( D ) IFNγ - primed wild type hGBP1 - KO HeLa stably expressing inactive mutant YFP - Caspase - 4C258S were infected with either cytosol - entering S . enterica Typhimurium mutant ΔsifA ( MOI = 25 ) or with S . flexneri ΔospC3ΔipaH9 . 8 ( MOI = 6 ) , a mutant lacking Caspase - 4 antagonist OspC3 as well as hGBP1 antagonist IpaH9 . 8 . were fixed at 4 hpi ( ST ΔsifA ) or at 2 hpi ( Sf ΔospC3ΔipaH9 . 8 ) stained for endogenous hGBP1 . YFP - Caspase - 4C258S associated were quantified mean ± of combined data from three independent experiments are shown . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-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

(D) IFNγ-primed wild type and hGBP1-KO HeLa cells stably expressing inactive mutant YFP-Caspase-4C258S were infected with either cytosol-entering S. enterica Typhimurium mutant ΔsifA (MOI = 25) or with S. flexneri ΔospC3ΔipaH9.8 (MOI = 6), a mutant strain lacking Caspase-4 antagonist OspC3 as well as hGBP1 antagonist IpaH9.8. Cells were fixed at 4 hpi (ST ΔsifA) or at 2 hpi (Sf ΔospC3ΔipaH9.8) and stained for endogenous hGBP1. YFP-Caspase-4C258S associated bacteria were quantified and mean ± SEM of combined data from three independent experiments are shown. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( B were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( B ) Unprimed HeLa were infected with wild type or rfaL S . flexneri . Total unipolar localization of IcsA as well as actin tail formation were quantified at 1 hpi . Significance was determined by unpaired two - t - . n . s . , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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

(B Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (B) Unprimed HeLa cells were infected with wild type or rfaL S. flexneri. Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi. Significance was determined by unpaired two-tailed t-tests. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( C - mCherry - hGBP1 was expressed in HeLa hGBP1 - KO infected with S . flexneri ΔipaH9 . 8 stained for IcsA , N - WASP Significance was determined by unpaired t - , two - . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (C- mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for IcsA, N-WASP Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . D ) mCherry - hGBP1 was expressed in HeLa hGBP1 - KO infected with S . flexneri ΔipaH9 . 8 stained for Arp2 , F - actin at 2 . 5 hpi . Significance was determined by unpaired t - , two - . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. D) mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for Arp2, and F-actin at 2.5 hpi. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( E ) IFNγ - primed unprimed wild type hGBP1 - KO HeLa were infected with S . flexneri ΔipaH9 . 8 subcellular localization of IcsA , N - WASP , Arp2 , F - actin was assessed quantified at 2 . 5 hpi . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (E) IFNγ-primed and unprimed wild type and hGBP1-KO HeLa cells were infected with S. flexneri ΔipaH9.8 and subcellular localization of IcsA, N-WASP, Arp2, and F-actin was assessed and quantified at 2.5 hpi. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( B ) Starvation - induced autophagosomes ( AP ) autolysosomes ( AL ) in fat ( functional analogue of liver ) of a fruit fly larva . Note that APs contain intact cytoplasm , whereas contents of ALs show various stages of degradation .

O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-TISSUE O O O O B-TISSUE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O

(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, whereas the contents of ALs show various stages of degradation.


( C ) Liver of starved mice carrying a fluorescently tagged LC3 transgene , labeling cup - shaped ring - shaped structures that correspond to IMs autophagosomes , respectively . courtesy of Ryan Scott ( B ) Dr . Noboru Mizushima ( C ) .

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

(C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively. Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).


( A ) Northern blot experiments show expression of RsaI during growth phase in HG001 in MHB medium with or without addition of 1 , 5 g / L of D - glucose . Glucose was added either at beginning of growth ( + glucose , left panel ) or after 3h of growth ( right panel ) .

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

(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).


( B ) Northern blot experiment shows expression of RsaI during growth phase in HG001 , ∆ ccpA mutant , ∆ codY mutant , in MHB medium with ( + ) or without ( - ) addition of 1 , 5 g / L of D - glucose .

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

(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.


( C ) Northern blot experiment shows expression of RsaI in HG001 ∆ ccpA mutant strains . Total was prepared after 2 , 3 , 4 , 5 6h of culture in BHI medium at 37 ° C .

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

(C) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ccpA mutant strains. Total RNA was prepared after 2, 3, 4, 5 and 6h of culture in BHI medium at 37°C.


( D ) Northern blot of RsaI in HG001 grown in MHB medium with or without addition of 1 g / L of glucose , fructose or xylose . For experiments , loading controls were done using expression of 5S rRNA ( 5S ) as revealed after hybridization of membranes with a specific probe . However for these controls , we used aliquots of same preparations but migration of samples was performed in parallel to experiments on a separate agarose gel because RsaI 5S rRNA have very similar sizes .

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

(D) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.


( B ) Gel retardation assays show formation of between RsaI icaR , glcU _ 2 fn3K mRNAs . 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 3 ( RsaI mut3 , deletion of two G - track motifs ) , RsaI mutant 4 ( RsaI mut4 , deletion of C / U rich unpaired interhelical region ) were incubated with increasing concentrations of mRNAs : lane 1 , 0 nM ; lane 2 , 25 nM ; lane 3 , 50 nM ; lane 4 , 100 nM lane 5 , 200 nM . Below gels , predicted interactions between RsaI its targets are shown . Translation start codons are in green SD is for Shine Dalgarno . Graphs represented % of formed between either RsaI or its two mutant forms ( RsaI mut3 RsaI mut4 ) target mRNA ( icaR , glcU _ 2 , fn3K ) as function of mRNA concentrations .

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

(B) Gel retardation assays show the formation of the complex between RsaI and icaR, glcU_2 and fn3K mRNAs. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 3 (RsaI mut3, deletion of the two G-track motifs), and RsaI mutant 4 (RsaI mut4, deletion of the C/U rich unpaired interhelical region) were incubated with increasing concentrations of mRNAs: lane 1, 0 nM; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM and lane 5, 200 nM. Below the gels, the predicted interactions between RsaI and its targets are shown. Translation start codons are in green and SD is for Shine Dalgarno sequence. Graphs represented the % of complex formed between either RsaI or its two mutant forms (RsaI mut3 and RsaI mut4) and the target mRNA (icaR, glcU_2, fn3K) as the function of mRNA concentrations.


( C ) Gel retardation assays show formation of between RsaI RsaG . 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 2 ( RsaI mut2 ) , RsaI mutant 4 ( RsaI mut4 ) , RsaI mutant 1 ( RsaI mut1 ) were incubated with increasing concentrations of RsaG given in nM on top of autoradiographies .

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

(C) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.


( A ) Toe - print assays showing effect of RsaI on formation of ribosomal initiation of glcU _ 2 fn3K mRNAs , respectively . Lane 1 : incubation control of mRNA alone ; lane 2 : incubation control of mRNA with 30S subunits ; lane 3 : incubation control of mRNA with RsaI ; lane 4 : formation of ribosomal initiation containing mRNA , 30S initiator tRNAfMet ( tRNAi ) ; lanes 5 to 9 : formation of initiation in presence of increasing concentrations of RsaI , respectively : 50 nM ( lane 5 ) , 100 nM ( lane 6 ) , 150 nM ( lane 7 ) , 300 nM ( lane 8 ) , 400 nM ( lane 9 ) . Lanes T , A , C , G : sequencing ladders . Shine Dalgarno ( SD ) , start site of translation ( START ) toe - printing signals ( + 16 ) are indicated . At bottom of gels are shown predicted interactions between RsaI its targets . Translation start codons are in green , Shine Dalgarno ( SD ) is underlined , arrowheads depict toe - printing signals .

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

(A) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals.


( B ) β - galactosidase activity ( Miller Units ) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter : PrpoB : : glcU : : lacZ : : rsaI , PrpoB : : fn3K : : lacZ : : rsaI , PrpoB : : treB : : lacZ : : rsaI , PrpoB : : HG001 _ 1242 : : lacZ : : rsaI , PrpoB : : HG001 _ 2520 : : lacZ : : rsaI expressed in mutant HG001 - ∆ rsaI . same constructs were made in absence of rsaI gene . For fn3K - lacZ fusion , we also used an additional construct PrpoB : : fn3K : : lacZ : : rsaImut4 expressing both fusion FN3K - LacZ RsaI mut4 . β - galactosidase activity was normalized for density results represented mean of four independent experiments . error bars are standard deviations statistical significance was determined using Student ' s t - . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 , ns is for not significant .

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

(B) The β-galactosidase activity (Miller Units) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter: PrpoB::glcU::lacZ::rsaI, PrpoB::fn3K::lacZ::rsaI, PrpoB::treB::lacZ::rsaI, PrpoB::HG001_1242::lacZ::rsaI, and PrpoB::HG001_2520::lacZ::rsaI expressed in the mutant strain HG001-∆rsaI. The same constructs were made in the absence of rsaI gene. For the fn3K-lacZ fusion, we also used an additional construct PrpoB::fn3K::lacZ::rsaImut4 expressing both the fusion FN3K-LacZ protein and RsaI mut4. The β-galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments. The error bars are standard deviations and the statistical significance was determined using the Student's t-test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, ns is for not significant.


( B ) Gel retardation assays show formation of between RsaI icaR full , icaR SUBST , icaR - 5 ' UTR , icaR - 3 ' UTR . 5 ' end - labeled of RsaI was incubated with increasing concentrations of various mRNAs . UTR is for untranslated region , SUBST stands for substitution of UCCCCUG by AGGGGAC . This mutation , which is located in 3 ' UTR of icaR , significantly destabilizes long - range interaction , enhances icaR translation . Lane C represents binding between radiolabelled RsaI full - icaR mRNA ( 50 nM ) . Due to rather short migration of gel , full - mRNA is still observed in pocket .

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

(B) Gel retardation assays show the formation of the complex between RsaI and icaR full length, icaR SUBST, icaR-5'UTR, and icaR-3'UTR. The 5' end-labeled of RsaI was incubated with increasing concentrations of the various mRNAs. UTR is for untranslated region, SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC. This mutation, which is located in the 3'UTR of icaR, significantly destabilizes the long-range interaction, and enhances icaR translation. Lane C represents binding between radiolabelled RsaI and full-length icaR mRNA (50 nM). Due to the rather short migration of the gel, the full-length mRNA is still observed in the pocket.


( C ) In vivo effect of RsaI on PIA - PNAG synthesis in S . aureus wild - type ( WT ) 132 , ∆ rsaI mutant , carrying a deletion of icaR 3 ' UTR ( ∆ 3 ' UTR ) . This last mutant has been transformed with pES plasmids expressing rsaI or rsaI mut5 . PIA - PNAG exopolysaccharide biosynthesis was quantified using dot - blot assays . Serial dilutions ( 1 / 5 ) of samples were spotted onto nitrocellulose membranes PIA - PNAG production was detected with specific anti - PIA - PNAG antibodies . RsaI was detected in same samples by Northern blot using a probe directed against RsaI . Ethidium bromide of rRNA was used as loading controls of same gel .

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

(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.


( A ) Ternary formation between RsaI , various mRNAs ( glcU _ 2 , HG001 _ 00942 HG001 _ 1242 ) , RsaG . 5 ' - end labeled RsaI was incubated with increasing concentrations of target mRNA alone or in presence of 50 nM of RsaG . various complexes are notified on sides of autoradiographies .

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

(A) Ternary complex formation between RsaI, various mRNAs (glcU_2, HG001_00942 and HG001_1242), and RsaG. The 5'-end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG. The various complexes are notified on the sides of the autoradiographies.


( B ) RsaG does not form stable complexes with glcU _ 2 HG001 _ 01242 . Binding assays were done in presence of 5 ' end - labeled RsaG either cold RsaI ( 300 nM ) or increasing concentrations of glcU _ 2 HG001 _ 01242 .

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

(B) RsaG does not form stable complexes with glcU_2 and HG001_01242. Binding assays were done in the presence of 5' end-labeled RsaG and either cold RsaI (300 nM) or increasing concentrations of glcU_2 and HG001_01242.


( C ) Measurements of half - lives of RsaI RsaG in HG001 - ∆ rsaG or HG001 - ∆ rsaI mutant strains using Northern blot experiments . were treated with rifampicin at 4h of growth total RNAs were extracted after 2 , 4 , 8 , 15 , 30 60 min at 37 ° C in BHI . 5S rRNA was probed to quantify yield of RNAs in each lane using same samples , which were however run on two different gels . Calculated half - lives are shown beneath autoradiographies are average of 2 experiments . data were normalized to 5S rRNA .

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

(C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.


B Hops − / − mouse genotyping expression . Genotyping by PCR using genomic showed a 242bp band for wild type ( Hops + / + ) allele a 331bp band for mutant ( Hops − / − ) allele . NTC is Null Template Control ( left panel ) . Genotyping by real time PCR confirmed absence of Hops mRNA in Hops − / − mice ( middle panel ) . extracts from Hops + / + Hops − / − MEFs were analysed by Western Blot using anti - HOPS antibody anti - α - tubulin antibody as loading control ( right panel ) . Data information : in B data are presented as mean ± SD .

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

B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD.


C Offspring distribution . Different HOPS genotypes at weaning ( approximately postnatal day 21 ) from crosses between homozygous knockout mice heterozygous ( n = 1525 pups ) . Data information : In C χ2 was performed to determine significant differences between expected observed ratios of Hops − / − to Hops + / − mice . P < 0 . 001 , by two Student ' s t - .

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

C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups). Data information: In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.


A Reduced caspase - 3 activation in Hops − / − mice upon apoptotic stimulus . Hops + / + Hops − / − mice were treated with etoposide for indicated times thymus , spleen , testis liver were harvested Tissue - Tek ® O . C . T . embedded . Sections were immunostained with anti - caspase - 3 antibody followed by appropriate secondary antibody labelling ( green ) . graphs display relative positive number . Scale bars , 10µm . Data information : experiments were performed three times in three different mice per condition a representative example is shown . In A data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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

A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS affects p53 increase after stress in vivo . Whole - homogenates from organs , were immediately prepared immunoblotted with antibodies to detect p53 GAPDH . p53 were semi - quantified using GAPDH as loading control relative p53 at time 0 were assumed as 1 . Data information : experiments were performed three times in three different mice per condition a representative example is shown . In B data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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

B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


A Thymocytes from Hops + / + Hops − / − mice were treated with dexamethasone ( Dex ) apoptotic were measured by flow cytometry . flow cytometry plots are shown on left data represented as a histogram on right . Data information : experiments were performed three times in from three different mice per condition . In A data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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

A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS was pivotal for apoptosis induction . Hops + / + Hops − / − MEFs were subjected to etoposide ( Eto ) apoptosis was analysed by flow cytometry ( left ) showed as a graph ( right ) . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown . In data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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

B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


C HOPS overexpression in MEFs promotes apoptosis . Hops + / + Hops − / − MEFs were transiently transfected with an empty vector ( pEGFP - N1 ) or with a Hops encoding vector ( pEGFP - N1 - Hops ) . apoptotic were measured by flow cytometry ( left ) on GFP positive negative populations showed as a graph ( right ) . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown . In C data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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

C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


D HOPS affects p53 following stress in vitro . Hops + / + Hops − / − MEFs were treated with etoposide harvested at indicated time points . p53 were semi - quantified using α - tubulin as loading control relative p53 at 0 µM dosage were assumed as 1 . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown .

O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-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 B-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O O

D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.


A HOPS loss p53 half - life . Hops + / + Hops − / − MEFs untransfected or transfected with HOPS , were analysed by immunoblotting following cycloheximide treatment for indicated time ( left panel ) . p53 were semi - quantified using GAPDH as loading control relative p53 at time 0 were assumed as 100 % ( right panel ) . Data information : experiments in MEFs were performed three times . Representative panels are shown . In A data are presented as mean ± SD .

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

A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A data are presented as mean ±SD.


B p19Arf - independent HOPS control of p53 half - life . p19Arf − / − MEFs untransfected ( CTRL ) , transfected with empty vector ( pSGV ) or HOPS were treated with cycloheximide for indicated times subjected to immunoblot with anti - p53 , anti - HOPS anti - GAPDH antibodies ( left panel ) . p53 were semi - quantified as above ( right panel ) . Data information : experiments in MEFs were performed three times . Representative panels are shown . In B data are presented as mean ± SD .

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

B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In B data are presented as mean ±SD.


C HOPS p53 ubiquitination . p53 , HDM2 , His - tagged ubiquitin were co - expressed in H1299 with HOPS or HOPS - G176A or HOPS - K126A mutants treated with MG132 . p53 ubiquitination rate was evaluated by elution using Ni2 + - NTA His - bind resin followed by immunoblotting with anti - p53 indicated antibodies .

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

C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.


D HOPS depletion HOPS - G176A mutant affect p53 ubiquitination . Hops + / + Hops − / − MEFs untransfected Hops − / − MEFs transfected with HOPS or HOPS - G176A mutant were co - expressed with p53 , HDM2 His - tagged ubiquitin . were treated with MG132 ubiquitinated p53 fraction was recovered as above . Immunodetection of ubiquitinated p53 was performed using anti - p53 antibody samples were tested with indicated antibodies . Data information : experiments in MEFs were performed three times . Representative panels are shown .

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

D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


E HOPS - G176A mutant ability to stabilize p53 . Hops − / − MEFs were transfected with HOPS - G176A , subjected to cycloheximide treatment for indicated times analysed by immunoblotting using indicated antibodies . Data information : experiments in MEFs were performed three times . Representative panels are shown .

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

E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


A binding in RKO . PLA showed HOPS / p53 interaction with or without etoposide treatment at indicated time . Red spots showed a single interaction . counterstaining was performed with DAPI . α - Tubulin - FITC antibody was used to identify edge . Background control was performed according to manufacturer ' s instruction . Scale bars , 10µm . graph represents binding increase . Non treated binding was assumed as 1 . Data information : experiments were performed three times , representative examples are shown . In A data are presented as mean ± SD .

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

A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.


B Biochemical of - distribution . RKO were treated with etoposide , collected after 2 4 hours processed to obtain fractions . Aliquots relative to same number per fraction were immunoblotted using anti - HOPS anti - p53 antibodies . GAPDH Lamin B1 are purity controls of fraction respectively .

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

B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.


C HOPS p53 binding in cytoplasm following apoptosis induction . RKO untreated or treated with etoposide as above were used to isolate fractions . WCL fractions were immunoprecipitated with anti - HOPS antibody subsequently immunoblotted using anti - p53 antibody .

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

C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.


A HOPS localization at mitochondria . RKO were stained by MitoTracker ™ ( red ) , immediately fixed stained to visualize HOPS ( green ) . Nuclei were DAPI stained ( blue ) . Samples were analysed with microscope representative were shown . Scale bars , 10µm .

O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O

A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.


B RKO were lysed processed for mitochondria isolation . WCL , cytoplasm mitochondria were analysed using anti - HOPS antibody . GAPDH , Lamin B1 Tom20 are purity controls of mitochondrial fraction .

O B-CELL_LINE O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O

B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.


C HOPS p53 translocation to mitochondria . RKO were treated with etoposide as indicated processed for mitochondrial extraction subsequent immunoblotting for p53 , HOPS reported extraction purity controls .

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

C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.


D HOPS / p53 binding at mitochondria . RKO were treated with etoposide subjected to mitochondrial purification , subsequent immunoprecipitation using anti - HOPS antibody . Samples were immunoblotted to detect p53 , HOPS reported controls .

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_LINE O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O

D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.


E HOPS p53 mitochondrial translocation in vivo . Hops + / + Hops − / − mice were treated with etoposide thymus , spleen testis were harvested . Organs were homogenized mitochondrial fraction were isolated analysed by immunoblotting versus p53 . GAPDH Tom20 are purity controls . p53 mitochondrial were semi - quantified using Tom20 as loading control p53 at time 0 were assumed as 1 . Data information : assays involving were performed three times in three different mice per condition . In E data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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

E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


F p53 - dependent mitochondrial potential ( Δψm ) induction in MEFs . Hops + / + Hops − / − MEFs were treated with etoposide then incubated with JC - 1 vital dye . were collected by microscopy representative are shown ( left panel ) . Scale bars , 10µm . was analysed by TECAN plate reader plotted in graph ( right panel ) . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times . In F data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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

F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


G p53 MOMP induction in HOPS deficiency . Mitochondria from Hops + / + Hops − / − MEFs , were purified incubated with p53 recombinant . Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting . Samples were tested to detect p53 as control Tom20 as mitochondria structural marker . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times .

O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O

G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.


A HOPS expression p53 accumulation . HOPS was overexpressed in RKO were fixed stained with antibody against p53 ( green ) . Nuclei were stained with DAPI ( blue ) . Samples were analysed with microscope merged are shown ( right panel ) . Scale bars , 10µm . were scored for p53 localization . results are shown as of versus p53 localization in untransfected HOPS transfected ( left panel ) . Data information : experiments detailed above were performed three times , representative panels are shown . In A data are presented as mean ± SD .

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY 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

A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.


B Biochemical of HOPS intracellular localization . RKO were transfected or not with HOPS fractionated into component . Aliquots relative to same number per fraction were immunoblotted using anti - p53 anti - HOPS antibodies . GAPDH Lamin B1 are purity controls of fraction respectively . Data information : experiments detailed above were performed three times , representative panels are shown .

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

B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


HOPS p53 binding to importin α . Untransfected or HOPS transfected RKO were treated with etoposide each sample was evaluated by immunoprecipitation with anti - p53 analysed by immunoblotting using anti - importin α antibody . Arrowhead indicates importin α specific signal . Data information : experiments detailed above were performed three times , representative panels are shown .

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

HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


D PCAF - mediated p53 acetylation . p53 PCAF Flag - tagged were co - expressed in H1299 with or without HOPS treated with trichostatin A nicotinamide . p53 acetylation were evaluated by immunoprecipitation with anti - p53 antibody followed by immunoblotting with anti - acetylated - Lysine ( AcK ) , anti - p53 , anti - Flag , anti - HOPS anti - α - tubulin antibodies . Data information : experiments detailed above were performed three times , representative panels are shown .

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

D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


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

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

(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 infected with control retroviruses with empty vector U2OS overexpressing siRNA - resistant HA - GRWD1 were treated analyzed as above .

O O O O B-CELL_LINE O O O B-ORGANISM O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O

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

O O O B-CELL_LINE O O O O O 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 B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O

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

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O 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 O O

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

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

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

O O O B-CELL_LINE O O O O O 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 B-EXP_ASSAY O O O O

(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 treated as above were stained with propidium iodide subjected to flow cytometry .

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

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


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

O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O B-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 B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O

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

O O O B-CELL_LINE O 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

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

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

(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 were immunoprecipitated with anti - GRWD1 antibody or control IgG . Immunoprecipitates ( IPs ) 2 . 7 % of inputs were immunoblotted with indicated antibodies .

O O O O O O B-CELL_LINE O B-EXP_ASSAY O O O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O

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

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

(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 bound analyzed by immunoblotting with indicated antibodies . I , input . P , pull - down .

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

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

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

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

O O 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 O O O O O O O B-SMALL_MOLECULE O O O O O O 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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY 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 were co - transfected with 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 analyzed by immunoblotting with indicated antibodies . GFP serves as a control to show equal transfection efficiencies .

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

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

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

(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 to detect MDM2 autoubiquitination in H1299 . Lysates were prepared from H1299 transfected with 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 , then immunoprecipitated with anti - MDM2 antibody . Immunoprecipitates ( IPs ) inputs were immunoblotted with indicated antibodies .

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

(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 with anti - Omni probe antibody . Recombinant p53 was incubated with E1 , E2 ( UbcH5a ) , His - ubiquitin , ATP , GST - RPL11 , GRWD1 - His , immunopurified His - Xpress - MDM2 or control immunoprecipitates at 30 ℃ for 120 min as indicated . samples were resolved by SDS - PAGE followed by immunoblotting with indicated antibodies .

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

(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 introduced effect on p53 . HFF2 / T stably expressing HA - GRWD1 , E7 , KRAS G12V were established with retroviral vectors analyzed by immunoblotting with indicated antibodies . * , endogenous RAS .

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

(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 C ) Anchorage - independent growth of HFF2 / T overexpressing E7 with or without HA - GRWD1 . Three after seeding , were acquired colonies > 100 µm in diameter were counted . Representative 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 .

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

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

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

(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 expressing E7 KRAS G12V with or without HA - GRWD1 . were subcutaneously injected into nude mice ( 1 x 106 / mouse ) , was measured . Average volumes , with SDs , are shown ( n = 5 ) .

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

(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 with wild type p53 but not with mutated p53 . data were obtained from TCGA databases ( cBioPortal for Cancer Genomics ; http : / / www . cbioportal . org ) . For detail , see Materials Methods .

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

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

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

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

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

(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 were co - transfected with 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 , then analyzed by immunoblotting with indicated antibodies . GFP serves as a control to show equal transfection efficiencies .

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

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

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

(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 F ) Anchorage - independent growth of HFF2 / T overexpressing E7 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 are shown in ( E ) . Scale bars , 500 um . Average numbers of colonies ( > 200 um in diameter ) per photographed with SDs are shown in ( F ) . Seventy randomly selected areas were counted .

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


collected in GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by N - terminus targeting properties on theoretical expectation summarized in ( B ) with MRPs possessing MTS - like N - termini in panel ( C )

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

Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) with the MRPs possessing MTS-like N-termini in panel (C)


collected in GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by N - terminus targeting properties on theoretical expectation summarized in ( B ) MRPs with intermediate phenotype in panel ( D ) MRPs without N - terminal signal in panel ( E ) , for each MRP a yeast gene name new nomenclature name is shown on left . Scale bar for is 5 µm .

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

Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) MRPs with intermediate phenotype in panel (D) and MRPs without N-terminal signal in panel (E), for each MRP a yeast gene name and new nomenclature protein name is shown on the left. Scale bar for all micrographs is 5 µm.


( A ) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by microscopy with MitoTracker Orange straining . Scale bar for is 10 µm .

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

(A) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm.


( B ) Characterization of Mrp17 translocation signal using an in vitro import : shown are autoradiographs of full - Mrp17 or its truncations fused to DHFRmut , translated in vitro with radiolabeled - , incubated with isolated yeast mitochondria for 2 , 5 , or 10 min , treated with proteinase K ( PK ) to remove nonimported visualized by 16 % SDS - PAGE / autoradiography . As a negative control , mitochondria were treated with valinomycin , antimycin oligomycin ( VAO ) that eliminate potential . For comparison , 20 % of used per import reaction was loaded on first lane .

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

(B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.


( B ) In vitro mitochondria translocation capacity of WT Mrp17 , Mrp17K - R with lysines ( K ) substituted with arginines ( R ) , Mrp17K - A with lysines ( K ) substituted with alanines ( A ) fused to DHFRmut . Import was performed as described in Fig . 2 legend .

O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O

(B) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend.


( E ) Mrp17 variants rescue ∆ mrp17 growth defect on respiratory media : indicated variants or empty vector ( EV ) were introduced in WT yeast then genomic MRP17 was disrupted by knock out , resulting mutants were serially diluted 10 × spotted on media containing glucose or glycerol as a sole carbon source .

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

(E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.


( A ) in vitro import of Mrp17 - DHFRmut is sensitive to elimination of outer mitochondrial by trypsinization ( left ) , more so than model import substrate Atp1 ( right ) . Mitochondria were incubated with indicated concentrations of trypsin import was performed as described in legend for Fig . 2 . Proteinase K ( PK ) was not added to sample without mitochondria . ( B ) In vitro translocation of Mrp17 - DHFRmut into mitochondria isolated from WT ∆ tom20 yeast showing reduced translocation in ∆ tom20 background .

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

(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria. (B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background.


( G ) , ( H ) Import of Mrp17 - DHFRmut control Atp1 Aac1 into WT , tim17 - ts ( G ) tim22 - ts ( H ) mitochondria . Import was performed at indicated temperatures , proteinase K was added to samples except loading control in first lane of each autoradiograph ( 20 % of amount used for each import reaction ) . full autoradiograph for Aac1 with molecular markers is shown in Appendix Figure S6D .

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

(G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.


( C ) Expression of structurally conserved RPs fused to GFP in yeast ( Full in al channels are shown on Fig . EV5 ) . Scale bar is 10 µm .

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

(C) Expression of structurally conserved bacterial RPs fused to GFP in yeast cells (Full micrographs in al channels are shown on Fig. EV5). Scale bar is 10 µm.


( E ) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria . Lanes are labeled as described in Fig . 2 legend .

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

(E) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria. Lanes are labeled as described in Fig. 2 legend.


( B ) On right : MRP N - termini structural environment ( solvent exposure ) : average number of atoms belonging to same , other chains or in 13Å radius from first structured acid Cα atom , plotted separately for with their most N - terminus appearing in structure vs with precisely MTS , other which have a longer unstructured extension ( see classification legend on bottom ) calculated on PDB : 5MRC ; on right : same as on left but only coordination number ( number of Cα atoms in 13Å radius ( Hamelryck , 2005 ) ) is calculated , * * p - value < 0 . 01 ( Mann - Whitney ) , absolute values were measured without replicates ; on bottom : MRPs were divided in three groups depending on relative position of MTS structured residues : " No MTS ( str ) " - structure starts at very N - terminus without a cleavable MTS , " Precise MTS " - MTS is off 1 - 2 before structure starts , " Other " - unstructured uncleaved present before structure start .

O O O O O O O O O O O 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 I-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 B-SMALL_MOLECULE I-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 O 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) On the right: MRP N-termini structural environment (solvent exposure): average number of atoms belonging to the same protein, other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom, plotted separately for proteins with their most N-terminus appearing in the structure vs proteins with precisely cleaved MTS, and other proteins which have a longer unstructured extension (see the classification legend on the bottom) calculated based on PDB:5MRC; on the right: same as on the left but only coordination number (number of protein Cα atoms in the 13Å radius (Hamelryck, 2005)) is calculated, ** p-value<0.01 (Mann-Whitney test), absolute values were measured without replicates; on the bottom: MRPs were divided in three groups depending on relative position of the MTS and structured residues: "No MTS (str)" - structure starts at the very N-terminus without a cleavable MTS, "Precise MTS" - MTS is cleaved off 1-2 amino acids before the structure starts, "Other" - unstructured uncleaved amino acids present before the structure start.


mRNA expression of CiTLR5a - HA ( D ) , CiTLR5b - HA ( D ) , CiTLR5a - GFP ( E ) , CiTLR5b - GFP ( E ) plasmids in FHM were examined by qRT - PCR ( upper panel ) with EF1α gene as an internal control by immunoblotting ( IB ) ( lower panel ) with β - tubulin as a reference ( n = 4 ) . Data information : data are presented as mean ± SD . two - Mann - Whitney U - was used for ( D ) ( E ) . n represents biological replicates . * * * P < 0 . 001 .

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

mRNA and protein expression of CiTLR5a-HA (D), CiTLR5b-HA (D), CiTLR5a-GFP (E), and CiTLR5b-GFP (E) plasmids in FHM cells were examined by qRT-PCR (upper panel) with EF1α gene as an internal control and by immunoblotting (IB) (lower panel) with β-tubulin as a reference (n = 4). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D) and (E). n represents biological replicates. ***P < 0.001.