J , Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C - deprivation . data are means ± s . d . of 3 biological replicates .

J, Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C-deprivation. data are means ± s.d. of 3 biological replicates.


C , ChIP - qPCR analysis of ARF2 - GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4 - 2 pif5 - 10 backgrounds . In C fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4-2 pif5-10 backgrounds. In C fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


D , RT - qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , pif4 - 2 pif5 - 10 , and pARF2 : ARF2 - GFP OEs in the pif4 - 2 pif5 - 10 background before and after 4 d C - deprivation . Fold changes were calculated with respect to the expression level in the WT before C - deprivation . In D data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. In D data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


E , ChIP - qPCR analysis of PIF5 - GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2 - 20 backgrounds . In fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, ChIP-qPCR analysis of PIF5-GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2-20 backgrounds. In fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


F , RT - qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background before and after 4 d C - deprivation . Fold changes were calculated as in D . In F , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 4 d C-deprivation. Fold changes were calculated as in D. In F, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


G , Hypocotyl elongation in seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background . Seedlings were grown under continuous light for one week .

O O B-TISSUE O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O

G, Hypocotyl elongation in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background. Seedlings were grown under continuous light for one week.


H , RT - qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G . Fold changes were calculated with respect to the expression level in the WT . Color legends in H are the same as in F . In H , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RT-qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G. Fold changes were calculated with respect to the expression level in the WT. Color legends in H are the same as in F. In H, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


J , ChIP - qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2 - 20 backgrounds . In J , fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, ChIP-qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2-20 backgrounds. In J, fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


A , RT - qPCR analysis of ARF2 expression levels in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , mateq , and pARF2 : ARF2 - GFP OEs in the mateq background . Fold changes were calculated with respect to the expression level in the WT . B , RT - qPCR analysis of PIF5 expression levels in 7 - d - old seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , mateq , and pPIF5 : PIF5 - GFP OEs in the mateq background . Fold changes were calculated as in A . In A , B , data are means ± s . d . of 3 biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O 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, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, mateq, and pARF2:ARF2-GFP OEs in the mateq background. Fold changes were calculated with respect to the expression level in the WT. B, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, mateq, and pPIF5:PIF5-GFP OEs in the mateq background. Fold changes were calculated as in A. In A, B, data are means ± s.d. of 3 biological replicates.


C , Seedlings of the same genotypes as in A before and after 6 d and 9 d C - deprivation . D , Seedlings of the same genotypes as in B before and after 6 d and 11 d C - deprivation .

O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

C, Seedlings of the same genotypes as in A before and after 6 d and 9 d C-deprivation. D, Seedlings of the same genotypes as in B before and after 6 d and 11 d C-deprivation.


E and F , Chlorophyll and total cellular protein content reduction in genotypes shown in A ( E ) after 6 d and 9 d C - deprivation and in genotypes shown in B ( F ) after 6 d and 11 d C - deprivation . Color legends in E are the same as in A . Color legends in F are the same as in B . data are means ± s . d . of 3 biological replicates .

O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) after 6 d and 9 d C-deprivation and in genotypes shown in B (F) after 6 d and 11 d C-deprivation. Color legends in E are the same as in A. Color legends in F are the same as in B. data are means ± s.d. of 3 biological replicates.


G and H , RT - qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A ( G ) and B ( H ) before and after 4 d C - deprivation . Fold changes were calculated with respect to the expression level in the WT before C - deprivation . Color legends in G are the same as in A . Color legends in are the same as in B . data are means ± s . d . of 3 biological replicates .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. Color legends in G are the same as in A. Color legends in are the same as in B. data are means ± s.d. of 3 biological replicates.


I , RT - qPCR analysis of PIL1 and YUC8 expression levels in 7 - d - old seedlings of the same genotypes as shown in B . Fold changes were calculated as in A . Color legends in I are the same as in B . data are means ± s . d . of 3 biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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, RT-qPCR analysis of PIL1 and YUC8 expression levels in 7-d-old seedlings of the same genotypes as shown in B. Fold changes were calculated as in A. Color legends in I are the same as in B. data are means ± s.d. of 3 biological replicates.


B , ChIP - qPCR analysis of ARF2 - GFP binding to the promoters of ABS3 . Fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 4 technical replicates . The experiment has been independently repeated 3 times with similar results .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ABS3. Fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 4 technical replicates. The experiment has been independently repeated 3 times with similar results.


C , pABS3 : GFP or pABS3 ∆ S2 : GFP was expressed in WT mesophyll protoplasts with or without p35S : ARF2 - HA . p35S : mCherry was co - transfected and served as a transfection control . Protoplasts were examined with fluorescence microscopy . Experiments were independently repeated 3 times with similar results . Bars , 100 μm . D , Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3 : GFP with or without p35S : ARF2 - HA . Data are median with interquartile range , n is indicated in parentheses , two - tailed Mann - Whitney test , n . s . , not significant , * * * * , p < 0 . 0001 .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-TISSUE B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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, pABS3:GFP or pABS3∆S2:GFP was expressed in WT mesophyll protoplasts with or without p35S:ARF2-HA. p35S:mCherry was co-transfected and served as a transfection control. Protoplasts were examined with fluorescence microscopy. Experiments were independently repeated 3 times with similar results. Bars, 100 μm. D, Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3:GFP with or without p35S:ARF2-HA. Data are median with interquartile range, n is indicated in parentheses, two-tailed Mann-Whitney test, n.s., not significant, ****, p<0.0001.


E , RT - qPCR analysis of ABS3 expression levels in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , pif4 - 2 pif5 - 10 , and pARF2 : ARF2 - GFP OEs in the pif4 - 2 pif5 - 10 background . Fold changes were calculated with respect to the expression level in the WT . Data are means ± s . d . of 3 biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O 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, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background. Fold changes were calculated with respect to the expression level in the WT. Data are means ± s.d. of 3 biological replicates.


( A ) Phenotypes of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on low - K + ( LK , 50 μM ) or high - K + ( HK , 5 mM ) medium for 7 d . Scale bar , 1 cm .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.


( B ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( D ) RT - qPCR analysis of KUP9 transcript levels in various materials . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 individual plants ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O

(D) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants).


( E ) and ( F ) K + content of the various plant materials tested in ( A ) . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 - 150 individual plants ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-ORGANISM O O O 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-ORGANISM O O

(E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).


( A ) Root meristem zones of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on LK or HK medium for 7 d . The meristem zone lengths are marked with red lines . Scale bars , 50 μm .

O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O

(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.


( B ) , ( C ) and ( D ) represent different zone lengths , different zone cell numbers , and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d . MEZ , meristem zone ; EZ , elongation zone ; MAZ , maturation zone . Data are means ± SE ( n = 10 - 15 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B), (C) and (D) represent different zone lengths, different zone cell numbers, and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d. MEZ, meristem zone; EZ, elongation zone; MAZ, maturation zone. Data are means ± SE (n = 10-15, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( A ) to ( F ) Expression of KUP9 as determined in the ProKUP9 : GUS line . GUS staining of 5 - day - old seedling ( A ) , root maturation zone ( B ) , transverse section of maturation zone ( C ) and ( D ) , primary root tip ( E ) , and root apex ( F ) . Ep , epidermis ; Co , cortex ; En , endodermis ; Pe , pericyle ; Xy , xylem ; Ph , phloem . Scale bars , 5 mm in ( A ) , 1 mm in ( B ) , 10 μm in ( C ) , 5 μm in ( D ) , 1 mm in ( E ) , and 20 μm in ( F ) .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F).


( G ) and ( H ) Expression of KUP9 protein as determined in the kup9 - 1 / ProKUP9 : KUP9 - GFP transgenic plants . Green fluorescence shows the subcellular localization of KUP9 - GFP . PI ( propidium iodide ) and ER tracker were used to indicate the positions of the plasma membrane ( PM ) and endoplasmic reticulum ( ER ) and are shown as red and cyan fluorescence , respectively . Scale bars for root tip , 10 μm in ( G ) and 5 μm in ( H ) . Scale bars for EZ and MAZ , 20 μm .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O

(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.


( I ) Co - localization analyses of KUP9 - GFP with ER marker NIP1 ; 1 - mCherry in their crossing line . Scale bars , 20 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

(I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm.


( J ) Co - localization analyses of KUP9 - GFP with the ER marker BIP2 using immunological staining . Scale bars , 5 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.


( A ) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5 : GFP and ProQC25 : GUS . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bars , 10 μm .

O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm.


( B ) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 150 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O 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-ORGANISM O O O O O O O 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) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( A ) Auxin levels in root tips , as indicated by the ProDR5 : GFP reporter line . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bar , 10 μm . ( B ) Quantification of GFP fluorescence in the QC cells shown in ( A ) . Data are means ± SE ( n = 20 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM 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) Auxin levels in root tips, as indicated by the ProDR5:GFP reporter line. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bar, 10 μm. (B) Quantification of GFP fluorescence in the QC cells shown in (A). Data are means ± SE (n = 20, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( A ) Phenotypes of various plants . Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d . Scale bar , 1 cm .

O O O B-EXP_ASSAY O O B-ORGANISM O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(A) Phenotypes of various plants. Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d. Scale bar, 1 cm.


( C ) QC cell observation using the QC marker line ProWOX5 : GFP . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium with or without NAA ( 100 nM ) for 96 h . Scale bar , 10 μm .

O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(C) QC cell observation using the QC marker line ProWOX5:GFP. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium with or without NAA (100 nM) for 96 h. Scale bar, 10 μm.


( D ) QC division rates of the plants tested The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM 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-ORGANISM O O O O O O O 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) QC division rates of the plants tested The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProWOX5 : AMI1 ( A ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

B-TISSUE I-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProKUP9 : AMI1 ( C ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


( A ) Expression of KUP9 - GFP in BY - 2 tobacco cells . HDEL - mCherry was used as an ER marker . Scale bar , 10 μm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O

(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm.


( B ) 3H - IAA accumulation in the BY - 2 cells expressing the indicated proteins . PIN4 - GFP and KUP10 - GFP were used as positive control and negative control , respectively . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 3 - 4 mL suspension cells ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( C ) Expression of KUP9 - GFP and OST4 - GFP - KUP9 in Xenopus oocytes . Scale bar , 100 μm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE O O O O O O O

(C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm.


( D ) 3H - IAA accumulation in the oocytes expressing KUP9 - GFP and PIN4 at the indicated times . PIN4 was used as a positive control . The oocytes were incubated in the bath solution containing 3H - IAA for the indicated times . Data are means ± SE ( n = 6 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).


( E ) IAA content in the oocytes expressing the indicated proteins . The oocytes were injected with the same amount of IAA and incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( F ) K + efflux activity of the oocytes expressing KUP9 and NRT1 . 5 . The oocytes were not injected with IAA . The oocytes were incubated in K + - free bath solution for 6 h . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O 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-CELL_TYPE O O

(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( G ) K + efflux activity of the oocytes expressing the indicated proteins . The oocytes were injected with H2O , IAA , or BA . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( H ) K + efflux activity of the oocytes expressing the indicated proteins . IAA was injected into the oocytes . Then the oocytes were incubated in K + - free bath solutions at different pH for 6 h . Data are means ± SE ( n = 3 - 6 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O 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-CELL_TYPE O O

(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).


( I ) IAA efflux activity of oocytes expressing the indicated proteins under different external K + conditions ( - K , 0 mM ; + K , 50 mM ) . The oocytes were injected with the same amount of IAA , and then incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O 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) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( A ) K + levels of various materials using the fluorescent dye Asante Potassium Green - 2 ( AM ) . Scale bar , 50 μm . ( B ) Quantification of K + concentration under LK and HK conditions . Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O 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) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm. (B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( C ) to ( E ) The contents of free IAA ( C ) , IA - Asp ( D ) , and IA - Glu ( E ) in the root tips of wild type and the kup9 - 1 mutant . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 1000 - 1200 individual plants ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD 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-ORGANISM O O O O O O O O O O O O O O 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) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.


( F ) Phenotype of the kup9 - 1 / ProKUP9 : PIN8 transgenic lines . Seeds were germinated and grown on LK or HK medium for 7 d . Scale bar , 1 cm .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

(F) Phenotype of the kup9-1/ProKUP9:PIN8 transgenic lines. Seeds were germinated and grown on LK or HK medium for 7 d. Scale bar, 1 cm.


( G ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( H ) QC division rates of the indicated plants . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(H) QC division rates of the indicated plants. The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance


( A ) Beclin 1 ( BCN1 ) interactors identified by yeast two ‐ hybrid technology . Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars . Numbers refer to amino ‐ acid positions .

O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) Beclin 1 (BCN1) interactors identified by yeast two‐hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino‐acid positions.


( B ) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two ‐ hybrid system . Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 ( numbers on the right refer to the amount of yeast clones identified for each fragment ) . The minimal domain required for the interaction is referred to as Beclin ‐ binding domain ( BBD ) .

O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two‐hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin‐binding domain (BBD).


( C ) Constructs derived from TAB2 and TAB3 used in this study .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O

(C) Constructs derived from TAB2 and TAB3 used in this study.


( D , E ) Co ‐ immunoprecipitation of TAB2 or TAB3 with BCN1 . The indicated constructs , namely HA ‐ tagged and T7 ‐ tagged TAB2 , and TAB3 constructs in ( D ) and ( E ) , respectively , and Flag ‐ tagged Beclin 1 ( Flag - BCN1 ) were transfected into HeLa cells alone or in combination . Twenty ‐ four hours later , TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA ( D ) or T7 ( E ) and the precipitate was separated by SDS - PAGE and revealed with an antibody specific for Flag .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O

(D, E) Co‐immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA‐tagged and T7‐tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag‐tagged Beclin 1 (Flag-BCN1) were transfected into HeLa cells alone or in combination. Twenty‐four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS-PAGE and revealed with an antibody specific for Flag.


( F ) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3 . HeLa cells were subjected to autophagy induction with starvation conditions , 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1 , TAK1 , TAB2 and TAB3 . Results in ( E ) and ( F ) are representative for three independent experiments .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

(F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.


( A , B ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa cells were co ‐ transfected with a GFP - LC3 ‐ encoding construct plus pcDNA3 . 1 ( empty vector ) , or plasmids for the expression of WT TAK1 ( TAK1WT ) or the DN TAK1K63W mutant . One day later , cells were either left untreated ( control ) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) , followed by immunoblotting for the detection of TAK1 and endogenous LC3 ( A ) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP - LC3 puncta ( GFP - LC3VAC cells ) ( B ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control cells ) . GAPDH levels were monitored to ensure equal loading .

O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP-LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP-LC3 puncta (GFP-LC3VAC cells) (B) (mean values±s.d., n=3; *P0.01 versus control cells). GAPDH levels were monitored to ensure equal loading.


( C , D ) Inhibition of autophagy by knockdown of VPS34 , Beclin 1 ( BCN1 ) and TAK1 . siRNAs that effectively deplete VPS34 , BCN1 and TAK1 were co ‐ transfected with a GFP - LC3 ‐ encoding plasmid in HeLa cells . Autophagy was then induced as in ( A ) and the frequency of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control cells ) was determined .

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(C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co‐transfected with a GFP-LC3‐encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus control cells) was determined.


( E , F ) The same setting shown in ( C , D ) was performed with U2OS cells and FYVE - RFP ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus control cells ) .

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(E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE-RFP (mean values±s.d., n=3; *P0.01, **P0.001 versus control cells).


( A , B ) Detection of autophagic GFP - LC3 + puncta . HeLa or U2OS cells stably expressing GFP - LC3 were transfected with siRNAs targeting TAK1 , TAB1 , TAB2 or TAB3 or with a control siRNA ( siUNR ) . One day later , the subcellular localization and abundance of GFP - LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy . Representative images are shown in ( A ) ( HeLa cells ) and quantitative results ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) are depicted in ( B ) ( U2OS cells ) .

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(A, B) Detection of autophagic GFP-LC3+ puncta. HeLa or U2OS cells stably expressing GFP-LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP-LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells) are depicted in (B) (U2OS cells).


( C ) Lipidation of LC3 induced by TAB2 or TAB3 knockdown . Representative immunoblots showing the conversion of non ‐ lipidated LC3 ( LC3 ‐ I ) to its lipidated variant ( LC3 ‐ II ) as well as SQSTM1 / p62 protein levels are shown . GAPDH levels were monitored to ensure equal loading .

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(C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non‐lipidated LC3 (LC3‐I) to its lipidated variant (LC3‐II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading.


( D , E ) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy . Representative images of HeLa cells transfected with siUNR or with TAB2 ‐ or TAB3 ‐ targeting siRNAs are shown in ( D ) , and quantitative results are depicted in ( E ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2‐ or TAB3‐targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells).


( F ) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with siRNAs specific for TAB2 or TAB3 and / or with cDNAs coding for full ‐ length HA ‐ tagged TAB2 ( HA - TAB2 ) or TAB3 ( HA - TAB3 ) . Twenty ‐ four hours later , the frequency of cells exhibiting > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full‐length HA‐tagged TAB2 (HA-TAB2) or TAB3 (HA-TAB3). Twenty‐four hours later, the frequency of cells exhibiting >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( G ) U2OS cells stably expressing FYVE - RFP were transfected with siUNR , or with siRNAs specific for TAB2 , TAB3 , VPS34 , Beclin 1 ( BCN1 ) and TAK1 , in the indicated combinations . Forty ‐ eight hours later , the percentage of cells with RFP - FYVE + puncta cells was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(G) U2OS cells stably expressing FYVE-RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty‐eight hours later, the percentage of cells with RFP-FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( A - D ) Impact of bafilomycin A1 ( BafA1 ) on the induction of GFP - LC3 + puncta by TAB2 and TAB3 depletion . HeLa cells stably expressing GFP - LC3 were transfected with a control siRNA ( siUNR ) or with siRNAs targeting TAB2 and TAB3 for 24 h . During the last 12 h of this period , BafA1 was optionally added . After fixation and permeabilization , LAMP2 was detected by immunofluorescence . Representative confocal microphotographs for the TAB2 siRNA are shown ( A ) , together with the profiles of colocalization of fluorescent signals ( B ) along the indicated direction ( α - ω ) . Columns in ( C ) represent the percentage of colocalization of GFP - LC3 and LAMP2 ( mean values ± s . d . ; * P0 . 01 versus siUNR ‐ transfected cells ) , as quantified in at least 50 cells for each condition . The frequency ( mean ± s . d . ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VACcells ) is plotted in ( D ) .

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(A-D) Impact of bafilomycin A1 (BafA1) on the induction of GFP-LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α-ω). Columns in (C) represent the percentage of colocalization of GFP-LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VACcells) is plotted in (D).


( E ) Impact of BafA1 on LC3 lipidation . MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3 ‐ I / LC3 ‐ II was determined by immunoblotting . GAPDH levels were monitored to ensure equal loading .

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(E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading.


( F , G ) Impact of autophagy ‐ relevant proteins and of the TAK1 ‐ IKK signalling axis on GFP - LC3 aggregation induced by the depletion of TAB2 or TAB3 . HeLa cells stably expressing GFP - LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins , alone or in combination , and 48 h later GFP - LC3VAC cells were quantified ( mean values ± s . d . , n = 4 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(F, G) Impact of autophagy‐relevant proteins and of the TAK1‐IKK signalling axis on GFP-LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP-LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP-LC3VAC cells were quantified (mean values±s.d., n=4; *P0.01 versus siUNR‐transfected cells).


( H ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa cells stably expressing GFP - LC3 were co ‐ transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding WT ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) together with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells )

O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O 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) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells stably expressing GFP-LC3 were co‐transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells)


( A ) Effects of full ‐ length TAB2 and TAB3 or their deletion mutants ( as in Figure 1C ) on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h , then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for 4 h . Finally , the frequency ( mean ± s . d . , n = 3 ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was assessed ( * P0 . 01 versus control cells transfected with the same plasmid ; # P0 . 01 versus pcDNA3 . 1 ‐ transfected cells treated with the same pro ‐ autophagic trigger ) .

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(A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency (mean±s.d., n=3) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (*P0.01 versus control cells transfected with the same plasmid; #P0.01 versus pcDNA3.1‐transfected cells treated with the same pro‐autophagic trigger).


( B ) U2OS cells stably expressing FYVE - RFP were co ‐ transfected with pcDNA3 . 1 or with vectors encoding TAB2C or TAB3C together with a control siRNA ( siUNR ) or with siRNAs specific for VPS34 , Beclin 1 ( BCN1 ) or TAK1 for 24 h , followed by the quantification of cells exhibiting FYVE - RFP + dots ( FYVE - RFP + ) . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(B) U2OS cells stably expressing FYVE-RFP were co‐transfected with pcDNA3.1 or with vectors encoding TAB2C or TAB3C together with a control siRNA (siUNR) or with siRNAs specific for VPS34, Beclin 1 (BCN1) or TAK1 for 24 h, followed by the quantification of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+). Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( C ) Inhibition of the interaction between endogenous TAB2 , TAB3 and BCN1 by C ‐ terminal fragments of TAB2 and TAB3 . Forty ‐ eight hours after transfection with pcDNA3 . 1 or plasmids coding for the indicated proteins , cells were lysed , TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected . GAPDH levels were monitored to ensure equal loading . This experiment has been done three times , yielding comparable results .

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(C) Inhibition of the interaction between endogenous TAB2, TAB3 and BCN1 by C‐terminal fragments of TAB2 and TAB3. Forty‐eight hours after transfection with pcDNA3.1 or plasmids coding for the indicated proteins, cells were lysed, TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected. GAPDH levels were monitored to ensure equal loading. This experiment has been done three times, yielding comparable results.


( D ) Mechanisms of autophagy induction by the Beclin ‐ binding domain ( BBD ) ‐ containing C ‐ terminal fragments of TAB2 and TAB3 . HeLa cells were transfected with pcDNA3 . 1 , TAB2C ‐ or TAB3C ‐ encoding plasmids in combination with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(D) Mechanisms of autophagy induction by the Beclin‐binding domain (BBD)‐containing C‐terminal fragments of TAB2 and TAB3. HeLa cells were transfected with pcDNA3.1, TAB2C‐ or TAB3C‐encoding plasmids in combination with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( A ) Determination of the TAB ‐ binding domain ( TBD ) within Beclin 1 ( BCN1 ) . Yeast two ‐ hybrid technology was used to screen for positive ( + ) or negative interactions ( − ) between BCN1 fragments and full ‐ length TAB2 .

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(A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2.


( C ) Confirmation of the TBD by immunoprecipitation . The indicated constructs were transfected into HeLa cells , followed by lysis , immunoprecipitation of HA ‐ tagged full ‐ length TAB2 or TAB3 and detection of His ‐ tagged constructs .

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(C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs.


( D , E ) Inhibition of the interaction between endogenous BCN1 , TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD . HeLa cells were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the TBD or a His - tagged Beclin 1 variant lacking the TBD ( His - BCN1ΔTBD ) , followed by co ‐ immunoprecipitation of endogenous TAB2 ( D ) or TAB3 ( E ) and detection of BCN1 .

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(D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His-tagged Beclin 1 variant lacking the TBD (His-BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1.


( F ) Induction of autophagy by the TBD . HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or constructs encoding the indicated BCN1 variants . After 24 h , Flag ‐ tagged proteins were detected by immunoblotting and the frequency of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was assessed ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus pcDNA3 . 1 ‐ transfected cells ) .

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(F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells).


( G , H ) Mechanisms of autophagy induction by the TBD . ( G ) U2OS cells stably expressing RFP - FYVE were transfected pcDNA3 . 1 or plasmids for the expression of TBD , BCN1 or BCN1ΔTBD in combination with a control siRNA ( siUNR ) or siRNAs that effectively deplete VPS4 , BCN1 and TAK1 . Forty ‐ eight hours later , the percentage of cells exhibiting FYVE - RFP + dots ( FYVE - RFP + ) was assessed . ( G ) . Alternatively , HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or with a TBD ‐ encoding plasmids plus siUNR or siRNAs specific the indicated proteins . Forty ‐ eight hours later , the percentage of GFP - LC3VAC cells was determined ( H ) . Results are mean values ± s . d . ( n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(G, H) Mechanisms of autophagy induction by the TBD. (G) U2OS cells stably expressing RFP-FYVE were transfected pcDNA3.1 or plasmids for the expression of TBD, BCN1 or BCN1ΔTBD in combination with a control siRNA (siUNR) or siRNAs that effectively deplete VPS4, BCN1 and TAK1. Forty‐eight hours later, the percentage of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+) was assessed. (G). Alternatively, HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or with a TBD‐encoding plasmids plus siUNR or siRNAs specific the indicated proteins. Forty‐eight hours later, the percentage of GFP-LC3VAC cells was determined (H). Results are mean values±s.d. (n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( I ) Inhibition of TBD ‐ induced autophagy by dominant ‐ negative ( DN ) TAK1 . Cells were co ‐ transfected pcDNA3 . 1 or vectors encoding WT TAK1 ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) alone or together with a TBD ‐ encoding plasmid for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus pcDNA3 . 1 ‐ transfected cells ) .

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(I) Inhibition of TBD‐induced autophagy by dominant‐negative (DN) TAK1. Cells were co‐transfected pcDNA3.1 or vectors encoding WT TAK1 (TAK1WT) or a DN TAK1 variant (TAK1K63W) alone or together with a TBD‐encoding plasmid for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus pcDNA3.1‐transfected cells).


Comparison of SWATH - MS - based quantification to DDA - MS - based strategies ( MS1 XIC and spectral counting ) with regard to consistency ( as judged based upon protein - level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections ) and precision ( judged based on the correlation between sibling peptide SEC chromatograms ) of quantification and overall performance in error - controlled complex - centric query of CORUM complexes in the respective protein level chromatogram sets ( also see Appendix Figure S3C ) . TP , true positive assignments according to error model , P , all positives .

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Comparison of SWATH-MS-based quantification to DDA-MS-based strategies (MS1 XIC and spectral counting) with regard to consistency (as judged based upon protein-level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections) and precision (judged based on the correlation between sibling peptide SEC chromatograms) of quantification and overall performance in error-controlled complex-centric query of CORUM complexes in the respective protein level chromatogram sets (also see Appendix Figure S3C). TP, true positive assignments according to error model, P, all positives.


Peptide - level SEC chromatograms are grouped by UniprotKB identifier to detect co - elution signals indicative of protein elution ranges / peaks . Based on external size calibration of the apparent analyte molecular weight per SEC fraction , signals can be attributed to likely assembled or monomeric state ( also see Appendix Figure S4 ) . For TCPE , three distinct elution signals , numbered 1 - 3 , are detected , two in the assembled and one in the monomer elution range .

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Peptide-level SEC chromatograms are grouped by UniprotKB identifier to detect co-elution signals indicative of protein elution ranges/peaks. Based on external size calibration of the apparent analyte molecular weight per SEC fraction, signals can be attributed to likely assembled or monomeric state (also see Appendix Figure S4). For TCPE, three distinct elution signals, numbered 1-3, are detected, two in the assembled and one in the monomer elution range.


Proteins are observed eluting in 1 - 6 distinct peaks and with a wide range of apparent vs . monomeric molecular weight ratios ( distributions , left panels ) . The molecular weight ratios of the three peaks detected for TCPE ( displayed in A ) are indicated . Many of the proteins eluting in a single peak ( top panel and bar ) appear assembled . Proteins eluting multiple times ( 27 % of the proteins ) do so preferentially in the assembled range , suggesting frequent participation in multiple differently sized macromolecular assemblies ( lower panels and pie chart ) . For a list of all detected protein peaks , see Dataset EV3 .

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Proteins are observed eluting in 1-6 distinct peaks and with a wide range of apparent vs. monomeric molecular weight ratios (distributions, left panels). The molecular weight ratios of the three peaks detected for TCPE (displayed in A) are indicated. Many of the proteins eluting in a single peak (top panel and bar) appear assembled. Proteins eluting multiple times (27 % of the proteins) do so preferentially in the assembled range, suggesting frequent participation in multiple differently sized macromolecular assemblies (lower panels and pie chart). For a list of all detected protein peaks, see Dataset EV3.


Proteins observed in multiple assembled peaks ( n = 659 ) are enriched in components of the proteasome and other known large complex assemblies .

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Proteins observed in multiple assembled peaks (n = 659) are enriched in components of the proteasome and other known large complex assemblies.


For nearly half the CORUM complex hypotheses queried , two or more distinct subunit co - elution signals were detected ( See methods and Appendix information on CCprofiler ) .

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For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler).


Among the four distinct co - elution signals detected from the eight canonical CSN subunits ' chromatograms ( here with CSN7A , not CSN7B ) are two distinct signals indicating distinct co - elution of two different complex variants .

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Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants.


Distinct co - elution of holo - CSN ( observed at the expected fraction 35 ) and Mini - CSN CSN1 / 3 / 8 ( observed eluting offset only one fraction late , F45 , of the expected fraction , F44 ) . Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration . Coloring adapted to highlight subversion components and their partitioning across holo - and sub - complex . CSN1 / 3 / 8 interact and form a submodule within the CSN holo - complex structure ( PDB accession 4D10 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6A ) . Lower right panel , validation of distinct elution behavior of holo - CSN exclusive ( CSN7A ) and shared subunit ( CSN8 ) by immunoblotting . For full immunoblotting data ( CSN1 , CSN3 , CSN4 , CSN5 , CSN7A and CSN8 ) see Appendix Figure S7C ) .

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Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).


CSN1 / 3 / 8 display distinct fractionation patterns in co - fractionation experiments performed in other laboratories , specifically in orthogonal ion exchange fractionation of HEK293 lysates ( Wan et al . 2015 , upper panels ) and size exclusion chromatographic fractionation of U2OS lysates ( Kirkwood et al . 2013 , lower panel ) , in line with the CSN1 / 3 / 8 as distinct entity .

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CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity.


Baculoviral co - expression of human CSN1 , CSN3 and CSN8 in Sf21VM insect cells , with CSN8 N - terminally Strep ( II ) - and CSN1 & CSN3 N - terminally His6 - tagged , followed by affinity purification and SDS - PAGE displays banding pattern in line with the formation of a stable trimer CSN1 / 3 / 8 .

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Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.


Protein - level SEC chromatograms of the 22 canonical 26S proteasome subunits . Vertical black lines indicate the apexes of six distinct co - elution signals detected in complex - centric scoring ; two of which represent well - known co - occurring variants , the full 26S ( i ) and the 20S ( ii ) particle devoid the 19S lid and ATPase ( Indicated by structural models , PDB accession 5GJR ) and four of which , composed of predominantly 20S α and β subunits , appear at reduced size ( 222 - 107 kDa , fractions 39 , 40 , 42 and 46 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6B ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O 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-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein-level SEC chromatograms of the 22 canonical 26S proteasome subunits. Vertical black lines indicate the apexes of six distinct co-elution signals detected in complex-centric scoring; two of which represent well-known co-occurring variants, the full 26S (i) and the 20S (ii) particle devoid the 19S lid and ATPase (Indicated by structural models, PDB accession 5GJR) and four of which, composed of predominantly 20S α and β subunits, appear at reduced size (222 - 107 kDa, fractions 39, 40, 42 and 46). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6B).


Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells .

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Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells.


SECexplorer web - interface for querying custom protein sets for co - elution behavior in the SEC - SWATH - MS data , viewing chromatograms for interpretation and with algorithmic assistance .

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SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance.


Zoom in to high MW peak shoulder of holo - COP9 Signalosome ( compare Figure 5 ) , where defined co - elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate - bound and free pools of CSN holo - complex .

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Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex.


Estimation of the fraction of holo - CSN in the likely substrate - bound pool versus the free pool , with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder ( also see Appendix Figure S7 ) .

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Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).


B - G . ( B , D , and F ) E histograms of the RDH substrate in the absence of EXD2 ( gray ) and after 0 - ( black ) , 100 - ( purple ) , and 200 - s ( blue ) incubation with 200 ( B ) , 100 ( D ) , and 5 ( F ) nM EXD2 . ( C , E , and G ) Representative smFRET trajectories showing EXD2 at 200 ( C ) , 100 ( E ) , and 5 ( G ) nM cleaving RDH in real time . The fluorescence intensities of the donor and acceptor are colored green and red , respectively . The corresponding FRET efficiencies are shown below in black . The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 .

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B-G. (B, D, and F) E histograms of the RDH substrate in the absence of EXD2 (gray) and after 0- (black), 100- (purple), and 200-s (blue) incubation with 200 (B), 100 (D), and 5 (F) nM EXD2. (C, E, and G) Representative smFRET trajectories showing EXD2 at 200 (C), 100 (E), and 5 (G) nM cleaving RDH in real time. The fluorescence intensities of the donor and acceptor are colored green and red, respectively. The corresponding FRET efficiencies are shown below in black. The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9.


H . The FRET trajectories ' percentages show the FRET values ending above 0 . 9 and between 0 . 2 and 0 . 9 . The word " Surface " represents the experiment with EXD2 anchored on the glass surface . I . Statistics of the time for the FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 under indicated experimental conditions . In violin plots , light - blue dots represent mean , and black bars show the interquartile range ( IQR ) ( thick bars ) and 1 . 5 times IQR ( thin bars ) ( n = 122 , 91 , 78 , and 101 from left to right ) .

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H. The FRET trajectories' percentages show the FRET values ending above 0.9 and between 0.2 and 0.9. The word "Surface" represents the experiment with EXD2 anchored on the glass surface. I. Statistics of the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9 under indicated experimental conditions. In violin plots, light-blue dots represent mean, and black bars show the interquartile range (IQR) (thick bars) and 1.5 times IQR (thin bars) (n = 122, 91, 78, and 101 from left to right).


D . A representative gel showing that EXD2 ( 500 nM ) cleaves the Cy5 - RNA and Cy5 - RDH substrates .

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D. A representative gel showing that EXD2 (500 nM) cleaves the Cy5-RNA and Cy5-RDH substrates.


F . The kinetic rates k1 and k2 obtained from the double exponential fitting to the dwell - time histograms of Pauses 1 - 3 and their combination ( Total ) . Fit parameters and 95 % confidence intervals are shown .

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F. The kinetic rates k1 and k2 obtained from the double exponential fitting to the dwell-time histograms of Pauses 1-3 and their combination (Total). Fit parameters and 95% confidence intervals are shown.


A - F . ( A and D ) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC - RDH substrate under 1 mM MnCl2 ( A ) or the RDH substrate under 0 . 2 mM MnCl2 ( D ) . The solid red lines are the step - fitting results from an automated step - finding algorithm . ( B and E ) The corresponding TDP of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( B ) or RDH under 0 . 2 mM MnCl2 ( E ) . ( C and F ) The dwell - time histograms of combined pauses ( Total ) in the process of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( C ) or RDH under 0 . 2 mM MnCl2 ( F ) . The histogram fittings to a double exponential function are shown in red . The R2 of the double exponential function are 0 . 94 and 0 . 95 , respectively .

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A-F. (A and D) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC-RDH substrate under 1 mM MnCl2 (A) or the RDH substrate under 0.2 mM MnCl2 (D). The solid red lines are the step-fitting results from an automated step-finding algorithm. (B and E) The corresponding TDP of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (B) or RDH under 0.2 mM MnCl2 (E). (C and F) The dwell-time histograms of combined pauses (Total) in the process of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (C) or RDH under 0.2 mM MnCl2 (F). The histogram fittings to a double exponential function are shown in red. The R2 of the double exponential function are 0.94 and 0.95, respectively.


( B ) ARSA activity quantification ( nmol / h / mg ) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days . Data represent mean ± SD of 7 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against negative

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(B) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days. Data represent mean ±SD of 7 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against negative control


( E ) Analysis of different retinoids ' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug ( Adapalene 1 µM ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Difference against DMSO control : * *

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(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: **


( D ) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) simultaneously treated with 10 and 20 µM tazarotene and bexarotene , respectively , up to 21 days . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . Displayed are significance levels for the next significant difference between adjacent treatment times . # # # # p < 0 . 0001 . Difference against 0 days control : * p < 0 . 05 , * * * * p < 0 . 0001 .

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(D) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively, up to 21 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent treatment times. #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001.


( E ) Analysis and quantification of increased sulfatase activities different to ARSA , namely ARSB , GALNS , and STS in MSD primary fibroblasts ( variant FGE Gly247Arg homozygous ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-DISEASE B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Analysis and quantification of increased sulfatase activities different to ARSA, namely ARSB, GALNS, and STS in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001, **** p<0.0001.


( F ) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations ( FGE Gly247Arg , FGE Gly263Val , FGE Ala279Val , FGE Arg349Trp ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s test for multiple comparisons . * * * * p < 0 . 0001 .

O O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations (FGE Gly247Arg, FGE Gly263Val, FGE Ala279Val, FGE Arg349Trp) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001.


( G ) Quantification of ARSA activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 6 independent experiments ( biological replicates ) . Unpaired t - test . * * * * p < 0 . 0001 . ( H ) Quantification of SGSH activity in MSD patient - derived iPSCs differentiated into NPCs and unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . Unpaired t - test . * p < 0 . 05 , * * * p < 0 . 001 .

O O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O B-DISEASE B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) Quantification of ARSA activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 6 independent experiments (biological replicates). Unpaired t-test. **** p<0.0001. (H) Quantification of SGSH activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 3 independent experiments (biological replicates). Unpaired t-test. * p<0.05, *** p<0.001.