e RT - qPCR - based validation of three differentially expressed genes , upon CHIR stimulation in WT , dBcat or d4TCF cell lines . The data represent the mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value < 0 . 05 , and ( * * ) a p - value <

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e RT-qPCR-based validation of three differentially expressed genes, upon CHIR stimulation in WT, dBcat or d4TCF cell lines. The data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<0.05, and (**) a p-value<


b , c RT - qPCR - based validation of selected TCF - independent β - catenin - dependent target genes ( B ) upregulated or ( C ) downregulated in d4TCF cells ( TCF - independent ) treated with 10μM CHIR or DMSO for 24hr . Data represent the mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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b, c RT-qPCR-based validation of selected TCF-independent β-catenin-dependent target genes (B) upregulated or (C) downregulated in d4TCF cells (TCF-independent) treated with 10μM CHIR or DMSO for 24hr. Data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


( A , B ) β - catenin occupancy on regulatory regions of the prototypical Wnt target genes , AXIN2 and LEF1 ( A ) , and at the promoter regions of HOXC4 and ZNF503 ( B )

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(A, B) β-catenin occupancy on regulatory regions of the prototypical Wnt target genes, AXIN2 and LEF1 (A), and at the promoter regions of HOXC4 and ZNF503 (B)


( C ) β - catenin ChIP - seq enrichment over ENCODE TCF7L2 ( also known as TCF4 ) ChIP peaks in HEK293T cells . The data represent the mean ± 95 % C . I . obtained from three independent experiments ( N = 3 )

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(C) β-catenin ChIP-seq enrichment over ENCODE TCF7L2 (also known as TCF4) ChIP peaks in HEK293T cells. The data represent the mean ± 95% C.I. obtained from three independent experiments (N = 3)


b RT - qPCR validation of the β - catenin - GHOST response . A selection of 7 genes ( colour coded ) that are activated upon CHIR treatment in d4TCF , or in wild - type ( WT ) in combination with ICAT overexpression , but not in pentaKO cells - indicating that their regulation is dependent on β - catenin but can only occur in the absence of TCF / LEF - β - catenin interaction . The genotype and the treatment for each condition is indicated in the x - axis . Error bars show that standard deviation obtained from 3 independent

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b RT-qPCR validation of the β-catenin-GHOST response. A selection of 7 genes (colour coded) that are activated upon CHIR treatment in d4TCF, or in wild-type (WT) in combination with ICAT overexpression, but not in pentaKO cells - indicating that their regulation is dependent on β-catenin but can only occur in the absence of TCF/LEF-β-catenin interaction. The genotype and the treatment for each condition is indicated in the x-axis. Error bars show that standard deviation obtained from 3 independent experiments


c FOXO4 but not FOXO3 overexpression upregulates the β - catenin - GHOST target GADD45 both in CHIR - treated WT ( black bars ) or d4TCF ( grey bars ) cells . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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c FOXO4 but not FOXO3 overexpression upregulates the β-catenin-GHOST target GADD45 both in CHIR-treated WT (black bars) or d4TCF (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


d FOXO4 overexpression induced GADD45 transcription in WT ( black bars ) but not in dBcat ( grey bars ) cells . Samples were compared using Student " s t - test . Asterisks ( * ) indicate a p - value <

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d FOXO4 overexpression induced GADD45 transcription in WT (black bars) but not in dBcat (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


e Endogenous FOXO4 , when immunoprecipitated ( IP ) pulls down β - catenin . Overexpressed FOXO4 increases the amount of β - catenin detected in the immunoprecipitated , indicating physical

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e Endogenous FOXO4, when immunoprecipitated (IP) pulls down β-catenin. Overexpressed FOXO4 increases the amount of β-catenin detected in the immunoprecipitated, indicating physical association


f GADD45 is upregulated by CHIR in d4TCF cell ; this positive transcriptional regulation is blocked by FOXO4 - specific siRNA but not control scrambled siRNA . All experiments were done at least three times ( N = 3 ) . Samples were compared using Student " s t - test . Asterisks ( * * ) indicate a p - value < 0 . 02 ; ns , non - statistically significant change was

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f GADD45 is upregulated by CHIR in d4TCF cell; this positive transcriptional regulation is blocked by FOXO4-specific siRNA but not control scrambled siRNA. All experiments were done at least three times (N=3). Samples were compared using Student"s t-test. Asterisks (**) indicate a p-value<0.02; ns, non-statistically significant change was obs


B . Protein structural predictions for S . pombe Rbd2 ( blue ) and E . coli GlpG ( red ) . Protein structures were predicted using the Phyre2 server ( http : / / www . sbg . bio . ic . ac . uk / phyre2 ) . Rbd2 model structure was aligned to GlpG using PyMOL .

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B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.


E . Cells carrying a plasmid expressing rbd2 - 6xmCherry and either of two Golgi proteins dsc2 - 6xGFP ( dsc2 - 6xGFPrbd2 - 6xmCherry ) or Sec72 - 6xGFP ( Sec72 - 6xGFPrbd2 - 6xmCherry ) were imaged by confocal fluorescence microscopy .

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E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP(dsc2-6xGFPrbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFPrbd2-6xmCherry) were imaged by confocal fluorescence microscopy.


A . Wild - type ( WT ) , rbd2Δ or sre1Δ yeast ( 200 cells ) containing either empty vector ( EV ) or a plasmid expressing sre1N ( Sre1amino acids 1 - 440 ) were grown on rich medium in the presence or absence of cobalt chloride ( CoCl2 ) .

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A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).


B . Western blot was probed with anti - Sre1 IgG of phosphatase - treated , whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen . Dsc5 serves as a loading control and was detected by chemiluminescence . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre1 cleavage . Western blot was performed using anti - Sre1 or anti - FlagIgG of phosphatase - treated , whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen .

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C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.


D . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre2 cleavage by western blot probed with anti - Sre2 serum or anti - FlagIgG of phosphatase - treated , whole cell lysates from cells grown in the presence of oxygen .

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D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.


E . Sre1 cleavage assay was performed as in ( C ) . G128V , A127D and A186T mutant strains were obtained from our previous mutagenesis screening ( Stewart et al , 2012 ) . Dsc5 serves as a loading control .

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E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.


F . Sre2 cleavage assay for indicated yeast strains was performed as in ( D ) .

F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).


G . A model substrate of Sre2 containing amino acids 423 - 793 ( Sre2MS ) with an N - terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ , dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains . Whole cell lysates from indicated yeast strains were analyzed by western blot with anti - Flag antibody . Three independent isolates ( A - C ) are shown for each strain . Empty vector ( EV ) transformation serves as a negative control .

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G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.


A . Wild - type ( WT ) and the indicated mutant strains were grown for 3 hr at the non - permissive temperature ( 36 ° C ) to inactivate Mts3 . Phosphatase - treated cell lysates were analyzed by western blot with anti - Sre2 serum and imaged using chemiluminescence . P and N denote Sre2 precursor and cleaved nuclear forms , respectively .

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A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.


B . WT , rbd2Δ and sre2Δ cells were treated with bortezomib ( Bz ) for 3 hr , and phosphatase - treated , whole cell lysates were analyzed by western blot with anti - Sre2 serum .

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B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.


C . WT , dsc1Δ and rbd2Δ cells carrying either empty vector ( EV ) or a plasmid expressing 3xFlag - Sre2MS ( MS ) were treated with bortezomib ( Bz ) for 3 hr . Whole cell lysates were analyzed by western blot with anti - Flag antibody .

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C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.


D . Whole cell lysates from the indicated strains were analyzed by western blot with anti - Sre2 serum .

D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.


E . Catalytically dead Dsc1 E3 ligase ( dsc1 - C634A ) mutant and other indicated yeast strains were analyzed by western blot probed with anti - Sre2 serum and imaged using chemiluminescence .

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E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.


A . HumanHEK293T cells were transfected with plasmids expressing different 3xHA - tagged rhomboid proteases : S . pomberbd2 , S . pomberbd2 recoded for human expression , humanRHBDL2 , D . melanogaster Rho1 , and P . falciparumROM4 . Whole cell lysates were analyzed by western blot to detect the HA - tagged rhomboid [ asterisks indicate loading control bands detected with antibody ab179726 ( abcam ) ] .

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A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].


B . HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S . pombe rbd2 , D . melanogaster Rho1 , or P . falciparumROM4 . Conditioned media and cell lysates were analyzed by western blot . P and C denote precursor and cleaved products , respectively .

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B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.


C . HumanHEK293T cells were transfected with plasmids expressing the GFP - TatA - Flag substrate and the indicated rhomboid proteases . Whole cell lysates were analyzed by western blot . P , CN , CC denote precursor , N - terminal cleaved and C - terminal cleaved products , respectively .

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C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.


D . HumanHEK293T cells were transfected with plasmids expressing the indicated GFP - TatA - Flag substrates and either wild - type or mutant recoded S . pomberbd2 . Whole cell lysates were analyzed by western blot .

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D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot.


E . HumanHEK293T cells were transfected with plasmids expressing the indicated GFP - TatA - Flag substrates and different rhomboid proteases . Whole cell lysates were analyzed by western blot .

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E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.


F . HumanHEK293T cells were transfected with plasmids expressing the GFP - TatA ( A8L ) - Flag and recoded S . pomberbd2 . C - terminal cleavage products were purified using anti - Flag antibodies and purified protein was analyzed by mass spectrometry .

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F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.


B . Whole cell lysates from WT , rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P and N denote Sre2MS precursor and cleaved forms , respectively .

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B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.


D - E . Whole cell lysates from WT , rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P and N denote Sre2MS precursor and cleaved forms , respectively . Asterisks indicate non - specific , loading control bands , which were used to normalize the intensities of P and N in Figure 5F . Two independent isolates , A and B , are shown for each strain .

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D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.


F . Intensities of individual P and N bands from western blots were quantified using LI - COR software . Relative intensity of P and N to corresponding non - specific band ( asterisk ) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis . The ratio of N to P or relative P level was presented in bar graphs .

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F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.


G . The average ratio of N to P level for different Sre2MS mutants in wild - type cells ( Appendix Fig S2 ) was plotted . Cleavage was scored as normal if the ratio was > 2 .

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G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.


H . For Sre2MS mutants showing defects in cleavage , the average fold - increase in P level in dsc1Δ cells compared to WT cells from two isolates ( Appendix Fig S2 ) was plotted . Mutants were grouped into three phenotypic classes ( intermediate , dsc1Δ , and rbd2 ∆ ) according to observed precursor accumulation . Precursor accumulation was defined as dsc1Δ if < 1 . 5 ( orange ) , rbd2Δ if > 3 ( pink ) , and intermediate if in between ( blue ) .

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H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).


B . Recombinant proteins GST - fused Rbd2 C - terminus ( Rbd2200 - 251 ) , Dsc5 UBX ( Dsc5323 - 425 ) , Dsc2 UBA ( Dsc2298 - 372 ) , and GST - HA - V5 control were bound to GST magnetic beads and incubated with S . pombe cytosol fraction from wild - type cells . Equivalent amounts of unbound and bound fractions were probed for anti - Cdc48 , anti - ubiquitin and anti - GSTIgG .

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B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG.


C . GSTpulldown assay was performed as in ( B ) using truncated forms of Rbd2 C - terminus ; Rbd2200 - 251 , Rbd2200 - 225 , Rbd2225 - 251 and Rbd2200 - 240 . Unbound ( 1x ) and bound ( 5x ) fractions were analyzed by western

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C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting


D . Single residue mutants of conserved glycine residues in Rbd2200 - 251 ( G244R and G246R ) were tested for Cdc48 binding as in ( C ) . Western blots ( B - D ) were imaged using chemiluminescence .

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D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.


A . Detergent - solubilized yeast extracts from cells containing chromosomal Flag - tagged rbd2 or rbd2 - G246R in rbd2Δ background were prepared , and proteins associated with Rbd2 - Flag were immunopurified using anti - Flag magnetic beads . Equal amount of total ( lanes 1 - 3 ) along with 25x bound fractions ( lanes 4 - 6 ) were analyzed by western blot with anti - Cdc48 serum and anti - FlagIgG .

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A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-FlagIgG.


C . Biotin - phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods . WT cells or rbd2Δ cells carrying rbd2 - Flag - APEX2 ( rbd2 - F - AX ) or rbd2 - G246R - Flag - APEX2 ( G246R - F - AX ) plasmid were lysed after biotin - labeling reaction , and proteins were denatured by heating the cells in lysis buffer containing 1 % SDS . Biotinylated proteins were then enriched using streptavidin magnetic beads . Lysates and 50x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Cdc48 serum , or anti - FlagIgG .

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C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.


A . Yeast strains ( 200 cells ) containing chromosomal Flag - tagged rbd2 , rbd2 - G244R , rbd2 - G246R or rbd2 - S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride ( CoCl2 ) .

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A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


B . Western blot of phosphatase - treated , whole cell lysates from strains in ( A ) grown for 3 hr in the presence or absence of oxygen was probed with anti - Sre1 or anti - Flag IgG . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Sre2 cleavage was assayed by western blot using anti - Sre2 serum or anti - Flag IgG of phosphatase - treated , whole cell lysates from the indicated strains . N denotes Sre2 cleaved nuclear form .

C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.


D . Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag - Sre2MS were analyzed by western blot with anti - Flag antibody . Two independent isolates , A and B , are shown for each strain . Asterisk denotes non - specific band .

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D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band.


F . Yeast strains containing Flag - tagged rbd2 - SHP , rbd2 - SHP * , rbd2 - G246R - SHP , and rbd2 - G246R - SHP * were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in ( E ) . Indicated yeast strains were then assayed for Sre1 cleavage as in ( B ) .

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F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).


G . Indicated strains were assayed for Sre2 cleavage as in ( C ) .

G. Indicated strains were assayed for Sre2 cleavage as in (C).


H . Wild - type , sre2Δ or strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130S in rbd2Δ background ( lane 7 - 12 ) were treated with bortezomib ( Bz ) for 2 hr , and phosphatase - treated , whole cell lysates were analyzed by western blot with anti - Sre2 serum or anti - Flag IgG .

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H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.


A . Western blot of phosphatase - treated whole cell lysates from yeast strains carrying rbd2 - Flag - APEX2 ( rbd2 - F ) , rbd2 - G246R - Flag - APEX2 ( G246R ) or rbd2 - S130A - Flag - APEX2 ( S130A ) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti - Sre1 or anti - Flag IgG . P and N denote Sre1 precursor and cleaved nuclear forms , respectively .

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A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


B . Indicated yeast strains ( 200 cells ) containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background ( lane 3 - 5 ) or yeast strains ( 200 cells ) carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background ( lane 6 - 8 ) were grown on rich medium in the absence or presence of cobalt chloride ( CoCl2 ) .

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B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


C . Comparison of Rbd2 protein levels from overexpression system to endogenous expression . Western blot of whole cell lysates from yeast strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background and yeast strains carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background was probed with anti - FlagIgG . For quantification , the intensity of Flag band in each lane was normalized to the non - specific band ( asterisk ) .

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C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).


D . rbd2Δ cells expressing indicated plasmids of GFP - Anp1 or GFP - Sre2MS together with rbd2 - Flag - APEX2 , rbd2 - S130A - Flag - APEX2 or rbd2 - S130A / G246R - Flag - APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin - labeling reaction . After labeling termination , cells were lysed and denatured as described in Materials and Methods . Biotinylated proteins were then enriched using streptavidin magnetic beads . Lysates ( 1x input ) and 100x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Flag IgG , anti - Cdc48 serum , and anti - GFP IgG . Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample , then normalized to the Flag intensity in enriched sample . Relative intensities of biotinylated GFP level are shown in bar graph . Error bars denote standard deviation from three biological replicates .

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

D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.


C Immunofluorescent analyses of USP33 ( red ) , the hypoxia marker CA9 ( green ) , and the stem cell marker SOX2 ( gray ) in human primary GBMs . Arrows indicate the USP33 + / CA9 + / SOX2 + cells . Scale bar , 80 µm .

C Immunofluorescent analyses of USP33 (red), the hypoxia marker CA9 (green), and the stem cell marker SOX2 (gray) in human primary GBMs. Arrows indicate the USP33+/CA9+/SOX2+ cells. Scale bar, 80 µm.


E Immunofluorescent analyses of USP33 ( red ) and the hypoxic cell population labeled by pimonidazole ( green ) in intracranial xenografts derived from T3359 GSCs . Mice bearing intracranial xenografts were injected intravenously ( tail vein ) with 60 mg / kg of pimonidazole and were sacrificed 2 hours post injection . Hypoxic cells were labeled with the FITC - conjugated mouse monoclonal anti - pimonidazole antibody . Scale bar , 80 µm .

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E Immunofluorescent analyses of USP33 (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 hours post injection. Hypoxic cells were labeled with the FITC-conjugated mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 µm.


G Immunoblot analysis of USP33 , HIF2α and HIF1α protein levels in GSCs , NSTCs and NPCs in normoxic and hypoxic conditions . Hypoxia ( 5 % O2 ) relative to normoxia ( 20 % O2 ) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs . The mild hypoxia of 5 % O2 showed negligible induction of HIF1α in all cells .

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G Immunoblot analysis of USP33, HIF2α and HIF1α protein levels in GSCs, NSTCs and NPCs in normoxic and hypoxic conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of HIF1α in all cells.


A , Representative images of immunofluorescent analyses of USP33 ( red ) and HIF2α ( green ) in human primary GBMs . Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α , and counterstained with DAPI to show nuclei ( blue ) . Scale bar , 40 μm . ( n = 5 sections ; mean ± s . e . m . ) .

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A, Representative images of immunofluorescent analyses of USP33 (red) and HIF2α (green) in human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m.).


F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33 . 0 . 5 µg of HA - tagged HIF2α plasmids together with the increased amounts of GFP - tagged USP33 plasmids ( 0 , 0 . 1 , 0 . 2 or 0 . 3 μg ) were introduced into 293T cells . Forty - eight hours post transfection , levels of ectopic HIF2α - HA and GFP - USP33 were analyzed by immunoblot analysis . Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33 . G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type ( WT ) USP33 or the catalytically dead ( CD ) USP33 . HA - tagged HIF2α plasmids ( 0 . 5 µg ) were introduced into 293T cells alone or together with GFP - tagged WT or CD USP33 plasmids ( 0 . 2 µg ) . Levels of ectopic HIF2α - HA and GFP - USP33 were analyzed 48 hours post transfection . Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels .

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F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33. 0.5 µg of HA-tagged HIF2α plasmids together with the increased amounts of GFP-tagged USP33 plasmids (0, 0.1, 0.2 or 0.3 μg) were introduced into 293T cells. Forty-eight hours post transfection, levels of ectopic HIF2α-HA and GFP-USP33 were analyzed by immunoblot analysis. Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33. G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type (WT) USP33 or the catalytically dead (CD) USP33. HA-tagged HIF2α plasmids (0.5 µg) were introduced into 293T cells alone or together with GFP-tagged WT or CD USP33 plasmids (0.2 µg). Levels of ectopic HIF2α-HA and GFP-USP33 were analyzed 48 hours post transfection. Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels.


A , Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - HIF2α antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen .

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A, Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.


D Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment . GSCs were treated with CoCl2 ( 300 µM ) for 12 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia .

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D Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment. GSCs were treated with CoCl2 (300 µM) for 12 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia.


G Co - immunoprecipitation to detect the interaction between HIF2α and the wild - type ( WT ) or the catalytically dead ( CD ) USP33 . Ectopic HA - tagged HIF2α were expressed in 293T cells with GFP - tagged wild - type or catalytically dead USP33 . Cell lysates were subjected to immunoprecipitation with anti - HA antibodies . Co - immunoprecipitated products immunoblotted with the indicated antibodies . The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α .

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G Co-immunoprecipitation to detect the interaction between HIF2α and the wild-type (WT) or the catalytically dead (CD) USP33. Ectopic HA-tagged HIF2α were expressed in 293T cells with GFP-tagged wild-type or catalytically dead USP33. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies. Co-immunoprecipitated products immunoblotted with the indicated antibodies. The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α.


A Immunoblot analysis of the serine / threonine phosphorylation ( pS / T ) status of HIF2α in GSCs in response to 5 % oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , and then harvested for immunoprecipitation with anti - HIF2α antibodies or IgG control . Immunoprecipitated endogenous HIF2α were immunoblotted with anti - pan - pS / T antibodies . An increased pS / T of HIF2α was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen .

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A Immunoblot analysis of the serine/threonine phosphorylation (pS/T) status of HIF2α in GSCs in response to 5% oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours, and then harvested for immunoprecipitation with anti-HIF2α antibodies or IgG control. Immunoprecipitated endogenous HIF2α were immunoblotted with anti-pan-pS/T antibodies. An increased pS/T of HIF2α was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.


D Co - immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1 / 2 activity . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , then treated with the MEK inhibitor U0126 ( 10µM ) for 30 minutes to inhibit ERK1 / 2 activity , and harvested for immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . Relative to 20 % oxygen treatment , 5 % oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs . However , inhibition of ERK1 / 2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33 .

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D Co-immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1/2 activity. GSCs were cultured under 20% or 5% oxygen for 24 hours, then treated with the MEK inhibitor U0126 (10µM) for 30 minutes to inhibit ERK1/2 activity, and harvested for immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. Relative to 20% oxygen treatment, 5% oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs. However, inhibition of ERK1/2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33.


G Co - immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33 . Ectopic GFP - tagged USP33 along with HA - tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells . Cell lysates were subjected to immunoprecipitation with anti - HA agaroses Co - immunoprecipitated products were immunoblotted with the indicated antibodies . The S484A mutation largely attenuated the interaction of HIF2α with USP33 , indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33 .

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G Co-immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33. Ectopic GFP-tagged USP33 along with HA-tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells. Cell lysates were subjected to immunoprecipitation with anti-HA agaroses Co-immunoprecipitated products were immunoblotted with the indicated antibodies. The S484A mutation largely attenuated the interaction of HIF2α with USP33, indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33.


B Representative images of tumorsphere formation of T387 GSCs expressing USP33 - targeting shRNA ( shUSP33 ) or non - targeting shRNA ( shNT ) . Twenty - four hours after lentiviral infection , T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2 , 000 cells per well and cultured for 48 hours under normoxia ( 20 % O2 ) or hypoxia ( 5 % O2 ) . Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia . Scale bar , 200 μm .

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B Representative images of tumorsphere formation of T387 GSCs expressing USP33-targeting shRNA (shUSP33) or non-targeting shRNA (shNT). Twenty-four hours after lentiviral infection, T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2,000 cells per well and cultured for 48 hours under normoxia (20% O2) or hypoxia (5% O2). Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia. Scale bar, 200 μm.


E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours , and then split into 96 well plates at the concentration of 2 , 000 cells per well . Cell titer was determined by the Glo luminescent cell viability assay kit ( Promega ) at the indicated time points . Disruption of USP33 inhibited GSC growth in normoxia and hypoxia . The experiment has been repeated three times . The presented curves were based on three technical replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( mean ± s . e . m . ; two tailed unpaired t - test ) .

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E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours, and then split into 96 well plates at the concentration of 2,000 cells per well. Cell titer was determined by the Glo luminescent cell viability assay kit (Promega) at the indicated time points. Disruption of USP33 inhibited GSC growth in normoxia and hypoxia. The experiment has been repeated three times. The presented curves were based on three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (mean ± s.e.m.; two tailed unpaired t-test).


F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were attached to hESC - Qualified Matrix ( Corning ) in 6 - well plates at the concentration of 50 , 000 cells per well . Forty - eight hours post infection with shUSP33 or shNT lentiviruses , cells were stained for cleaved caspase 3 ( green ) and nuclei ( blue ) . Scale bar , 80 μm .

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F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were attached to hESC-Qualified Matrix (Corning) in 6-well plates at the concentration of 50,000 cells per well. Forty-eight hours post infection with shUSP33 or shNT lentiviruses, cells were stained for cleaved caspase 3 (green) and nuclei (blue). Scale bar, 80 μm.


C , Representative images of autonomous fluorescent signals of 4HRE - d2GFP in xenografts derived from or shUSP33 - or shNT - expressing T387 GSCs transduced with 4HRE - d2GFP . Scale bar , 200 μm .

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C, Representative images of autonomous fluorescent signals of 4HRE-d2GFP in xenografts derived from or shUSP33- or shNT- expressing T387 GSCs transduced with 4HRE-d2GFP. Scale bar, 200 μm.


E , Representative images of autonomous fluorescent signals of 4HRE - d2GFP ( green ) and immunofluorescent staining of the stem cell marker SOX2 ( red ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . More GFP signals were detected in the SOX2 + cells compared with the SOX2 - cells , suggesting a stronger hypoxia response in GSCs relative to NSTCs . Scale bar , 200 μm .

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E, Representative images of autonomous fluorescent signals of 4HRE-d2GFP (green) and immunofluorescent staining of the stem cell marker SOX2 (red) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. More GFP signals were detected in the SOX2+ cells compared with the SOX2- cells, suggesting a stronger hypoxia response in GSCs relative to NSTCs. Scale bar, 200 μm.


G , Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 ( red ) and autonomous fluorescent signals of 4HRE - d2GFP ( green ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . The GFP + population had much less apoptosis relative to the GFP - population , suggesting that hypoxia response inhibited cell apoptosis . Scale bar , 200 μm .

G, Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 (red) and autonomous fluorescent signals of 4HRE-d2GFP (green) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. The GFP+ population had much less apoptosis relative to the GFP- population, suggesting that hypoxia response inhibited cell apoptosis. Scale bar, 200 μm.


B Kaplan - Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from ( A ) . Disruption of USP33 extended the survival of mice bearing GSC - derived GBM tumors . ( n = 5 mice for each group ; shUSP33 # 42 vs shNT , p = 0 . 0185 ; shUSP33 # 45 vs shNT , p = 0 . 0027 ; two - tailed log - rank test ) .

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B Kaplan-Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from (A). Disruption of USP33 extended the survival of mice bearing GSC-derived GBM tumors. (n= 5 mice for each group; shUSP33#42 vs shNT, p = 0.0185; shUSP33#45 vs shNT, p = 0.0027; two-tailed log-rank test).


E , F Immunofluorescent analysis ( E ) and statistical quantifications ( F ) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT . Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts . Scale bar , 80 μm . ( n = 5 tumors for each group ; * * * , p < 0 . 001 ; mean ± s . e . m . ; two tailed unpaired t - test ) .

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E, F Immunofluorescent analysis (E) and statistical quantifications (F) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts. Scale bar, 80 μm. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).


( A ) Wild type ( left and middle panels ) and p53 - / - ( right panel ) HCT116 cells were transfected with control ( mixture of control DS scrambledNeg , siLuci , and siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 and 4 ) siRNAs for 36 h , treated with 5 nM actinomycin D , and harvested at the indicated times . Whole cell extracts were analyzed by immunoblotting with the indicated antibodies . Coomassie Brilliant Blue ( CBB ) staining serves as a loading control .

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(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control.


( B ) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA - resistant HA - GRWD1 were treated and analyzed as above .

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(B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above.


( C ) HCT116 cells were transfected with control ( mixture of control DS scrambledNeg , siLuci , and siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 and 4 ) siRNAs for 24 h , treated with 50 µg / ml cycloheximide , and harvested at the indicated times . Whole cell extracts were analyzed by immunoblotting with the indicated antibodies .

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(C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies.


( D ) HFF2 / T cells stably overexpressing HA - GRWD1 were established by retroviral infection . Cells were cultured in the presence or absence of actinomycin D ( 5 nM ) for 12h and then treated with or without MG132 ( 20uM ) for 6 h as indicated . Whole cell extracts were analyzed as above .

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(D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above.


( E ) Control and HA - GRWD1 - overexpressing HFF2 / T cells were first treated with actinomycin D ( 5nM ) for 12 h , then further treated with 50 ug / ml cycloheximide , and harvested at the indicated times . Whole cell extracts were analyzed by immunoblotting with the indicated antibodies . The means and SDs from two independent experiments are shown .

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(E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.


( A ) U2OS cells were transfected with control ( mixture of control DS scrambledNeg , siLuci , and siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 and 4 or siGRWD1 - 1 ) siRNAs for 24 h . Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies .

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(A) U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h. Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies.


B ) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry .

B) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry.


( C ) HCT116 cells treated with actinomycinD ( 5nM ) or vehicle ( DMSO ) for 12h were first extracted with Triton X - 100 to remove nucleoplasmic proteins , double - immunostained with anti - GRWD1 ( green ) and anti - fibrillarin ( red ) antibodies as a marker for nucleoli , and counterstained with DAPI . Scale bar , 20 um .

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(C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um.


( D ) HCT116 cells were transfected with control ( mixture of control siLuci and siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 and 4 or siGRWD1 - 1 ) siRNAs for 24 h and then immunostained as above . Scale bar , 20 um .

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(D) HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 um.


( A ) 293T cells were co - transfected with HA - GRWD1 ( 1 . 6 ug ) and RPL5 - FLAG ( 2 . 4 ug ) or RPL11 - FLAG ( 2 . 4 ug ) as indicated for 42h and subjected to immunoprecipitation with anti - FLAG antibody . Immunoprecipitates ( IPs ) and 3 . 125 % of inputs were immunoblotted with the indicated antibodies . * , immunoglobulin light chains .

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(A) 293T cells were co-transfected with HA-GRWD1 (1.6 ug) and RPL5-FLAG (2.4 ug) or RPL11-FLAG (2.4 ug) as indicated for 42h and subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitates (IPs) and 3.125% of inputs were immunoblotted with the indicated antibodies. *, immunoglobulin light chains.


( B ) Lysates from untransfected 293T cells were immunoprecipitated with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) and 2 . 7 % of inputs were immunoblotted with the indicated antibodies .

(B) Lysates from untransfected 293T cells were immunoprecipitated with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 2.7% of inputs were immunoblotted with the indicated antibodies.


( C ) HCT116 cells were treated with 5nM actinomycin D for 12 h or left untreated and subjected to immunoprecipitation with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) and 3 . 5 % of inputs were immunoblotted with the indicated antibodies .

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(C) HCT116 cells were treated with 5nM actinomycin D for 12 h or left untreated and subjected to immunoprecipitation with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 3.5% of inputs were immunoblotted with the indicated antibodies.


( D ) GST - RPL11 or GST alone was incubated with purified GRWD1 and bound proteins analyzed by immunoblotting with the indicated antibodies . I , input . P , pull - down .

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(D) GST-RPL11 or GST alone was incubated with purified GRWD1 and bound proteins analyzed by immunoblotting with the indicated antibodies. I, input. P, pull-down.


( E ) Cell lysates were prepared from 293T cells transfected with wild - type ( WT ) HA - GRWD1 - FLAG ( 12 µg ) or various N - terminal truncated mutants of HA - GRWD1 - FLAG ( 12 µg ) for 42h , and then subjected to GST pull - down assay with purified GST - RPL11 or GST . Bound proteins were analyzed by CBB staining ( GST and GSR - RPL11 ) and immunoblotting with the indicated antibodies . * , non - specific bands .

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(E) Cell lysates were prepared from 293T cells transfected with wild-type (WT) HA-GRWD1-FLAG (12 µg) or various N-terminal truncated mutants of HA-GRWD1-FLAG (12 µg) for 42h, and then subjected to GST pull-down assay with purified GST-RPL11 or GST. Bound proteins were analyzed by CBB staining (GST and GSR-RPL11) and immunoblotting with the indicated antibodies. *, non-specific bands.


( G ) HCT116 cells were transfected with RPL11 - FLAG expression vector ( 0 . 56 ug ) or empty vector ( 0 . 56 ug ) for 24 h and then treated with 5 nM actinomycinD or vehicle ( PBS ) for 12 h . The cells were first extracted with Triton X - 100 to remove nucleoplasmic proteins , double - immunostained with anti - GRWD1 antibody ( green ) and anti - FLAG M2 antibody ( red ) , and counterstained with DAPI . Scale bar , 20 um .

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

(G) HCT116 cells were transfected with RPL11-FLAG expression vector (0.56 ug) or empty vector (0.56 ug) for 24 h and then treated with 5 nM actinomycinD or vehicle (PBS) for 12 h. The cells were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 antibody (green) and anti-FLAG M2 antibody (red), and counterstained with DAPI. Scale bar, 20 um.


( A ) H1299 cells were co - transfected with the indicated expression vectors ( p53 , 7 . 5 ng ; HA - Ub , 0 . 5 ug ; His - Xpress - MDM2 , 2 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG , 1 . 5 ug ; GFP , 0 . 04 ug ) for 48 h and analyzed by immunoblotting with the indicated antibodies . GFP serves as a control protein to show equal transfection efficiencies .

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(A) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug; GFP, 0.04 ug) for 48 h and analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.


( B ) Lysates were prepared from 293T cells co - transfected with His - Xpress - MDM2 ( 1 . 2 ug ) , FLAG - RPL11 ( 1 . 8 ug ) , and HA - GRWD1 ( 1 ug ) as indicated for 42 h and then immunoprecipitated with anti - FLAG antibody . Immunoprecipitates ( IPs ) and 1 . 5 % of inputs were immunoblotted with the indicated antibodies .

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(B) Lysates were prepared from 293T cells co-transfected with His-Xpress-MDM2 (1.2 ug), FLAG-RPL11 (1.8 ug), and HA-GRWD1 (1 ug) as indicated for 42 h and then immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IPs) and 1.5% of inputs were immunoblotted with the indicated antibodies.


( C ) In vivo ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells . Lysates were prepared from H1299 cells transfected with the indicated expression vectors ( His - Xpress - MDM2 , 2 ug ; HA - Ub , 0 . 5 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG , 1 . 5 ug ) for 48 h , treated with proteasome inhibitors for 6 h before harvest , and then immunoprecipitated with anti - MDM2 antibody . Immunoprecipitates ( IPs ) and inputs were immunoblotted with the indicated antibodies .

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(C) In vivo ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells. Lysates were prepared from H1299 cells transfected with the indicated expression vectors (His-Xpress-MDM2, 2 ug; HA-Ub, 0.5 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG, 1.5 ug) for 48 h, treated with proteasome inhibitors for 6 h before harvest, and then immunoprecipitated with anti-MDM2 antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies.


( D ) In vitro ubiquitination of p53 by immunopurified MDM2 . His - Xpress - MDM2 was immunopurified from transfected 293T cells with anti - Omni probe antibody . Recombinant p53 was incubated with E1 , E2 ( UbcH5a ) , His - ubiquitin , ATP , GST - RPL11 , GRWD1 - His , and immunopurified His - Xpress - MDM2 or control immunoprecipitates at 30 ℃ for 120 min as indicated . The samples were resolved by SDS - PAGE followed by immunoblotting with the indicated antibodies .

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(D) In vitro ubiquitination of p53 by immunopurified MDM2. His-Xpress-MDM2 was immunopurified from transfected 293T cells with anti-Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His-ubiquitin, ATP, GST-RPL11, GRWD1-His, and immunopurified His-Xpress-MDM2 or control immunoprecipitates at 30℃ for 120 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.


( A ) Expression of the introduced proteins and the effect on p53 . HFF2 / T cells stably expressing HA - GRWD1 , E7 , and KRAS G12V were established with retroviral vectors and analyzed by immunoblotting with the indicated antibodies . * , endogenous RAS proteins .

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(A) Expression of the introduced proteins and the effect on p53. HFF2/T cells stably expressing HA-GRWD1, E7, and KRAS G12V were established with retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.


( B and C ) Anchorage - independent growth of HFF2 / T cells overexpressing E7 with or without HA - GRWD1 . Three weeks after seeding , images were acquired and colonies > 100 µm in diameter were counted . Representative images are shown in ( B ) . Scale bars , 500 um . Average numbers of colonies per field , with SDs , are shown in ( C ) . Six randomly selected areas were counted per sample .

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(B and C) Anchorage-independent growth of HFF2/T cells overexpressing E7 with or without HA-GRWD1. Three weeks after seeding, images were acquired and colonies >100 µm in diameter were counted. Representative images are shown in (B). Scale bars, 500 um. Average numbers of colonies per field, with SDs, are shown in (C). Six randomly selected areas were counted per sample.


( D and E ) Anchorage - independent growth of HFF2 / T cells overexpressing E7 and activated KRAS G12V with or without HA - GRWD1 . At 9 days after seeding , the colonies were analyzed as above . Representative images are shown in ( D ) . Scale bars , 500 um . The average numbers of colonies ( > 200 um in diameter ) per field , with SDs , are shown in ( E ) . Twenty randomly selected areas were counted .

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(D and E) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with or without HA-GRWD1. At 9 days after seeding, the colonies were analyzed as above. Representative images are shown in (D). Scale bars, 500 um. The average numbers of colonies (>200 um in diameter) per field, with SDs, are shown in (E). Twenty randomly selected areas were counted.


( F ) Tumorigenesis of HFF2 / T cells expressing E7 and KRAS G12V with or without HA - GRWD1 . Cells were subcutaneously injected into nude mice ( 1 x 106 cells / mouse ) , and tumor size was measured . Average tumor volumes , with SDs , are shown ( n = 5 ) .

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(F) Tumorigenesis of HFF2/T cells expressing E7 and KRAS G12V with or without HA-GRWD1. Cells were subcutaneously injected into nude mice (1 x 106 cells/mouse), and tumor size was measured. Average tumor volumes, with SDs, are shown (n=5).


( G ) High expression of GRWD1 is associated with poor prognosis of brain lower grade glioma patients with wild type p53 but not with mutated p53 . The data were obtained from the TCGA databases ( the cBioPortal for Cancer Genomics ; http : / / www . cbioportal . org ) . For detail , see Materials and Methods .

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(G) High expression of GRWD1 is associated with poor prognosis of brain lower grade glioma patients with wild type p53 but not with mutated p53. The data were obtained from the TCGA databases (the cBioPortal for Cancer Genomics; http://www.cbioportal.org). For detail, see Materials and Methods.


( A ) 293T cells were co - transfected with HA - GRWD1 wild - type ( WT ) ( 0 . 5 ug ) or HA - GRWD1 Δ1 - 136 ( 3 . 5 ug ) and RPL11 - FLAG ( 1 . 5 ug ) as indicated for 42h , and then immunoprecipitated with an anti - FLAG antibody . Immunoprecipitates ( IPs ) and 1 . 6 % of inputs were immunoblotted with the indicated antibodies .

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(A) 293T cells were co-transfected with HA-GRWD1 wild-type (WT) (0.5 ug) or HA-GRWD1 Δ1-136 (3.5 ug) and RPL11-FLAG (1.5 ug) as indicated for 42h, and then immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.


( B ) 293T cells were co - transfected with His - Xpress - MDM2 ( 0 . 6 ug ) , RPL11 - FLAG ( 0 . 9 ug ) , and HA - GRWD1 WT ( 0 . 5 ug ) or HA - GRWD1 Δ1 - 136 ( 4 . 5 ug ) as indicated for 42h and subjected to immunoprecipitation with an anti - FLAG antibody . Immunoprecipitates ( IPs ) and 1 . 6 % of inputs were immunoblotted with the indicated antibodies .

(B) 293T cells were co-transfected with His-Xpress-MDM2 (0.6 ug), RPL11-FLAG (0.9 ug), and HA-GRWD1 WT (0.5 ug) or HA-GRWD1 Δ1-136 (4.5 ug) as indicated for 42h and subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies.


( C ) H1299 cells were co - transfected with the indicated expression vectors ( p53 , 7 . 5 ng ; HA - Ub , 0 . 5 ug ; His - Xpress - MDM2 , 2 ug ; RPL11 - FLAG , 1 ug ; HA - GRWD1 - FLAG WT , 1 . 5 ug ; HA - GRWD1 - FLAG Δ1 - 136 , 1 . 5 ug ; GFP , 0 . 04 ug ) for 48 h , and then analyzed by immunoblotting with the indicated antibodies . GFP serves as a control protein to show equal transfection efficiencies .

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(C) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG WT, 1.5 ug; HA-GRWD1-FLAG Δ1-136, 1.5 ug; GFP, 0.04 ug) for 48 h, and then analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies.


( D ) HFF2 / T cells stably expressing HA - GRWD1 wild - type ( WT ) or Δ1 - 136 , E7 , and KRAS G12V were established using retroviral vectors and analyzed by immunoblotting with the indicated antibodies . * , endogenous RAS proteins .

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(D) HFF2/T cells stably expressing HA-GRWD1 wild-type (WT) or Δ1-136, E7, and KRAS G12V were established using retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins.


( E and F ) Anchorage - independent growth of HFF2 / T cells overexpressing E7 and activated KRAS G12V with HA - GRWD1 WT or Δ1 - 136 ( or without HA - GRWD1 ) . At 9 days after seeding , colonies were analyzed as above . Representative images are shown in ( E ) . Scale bars , 500 um . Average numbers of colonies ( > 200 um in diameter ) per photographed area with SDs are shown in ( F ) . Seventy randomly selected areas were counted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with HA-GRWD1 WT or Δ1-136 (or without HA-GRWD1). At 9 days after seeding, colonies were analyzed as above. Representative images are shown in (E). Scale bars, 500 um. Average numbers of colonies (>200 um in diameter) per photographed area with SDs are shown in (F). Seventy randomly selected areas were counted.


( A ) Morphological phenotypes of Wild type ( WT ) , mkk1 mkk2 and summ3 - 1 mkk1 mkk2 plants . Photos were taken on three - week - old soil - grown plants .

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(A) Morphological phenotypes of Wild type (WT), mkk1 mkk2 and summ3-1 mkk1 mkk2 plants. Photos were taken on three-week-old soil-grown plants.


( B ) Trypan Blue staining of cell death in the indicated genotypes .

(B) Trypan Blue staining of cell death in the indicated genotypes.


( C ) DAB staining of H2O2 accumulation in the indicated genotypes .

(C) DAB staining of H2O2 accumulation in the indicated genotypes.


( D - E ) Expression of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

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(D-E) Expression of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


( A ) Morphological phenotypes of WT , summ3 - 17 , mekk1 - 1 and summ3 - 17 mekk1 - 1 plants grown on soil for three weeks .

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(A) Morphological phenotypes of WT, summ3-17, mekk1-1 and summ3-17 mekk1-1 plants grown on soil for three weeks.


( B - C ) Expression levels of PR1 ( B ) and PR2 ( C ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( B ) - ( C ) .

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(B-C) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (B)-(C).


( D ) Morphological phenotypes of WT , mpk4 - 3 , summ3 - 1 mpk4 - 3 and summ3 - 2 mpk4 - 3 plants . Photos were taken on three - week - old soil grown plants .

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(D) Morphological phenotypes of WT, mpk4-3, summ3-1 mpk4-3 and summ3-2 mpk4-3 plants. Photos were taken on three-week-old soil grown plants.


( E - F ) Expression levels of PR1 ( E ) and PR2 ( F ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( E ) - ( F ) .

(E-F) Expression levels of PR1 (E) and PR2 (F) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (E)-(F).


( A ) Morphological phenotypes of transgenic plants over - expressing MEKK2 - FLAG protein in WT and summ3 - 17 background . Line 1 and 2 express MEKK2 - FLAG in WT background . Line 3 - 5 express MEKK2 - FLAG in the summ3 - 17 mutant background . The photograph shows four - week - old soil grown plants . Expression of MEKK2 - FLAG was detected by western blot using an anti - FLAG antibody .

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(A) Morphological phenotypes of transgenic plants over-expressing MEKK2-FLAG protein in WT and summ3-17 background. Line 1 and 2 express MEKK2-FLAG in WT background. Line 3-5 express MEKK2-FLAG in the summ3-17 mutant background. The photograph shows four-week-old soil grown plants. Expression of MEKK2-FLAG was detected by western blot using an anti-FLAG antibody.


( B - C ) Trypan Blue staining of cell death ( B ) in the indicated genotypes .

(B-C) Trypan Blue staining of cell death (B) in the indicated genotypes.


( B - C ) DAB staining of H2O2 accumulation ( C ) in the indicated genotypes .

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(B-C) DAB staining of H2O2 accumulation (C) in the indicated genotypes.


( D - E ) Expression levels of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

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(D-E) Expression levels of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


( A ) Quantification of luciferase activity in N . benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc . Data were collected three days after infiltration . The data are shown as mean ± SD ( n = 8 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated twice with similar results .

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(A) Quantification of luciferase activity in N. benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc. Data were collected three days after infiltration. The data are shown as mean ± SD (n=8) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated twice with similar results.


( B ) Phosphorylation of CRCK3G390R by MPK4 . MPK4 was immunoprecipitated from wild type or MPK4 : : MPK4 - FLAG . After incubation with [ γ - 32P ] ATP and the immunoprecipitated MPK4 - FLAG protein in protein kinase buffer , CRCK3G390R was separated by SDS - PAGE . The autoradiograph of the gel is shown in the top panel , and immunoblot analysis of MPK4 - FLAG levels is shown in the bottom panel . Myelin basic protein ( MBP ) was used as a positive control . This experiment was repeated three times with similar results .

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(B) Phosphorylation of CRCK3G390R by MPK4. MPK4 was immunoprecipitated from wild type or MPK4::MPK4-FLAG. After incubation with [γ-32P] ATP and the immunoprecipitated MPK4-FLAG protein in protein kinase buffer, CRCK3G390R was separated by SDS-PAGE. The autoradiograph of the gel is shown in the top panel, and immunoblot analysis of MPK4-FLAG levels is shown in the bottom panel. Myelin basic protein (MBP) was used as a positive control. This experiment was repeated three times with similar results.


( C ) Western blot analysis of CRCK3 - FLAG proteins in wild type and mpk4 - 3 . Total protein was extracted from two - week - old CRCK3 - FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and CRCK - FLAG proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . pCRCK3 , phosphorylated CRCK3 - FLAG protein .

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(C) Western blot analysis of CRCK3-FLAG proteins in wild type and mpk4-3. Total protein was extracted from two-week-old CRCK3-FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and CRCK-FLAG proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. pCRCK3, phosphorylated CRCK3-FLAG protein.


( D ) Western blot analysis of CRCK3 - FLAG and CRCK35S - 5A - FLAG proteins in transgenic plants of wild type background . Total protein was extracted from two - week - old plants grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and the FLAG - tagged proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . 5S - 5A stands for a change in five amino acids ( S172A , S181A , S188A , S195A , S202A ) .

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(D) Western blot analysis of CRCK3-FLAG and CRCK35S-5A-FLAG proteins in transgenic plants of wild type background. Total protein was extracted from two-week-old plants grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and the FLAG-tagged proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. 5S-5A stands for a change in five amino acids (S172A, S181A, S188A, S195A, S202A).


( A ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments .

(A) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3.FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.


( B ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 kinase domain . KD represents a truncation of SUMM3 with only the kinase domain ( amino acid 201 - 510 ) . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments .

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(B) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3 kinase domain. KD represents a truncation of SUMM3 with only the kinase domain (amino acid 201-510).FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.


A RT - qPCR reveals that miR - 210 was increased in lungs of mice with PH triggered by various conditions : VHL − / − as compared with VHL + / + mice ( N = 4 / group ) , * * * P < 0 . 0001 ( first graph ) ; hypoxia + SU5416 ( Hyp + SU5416 ) ( N = 6 / group ) as compared with normoxia + SU5416 ( Norm + SU5416 ) ( N = 7 / group ) , * * P = 0 . 0015 ( second graph ) ; Il6 transgenic versus littermate control mice ( N = 4 / group ) , * * P = 0 . 0097 ( third graph ) ; and S . mansoni - infected mice ( N = 4 ) compared with non - infected control mice ( N = 5 ) , * * * P < 0 . 0001 ( fourth graph ) .

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A RT-qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).


B From animal subjects in ( A ) , in situ hybridization ( ISH , purple stain ) revealed increased miR - 210 in < 100 - μm pulmonary vessels of mice suffering from PH , * P = 0 . 0493 for first graph , * * P = 0 . 0015 for second graph , * P = 0 . 0391 for third graph .

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B From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph.


C Representative ISH stain of miR - 210 in < 100 μm pulmonary vessels of mice ( bottom micrographs ) exposed to Hyp + SU5416 compared with Norm + SU5416 ( α - smooth muscle actin stain from serial sections , top row of micrographs ) .

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C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).


D Increased miR - 210 in < 200 - μm remodeled pulmonary vessels of patients suffering from PAH ( N = 19 , Supplementary Table S1 ) as compared with non - PAH donor control lung ( N = 10 ) . Serial staining with hematoxylin and eosin is displayed in the top row of micrographs ; quantification of miRNA ISH , right graph , * P = 0 . 0167 .

D Increased miR-210 in < 200-μm remodeled pulmonary vessels of patients suffering from PAH (N = 19, Supplementary Table S1) as compared with non-PAH donor control lung (N = 10). Serial staining with hematoxylin and eosin is displayed in the top row of micrographs; quantification of miRNA ISH, right graph, *P = 0.0167.


E Increased levels of miR - 210 in plasma drawn from the pulmonary circulation ( pulmonary capillary wedge position , PCWP ) of patients with elevated mean pulmonary arterial pressures ( mean PAP ≥ 25 mmHg ) compared with control subjects ( mPAP < 25 mmHg , N = 5 / group , demographics in Supplementary Table S2 ) , * P = 0 . 0357 .

E Increased levels of miR-210 in plasma drawn from the pulmonary circulation (pulmonary capillary wedge position, PCWP) of patients with elevated mean pulmonary arterial pressures (mean PAP ≥ 25 mmHg) compared with control subjects (mPAP < 25 mmHg, N = 5/group, demographics in Supplementary Table S2), *P = 0.0357.


F From animals in ( C ) immunohistochemistry ( IHC ) revealed that the miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in PH mice exposed to Hyp + SU5416 ( F , * * * P = 0 . 0002 ) .

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F From animals in (C) immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002).


G From humans in ( D ) , immunohistochemistry ( IHC ) revealed that the miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in human PAH patients ( G , * * * P = 0 . 0008 ) .

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G From humans in (D), immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in human PAH patients (G, ***P = 0.0008).


H , I miR - 210 expression ( H ) was increased ( * * * P = 0 . 0003 ) , and ISCU1 / 2 expression ( I ) was decreased ( * * * P < 0 . 0001 ) in PECAM + pulmonary vascular endothelial cells isolated from PH mice ( Hyp + SU5416 ) as compared with control ( Norm + SU5416 ) ( N = 4 / group , left bars ) .

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H, I miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+pulmonary vascular endothelial cells isolated from PH mice (Hyp + SU5416) as compared with control (Norm + SU5416) (N = 4/group, left bars).