grown as in a . Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels . Error bars represent s . d . calculated from five independent experiments .

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b, pho8Δ60 and pho8Δ60 atg1Δ; cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments.


c , GFP : : ATG8 and GFP : : ATG8atg1Δ ; cells were grown as in a . Cell samples were processed for western blot using anti - GFP antibody . Quantification is presented in Supplementary Fig . 3 .

c, GFP::ATG8 and GFP::ATG8atg1Δ; cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.


a , 4HA - Sae2 was immunoprecipitated ± VPA with anti - HA and subsequently ± anti - acetyl - Lysine . Eluate was analysed using anti - HA . Lane 1 : input Sae2 ; 2 : input Sae2 - HA - VPA ; 3 : as in 2 but + VPA ; 4 : 3 µl elution Sae2 - HA - VPA after anti - HAimmunoprecipitation ( IP ; input AcK - IP ) ; 5 : double amount of 4 ; 6 : IP anti - AcK elution from anti - HA IP of Sae2 - HA - VPA ; 7 : as in 6 but + VPA .

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a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA.


b , SAE2 : : PKerg6Δ cells were treated as in Fig . 2a . After 30 min induction , VPA and PMSF were added or not . Samples were processed for western blot using anti - PK .

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b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK.


c , Wild - type SAE2 : : PK , SAE2 : : PKatg1Δ and SAE2 : : PKatg19Δ cells were grown as in b . After 30 min induction , VPA was added or not and samples treated as in b .

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c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b.


d , Wild - type SAE2 : : PK cells were grown as in b . After 120 min induction , rapamycin ( 200 ng ml - 1 ) was added or not and samples were treated as in b .

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d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.


a , Survival of wild - type , rpd3Δ , hda1Δ and rpd3Δ hda1Δ strains after 4NQO , MMS and HU treatment . Error bars represent s . d . calculated from seven independent experiments .

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a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments.


b , HO was induced in G2 wild - type , hda1Δ , rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed .

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b, HO was induced in G2 wild-type, hda1Δ, rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed.


c , Wild - type SAE2 : : PK and hda1Δ rpd3Δ SAE2 : : PK cells were grown as in b and western blot was performed as in Fig . 4b .

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c, Wild-type SAE2::PK and hda1Δ rpd3Δ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b.


d , e , Wild - type SAE2 : : PK and gcn5Δ SAE2 : : PK strains were grown as in b and after 30 min of HO induction either VPA ( d ) or rapamycin ( e ) was added or not . Western blot was performed .

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d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed.


f , Percentage of viability of wild - type , atg1Δ , rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains . Error bars represent s . d . calculated from four independent experiments .

f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains. Error bars represent s.d. calculated from four independent experiments.


Immunofluorescence labeling of QSOX1 and a Golgi marker ( GM130 ) in epithelial cells isolated from murine colon . QSOX1 is green , GM130 is red , and DAPI staining of nuclei is blue in the merged image . Scale bar is 10 µm .

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Immunofluorescence labeling of QSOX1 and a Golgi marker (GM130) in epithelial cells isolated from murine colon. QSOX1 is green, GM130 is red, and DAPI staining of nuclei is blue in the merged image. Scale bar is 10 µm.


Top , average colon length of WT and QSOX1 KO mice following indicated treatments . For untreated mice , colon lengths were calculated for 3 WT and 3 KO . For treated mice , lengths were calculated from 5 mice except for the DSS - treated WT , for which 3 mice were analyzed . Error bars are standard deviation . Statistical analysis was performed using Tukey multiple comparison of means ( * * p < 0 . 01 ) . Administration of QSOX1 inhibitory antibody MAb316 . 1 ( αQSOX1 ) , did not enhance the sensitivity of WT colons to DSS ( p > 0 . 4 for MAb316 . 1 - treated WT vs . WT control antibody ( IgG ) - treated ; p > 0 . 5 for MAb316 . 1 - treated WT vs . WT without antibody treatment ) . Bottom , representative images of colons following DSS treatment . Scale bar is 1 cm .

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Top, average colon length of WT and QSOX1 KO mice following indicated treatments. For untreated mice, colon lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice except for the DSS-treated WT, for which 3 mice were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1 inhibitory antibody MAb316.1 (αQSOX1), did not enhance the sensitivity of WT colons to DSS (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS treatment. Scale bar is 1 cm.


Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well - packed goblet cell granules in WT and QSOX1 KO colons . Scale bar is 5 µm .

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Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well-packed goblet cell granules in WT and QSOX1 KO colons. Scale bar is 5 µm.


Alcian blue and PAS staining of reduced guanidine - insoluble mucins from WT and QSOX1 KO mice , separated on a 6 % polyacrylamide gel . Data information : each lane corresponds to a sample from one mouse .

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Alcian blue and PAS staining of reduced guanidine-insoluble mucins from WT and QSOX1 KO mice, separated on a 6% polyacrylamide gel. Data information: each lane corresponds to a sample from one mouse.


Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels . Data information : each lane corresponds to a sample from one mouse .

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Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.


Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels . Data information : each lane corresponds to a sample from one mouse .

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Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.


QSOX1 immunofluorescence ( green ) in control ( siControl ) and QSOX1 knockdown ( siQSOX1 ) MDA - MB - 231 cells . Blue is DAPI staining of nuclei . Scale bar is 20 µm . Data information : The results described in are representative of 3 experiments that were performed .

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QSOX1 immunofluorescence (green) in control (siControl) and QSOX1 knockdown (siQSOX1) MDA-MB-231 cells. Blue is DAPI staining of nuclei. Scale bar is 20 µm. Data information: The results described in are representative of 3 experiments that were performed.


Western blot analysis of the MUC2 N - terminal region and indicated cysteine mutants in supernatants of transfected cell cultures . No differences in disulfide - mediated dimerization were observed for any MUC2 variant between siC ( siControl ) and siQ ( siQSOX1 ) . Data information : The results described in G are representative of 3 experiments that were performed .

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Western blot analysis of the MUC2 N-terminal region and indicated cysteine mutants in supernatants of transfected cell cultures. No differences in disulfide-mediated dimerization were observed for any MUC2 variant between siC (siControl) and siQ (siQSOX1). Data information: The results described in G are representative of 3 experiments that were performed.


Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice . Lysates were treated with PEG - mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1 . As for panel A but using antibodies to St3gal1 . As for panel A but using antibodies to B3galt5 . Data information : For the experiments in panels A - C , equal amounts of total protein were applied to each gel lane , and no reducing agent was added . Experiments were repeated 3 times with fresh new lysates , and representative blots are shown .

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Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG-mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1. As for panel A but using antibodies to St3gal1. As for panel A but using antibodies to B3galt5. Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.


QSOX1 oxidizes St6gal1 in vitro . MAb492 . 1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG - mal additions ( 2PEG ) , whereas protein with two pairs of free cysteines acquires four PEG - mal additions ( 4PEG ) . The change in migration per two PEG - mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs . protein QSOX1 oxidation of St6gal1 mutants . The C350 - C361 disulfide ( the redox - active disulfide present in the C139A - C403A mutant ) is oxidized rapidly , while the C142 - C406 disulfide ( present in the C350A - C361A mutant ) is oxidized slowly . Data information : Experiments were repeated 3 times with fresh new lysates , and representative blots are shown .

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QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein QSOX1 oxidation of St6gal1 mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly. Data information: Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.