C BN ‐ PAGE confirms formation of MPCFERM MPCOX complexes after constitutive plasmid ‐ expression . Non ‐ specific reactivity of antibody is denoted by an asterisk ( * ) . It has to be noted that complexes migrate slightly faster than in Fig C , most likely because MPC are tagged with a single epitope tag as opposed to 3HA ‐ tag in Fig C .

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C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.


D Mpc1 is co ‐ immunoprecipitated with Mpc2 or Mpc3 . Mitochondria isolated from harboring appropriate expression plasmids grown in glycerol ‐ containing medium were lysed in 1 % digitonin , either Mpc2 ‐ Flag or Mpc3 ‐ Flag was immunoprecipitated . Mpc1 ‐ HA was detected by Western blot with an antibody directed against epitope tag . matrix aconitase ( Aco1 ) 40 ‐ kDa translocase of outer subunit ( Tom40 ) were not co ‐ immunoprecipitated .

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D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.


E , F Stoichiometry of Mpc1 complexes . Chemical cross ‐ linking indicates that Mpc1 / Mpc2 Mpc1 / Mpc3 form heterodimers of predicted molecular . DSG , disuccimidyl glutarate . Cross ‐ linking products with unknown partners are labeled with a question mark ( ? ) .

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E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).


A , B Determination of MPC topology by IASD labeling of isolated mitochondria expressing indicated single cysteine variants of Mpc1 ( A ) Mpc3 ( B ) . Mitochondria were treated with IASD as indicated . Negative [ quenching by pretreatment with DTT ( pre ‐ DTT ) ] positive [ lysis with 0 . 5 % Triton X ‐ 100 ( T ‐ X100 ) ] controls for labeling reaction are shown . As a further control , also translocase of inner subunit Tim23 is detected by Western blot , which contains three endogenous cysteines , of which are located in ‐ embedded regions . Labeling of an IMS ‐ exposed cysteine with IASD is indicated by a mobility shift on SDS - PAGE corresponding to molecular of IASD ( ˜ 500 Da ) .

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A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).


C Protease protection in mitochondria expressing Mpc1 ‐ GFP , Mpc2 ‐ GFP , or Mpc3 ‐ GFP . Intact mitochondria ( M ) , mitoplasts with a ruptured outer after hypo ‐ osmotic swelling ( Sw ) , or mitochondrial lysates with 0 . 5 % Triton X ‐ 100 ( Tr ) were treated with proteinase K ( PK ) . Loading controls are Tom70 ( outer ) , Tim23 ( inner ) , Mdh1 ( matrix ) . GFP ‐ fused Mpc2 Mpc3 are degraded in mitoplasts , whereas Mpc1 ‐ GFP is not .

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C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.


A MPCOX has higher transport activity than MPCFERM . uptake of 14C ‐ labeled pyruvate into intact mitochondria was measured in vitro . Mitochondria had been isolated from grown in glycerol ‐ containing medium expressing no subunit ( vectors ) , MPCFERM , MPCOX , Mpc2 , or Mpc3 . Imported pyruvate was quantified by re ‐ isolation of mitochondria subsequent scintillation counting after 1 , 2 , or 5 min of incubation with [ 14C ] ‐ pyruvate . B Imported [ 14C ] ‐ pyruvate as in ( A ) after 5 ‐ min incubation . difference between MPCOX MPCFERM ( * * P = 0 . 0037 ) was significant ( unpaired t ‐ ) .

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A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).


C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit ( vectors ) , MPCFERM , or MPCOX .

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C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit (vectors), MPCFERM, or MPCOX.


chimeric construct Mpc2C3 is expressed in isolated mitochondria , while Mpc3C2 is not . Mpc1 ‐ HA Flag ‐ tagged chimeric were detected by Western blotting in isolated mitochondria .

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The chimeric construct Mpc2C3 is expressed in isolated mitochondria, while Mpc3C2 is not. Mpc1‐HA and the Flag‐tagged chimeric proteins were detected by Western blotting in isolated mitochondria.


Mpc2C3 forms a carrier with Mpc1 , as analyzed by BN ‐ PAGE . When co ‐ expressed with Mpc1 , Mpc2C3 shows same pattern as Mpc3 , including 300K .

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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.


Pyruvate transport activity of Mpc1 / Mpc2C3 reaches ˜ 85 % of MPCOX activity . Uptake of [ 14C ] ‐ pyruvate into mitochondria isolated from yeastcells expressing indicated MPC combinations was measured in vitro . Difference between MPCFERM Mpc1 / Mpc2C3 is significant ( unpaired t ‐ ; * P = 0 . 02 ) . Means of n = 4 experiments are shown , with error bars representing .

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Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.


( C ) Interaction between hCAP - G hCAP - H . lysates co - expressing hCAP - G ( residues 1 - 478 , 554 - 900 ) hCAP - H ( residues 394 - 515 ) , either wild type ( WT ; lanes 2 , 10 13 ) , 3Q ( F463Q , F469Q F473Q ; lane 3 ) , 5Q ( F463Q , F469Q , F473Q , F501Q Y503Q ; lane 4 ) , 3A ( F463A , F469A F473A ; lane 5 ) , 5A ( F463A , F469A , F473A , F501A Y503A ; lane 6 ) or 2A ( F501A Y503A ; lane 11 ) , or a C - terminal deletion mutant ( 506 - 514 residues were deleted from 394 - 515 ; lane 14 ) were applied to Ni - NTA agarose resin , bound fraction was analyzed by SDS - PAGE . Alternatively , a lysate co - expressing mutant hCAP - G ( D647K ) wild - type hCAP - H was examined ( lane 7 ) . uninduced lysate was also used as a negative control ( lane 9 ) .

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(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).


( A ) Expression of condensin I subunits in insect . wild - type ( WT ) or IV - 5Q mutant CAP - H subunit was co - expressed with other four subunits ( GST - SMC4 , SMC2 , CAP - D2 CAP - G ) in insect . lysates were prepared subjected to SDS - PAGE , followed by immunoblotting with a mixture of antibodies against SMC2 SMC4 ( left panel ) or against CAP - D2 , - G , - H ( right panel ) .

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(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel).


( B ) Purification of WT IV - 5Q mutant condensin I complexes . samples purified through glutathione - affinity chromatography were subjected to SDS - PAGE analyzed by CBB ( left panel ) or immunoblotting with a mixture of antibodies as described above ( middle right panels ) .

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(B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels).


( C ) Add - back using WT mutant condensin I complexes . Xenopus extracts depleted of endogenous condensin complexes were supplemented with purified complexes ( from top to bottom ; WT , IV - 5Q , ∆ G , ∆ G [ IV - 5Q ] ) . supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes . samples were fixed labeled with an antibody against mSMC4 ( red ) . was counterstained with DAPI ( blue ) . data from a single representative experiment out of two repeats are shown . In experiment shown here , multiple were collected for condensin - depleted extracts supplemented with WT ( n = 17 ) , IV - 5Q ( n = 22 ) , ∆ G ( n = 20 ) ∆ G ( IV - 5Q ) ( n = 25 ) complexes . scale bar represents 10 μm .

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(C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.


( A ) Purification of hCAP - G hCAP - G - H subcomplexes : wild type ( WT ) , CAP - G K60D / R848E double mutant ( K60D / R848E ) CAP - G R168E mutant ( R168E ) . Purified samples were subjected to SDS - PAGE analyzed by CBB .

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(A) Purification of hCAP-G and hCAP-G-H subcomplexes: wild type (WT), CAP-G K60D/R848E double mutant (K60D/R848E) and CAP-G R168E mutant (R168E). Purified protein samples were subjected to SDS-PAGE and analyzed by CBB staining.


( B ) Double - stranded ( dsDNA ) single - stranded binding of hCAP - G . 30 - bp dsDNA was incubated with no ( lanes 1 ) or increasing amounts of WT hCAP - G ( WT ; lanes 2 - 4 ) . 30 - mer ssDNA was incubated with no ( lanes 5 ) or increasing amounts of WT hCAP - G ( WT ; lanes 6 - 8 ) .

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(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).


( C ) dsDNA binding for hCAP - G - H subcomplexes . 30 - bp dsDNA was incubated with no ( lanes 1 , 5 9 ) , increasing amounts of WT hCAP - G - H subcomplex ( WT ; lanes 2 - 4 ) , CAP - G K60D / R848E double mutant ( K60D / R848E ; lanes 6 - 8 ) or CAP - G R168E mutant ( R168E ; lanes 10 - 12 ) .

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(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).


(D) The ssDNA binding assay for the hCAP-G-H subcomplexes


W0 , W6 W12 refer to week of study . Serum FGF - 21 GDF - 15 before , during after treatment ( two - sided Wilcoxon signed - rank , P - value = 0 . 031 in significant cases ) ( see Fig . EV2 for details where P1 - P6 refer to ) .

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W0, W6 and W12 refer to the week of study. Serum FGF-21 and GDF-15 levels before, during and after treatment (two-sided Wilcoxon signed-rank test, P-value = 0.031 in all significant cases) (see Fig. EV2 for details where P1 - P6 refer to the individual patients).


Volcano plot showing differences in metabolite between 0 6 & 12 of treatment . Metabolites in histidine , alanine , aspartate glutamine metabolism , TCA cycle pathways are annotated at 10 % False Discovery Rate ( see Dataset EV1 for whole list of differentially regulated metabolites . Although some other metabolites showed a more pronounced difference , these did not fit into a recognised pathway ) . Metabolite labels are colour - coded according to identified KEGG pathways ( red = " Alanine , aspartate glutamate metabolism " , green = " Histidine metabolism " blue = " Citrate cycle ( TCA cycle ) " , with transition colour - code if metabolite belongs to more than one KEGG pathways ) .

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Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways).


Scatterplot depicting P - values from metabolites Pathway Enrichment ( y - axis ) impact values from Pathway Topology ( x - axis ) ( see Dataset EV2 for details ) . KEGG pathways at 10 % adjusted Holm - Bonferroni P - value are highlighted .

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Scatterplot depicting P-values from the metabolites Pathway Enrichment Analysis (y-axis) and impact values from Pathway Topology Analysis (x-axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm-Bonferroni P-value are highlighted.


Volcano plot of acyl - carnitine metabolites between 0 6 & 12 of treatment with highlighted differential metabolites at 10 % FDR .

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Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR.


Respiratory chain citrate synthase activity in skeletal muscle before after 12 of treatment . No significant differences were detected before after 12 of treatment by using two - sided Wilcoxon signed - rank with an empirical level of significance .

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Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two-sided Wilcoxon signed-rank test with an empirical level of significance.


P1 - P6 refer to . Representative examples of quadruple immunofluorescence of skeletal muscle fibres before after treatment . box in top right - corner indicates normal range , using two antibodies to NDUFB8 COX I .

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P1 - P6 refer to individual patients. Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right-hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I.


Quantitative quadruple immunofluorescence of skeletal muscle fibres in six showing a decrease ( two - sided Wilcoxon signed - rank with an empirical level of significance ) in proportion of IV deficient skeletal muscle fibres before after 12 of treatment ( empirical P - value = 0 . 048 ) .

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Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two-sided Wilcoxon signed-rank test with an empirical level of significance) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P-value = 0.048).


Hierarchical cluster of skeletal muscle - seq transcripts before after treatment showing no clear separation between untreated treated .

Hierarchical cluster analysis of skeletal muscle RNA-seq transcripts before and after treatment showing no clear separation between untreated and treated patients.


Volcano plot depicting differentially expressed - seq genes at 10 % FDR after 12 of treatment . Significantly different mammalian mitochondrial genes ( MitoCarta 2 . 0 ) are annotated .

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Volcano plot depicting differentially expressed RNA-seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated.


level m . 3243A > G in blood , urinary sediment skeletal muscle . No differences were detected by using two - sided Wilcoxon signed - rank with an empirical level of significance . W = week of study .

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Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two-sided Wilcoxon signed-rank test with an empirical level of significance. W = week of the study.


Cardiac parameters before after treatment ( two - sided Wilcoxon signed - rank with an empirical level of significance ) . ES = end - systolic volume ( ml ) ( empirical P - value = 0 . 047 ) index ( ml / m2 ) ( empirical P - value = 0 . 046 ) , measured by MRI . Horizontal lines on ( B ) , ( F ) ( G ) refer to statistical comparisons of mean values , where ns = non - significant , * P - value ≤ 0 . 05 . For normal reference ranges see ( Hollingsworth et al . , 2012 )

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Cardiac parameters before and after treatment (two-sided Wilcoxon signed-rank test with an empirical level of significance). ES = end-systolic volume (ml) (empirical P-value = 0.047) and index (ml/m2) (empirical P-value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05. For normal reference ranges see (Hollingsworth et al., 2012)


B - C ' ) Immunofluorescence of active - caspase 3 in ONL of P347S P347S / miR - 181a / b + / - . D , E ) TUNEL in ONL of P347S P347S / miR - 181a / b + / - . F ) of immunofluorescence in ( B - C ' ) ( N = 4 eyes P347S N = 6 eyes P347S / miR - 181a / b + / ) . G ) of TUNEL in ( D , E ) ( N = 3 eyes / genotype ) .

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

B-C') Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/-. D, E) TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/-. F) Quantification of immunofluorescence analysis in (B-C') (N=4 eyes P347S and N=6 eyes P347S/ miR-181a/b+/). G) Quantification of TUNEL analysis in (D, E) (N=3 eyes/genotype).


D - P ) Immunofluorescence showed amelioration of Rhodopsin localization ( D - F ' ; arrowheads ) ; C - arrestin expression ( G - I ' ) PR outer segment ( OS ) inner segment ( IS ) structures , as determined , respectively , by PNA ( J - L ' ) ; Phalloidin ( M - O ' ' ) in P347S / miR - 181a / b + / - vs P347S eyes at p30 . M ' ' - O ' ' show higher magnification of M - O . Scale bars 25 μm in D - O ' ; 5μm in M ' ' - O ' ' . densitometry of each is reported in ( P ) , N = 3 eye / genotype for each . WT vs P347S p values are reported in red , P347S vs P347S / miR - 181a / b + / - p values are reported in black .

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

D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black.


D - L ) Immunofluorescence of Rhodopsin ( D - E ' ) , C - arrestin ( F - G ' ) , PNA ( H - I ' ) ; Phalloidin ( J - K ' ' ) in P347S P347S / miR - 181a / b + / - retinas at p90 . J ' ' - K ' ' show higher magnification of J - K . Scale bars 25 μm in D - K ' ; 10μm in J ' ' - K ' ' .

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

D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''.


Immunofluorescence of Rhodopsin C - arrestin PNA Phalloidin in P347S P347S / miR - 181a / b + / - retinas at p90 . densitometry of each is reported in ( L ) , N = 3 eye / genotype / .

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

Immunofluorescence analysis of Rhodopsin C-arrestin PNA and Phalloidin staining in P347S and P347S/miR-181a/b+/- retinas at p90. Fluorescence densitometry quantification of each staining is reported in (L), N=3 eye/genotype/staining.


M ) Measurement of P347S P347S / miR - 181a / b + / - ONL thickness ( N ≥ 3 eyes / genotype ;

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

M) Measurement of P347S and P347S/miR-181a/b+/- ONL thickness (N≥3 eyes/genotype;


N ) Graphical representation of results of OKR by optokinetic tracking assays reported as cycles / degree . Visual acuity is preserved in P347S / miR - 181a / b + / - with respect to P347S ( N = 4 / genotype ) .

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

N) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles/degree. Visual acuity is preserved in P347S/miR-181a/b+/- animals with respect to P347S (N=4 animals/genotype).


B - F ) Electron microscopy shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT amelioration of mitochondrial phenotype in P347S / miR - 181a / b + / - vs P347S PRs at p12 p30 ( N = 2 / genotype ) . M , mitochondria . G - I ) quantitative of mitochondrial phenotype is expressed as measurement of mitochondria perimeter ( G ) , mitochondria number ( H ) as of mitochondria per analyzed field ( I ) . N = 2 / genotype .

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

B-F) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S/miR-181a/b+/- vs P347S PRs at p12 and p30 (N=2 animals/genotype). M, mitochondria. G-I) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter (G), mitochondria number (H) and as the ratio of mitochondria area per the analyzed field area (I). N=2 animals/genotype.


A ) WB of Drp1 , one of key involved in mitochondrial fission pathway , in optic cup of WT , P347S DMSO - treated , P347S / miR - 181a / b + / - DMSO - treated of P347S / miR - 181a / b + / - FEDRATINIB - treated ( an inhibitor of JAK2 / STAT3 pathway ) mice at p30 .

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

A) WB analysis of Drp1, one of the key proteins involved in the mitochondrial fission pathway, in the optic cup of WT, P347S DMSO-treated, P347S/miR-181a/b+/- DMSO-treated and of P347S/miR-181a/b+/- FEDRATINIB-treated (an inhibitor of the JAK2/STAT3 pathway) mice at p30.


B ) revealed that Drp1 are increased in P347S with respect to WT , partially rescued in P347S / miR - 181a / b + / - . Data are normalized to Gapdh . N ≥ 4 eye / genotype . Please note that compared bands from WT , P347S , P347S / miR - 181a / b + / - P347S / miR - 181a / b + / - with FEDRATINIB samples are from same blots , which were cropped shown organized in panel for sake of data presentation clarity ( see source data ) .

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

B) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT, and partially rescued in P347S/miR-181a/b+/-. Data are normalized to Gapdh. N ≥ 4 eye/genotype. Please note that all compared bands from WT, P347S, P347S/miR-181a/b+/- and P347S/miR-181a/b+/- with FEDRATINIB samples are from the same blots, which were cropped and shown organized in the panel for the sake of data presentation clarity (see source data).


C - D ) qRT - PCR reveals decreased of Drp1 transcript increased of Stat3 Jak2 transcripts in eyes of P347S / miR - 181a / b + / - vs those of P347S . N ≥ 4 eyes / genotype .

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

C-D) qRT-PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S/miR-181a/b+/- vs those of P347S animals. N≥4 eyes/genotype.


F ) of WB in A reveals decreased of Drp1 in P347S / miR - 181a / b + / - DMSO - treated vs P347S increased in P347S / miR - 181a / b + / - FEDRATINIB - treated ex - vivo retinas . N ≥ 4 eyes / genotype .

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

F) Quantification of WB in A reveals decreased levels of Drp1 in P347S/miR-181a/b+/- DMSO-treated vs P347S and increased levels in P347S/miR-181a/b+/- FEDRATINIB-treated ex-vivo retinas. N≥4 eyes/genotype.


( D - U ) Immunofluorescence showed amelioration of Rhodopsin localization ( p30 D - G ' , p70 L - O ' ; arrowheads ) C - arrestin ( p30 H - K ' , p70 P - S ' ; arrowheads ) expression in Sponge - miR - 181a / b - injected eyes vs corresponding controls also highlighting improvement of OS structure . At both time points effect of amelioration was more significant evident when AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b was used . Scale bars are 25 μm . densitometry of each is reported in T for p30 in U for p70 , N = 3 eye / treatment / .

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

(D-U) Immunofluorescence analysis showed amelioration of Rhodopsin localization (p30 D-G', p70 L-O'; white arrowheads) and C-arrestin (p30 H-K', p70 P-S'; white arrowheads) expression in Sponge-miR-181a/b-injected eyes vs the corresponding controls also highlighting improvement of the OS structure. At both time points the effect of amelioration was more significant and evident when the AAV2/8.CMV.GFP-Sponge-miR-181a/b was used. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70, N=3 eye/treatment/staining.


A - I ' ) Immunofluorescence on central retinal sections at p30 showed ONL thickness increase , amelioration of Rhodopsin localization ( A - D ' ) C - arrestin expression ( E - H ' ) in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected vs AAV2 / 8 . CMV . GFP - injected rd10 eyes . Scale bars are 25 μm . densitometry of each is reported in I , N = 3 eye / treatment / .

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

A-I') Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase, amelioration of Rhodopsin localization (A-D') and C-arrestin expression (E-H') in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected vs AAV2/8.CMV.GFP-injected rd10 eyes. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in I, N=3 eye/treatment/staining.


of retinal function on pupillary responses ( PLR ) Water Maze . ( J ) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b than in AAV2 / 8 . CMV . GFP - injected rd10 eyes , at both 10 lux 100 lux stimuli ( N ≥ 6 eyes / treatment N = 9 eyes / treatment , respectively ) . Data are presented as mean ± . Student ' s t - , unpaired .

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

Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (J) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2/8.CMV.GFP-Sponge-miR-181a/b than in AAV2/8.CMV.GFP-injected rd10 eyes, at both 10 lux and 100 lux intensity stimuli (N≥6 eyes/treatment and N=9 eyes/treatment, respectively). Data are presented as mean ± SEM. Student's t-test, unpaired.


of retinal function on pupillary responses ( PLR ) Water Maze . ( K ) Water Maze highlighted a shorter latency in reaching platform in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected respect to control - injected ones ( N ≥ 6 eyes / treatment ) .

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Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (K) Water Maze test highlighted a shorter latency in reaching the platform in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected animals respect to control-injected ones (N≥6 eyes/treatment).


Detection of phosphorylated 17K in BYDV - infected using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . dpi , days after BYDV inoculation ; P , phosphorylated 17K bands ; λ - PP , λ - phosphatase .

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

Detection of phosphorylated 17K in BYDV-infected cells using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. dpi, days after BYDV inoculation; P, phosphorylated 17K bands; λ-PP, λ-phosphatase.


Active HvSnRK1 , but not its total amount , was increased in BYDV - infected barley as compared to mock controls . P - SnRK1 or SnRK1 , enzymatically active SnRK1 ( with T172 phosphorylation ) or unphosphorylated SnRK1 .

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

Active HvSnRK1, but not its total amount, was increased in BYDV-infected barley plants as compared to the mock controls. P-SnRK1 or SnRK1, enzymatically active SnRK1 (with T172 phosphorylation) or unphosphorylated SnRK1 protein.


of 17K phosphorylation by HvGRIK1 - HvSnRK1 kinases in vitro . kinase reactions were set up by mixing desired GST fusions of 17K , 17K3A , 17K5A , HvGRIK1 , HvSnRK1 kinase domain ( KD ) , with reaction products analyzed using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . P , phosphorylated 17K bands ; arrowhead , unphosphorylated 17K .

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

Analysis of 17K phosphorylation by HvGRIK1-HvSnRK1 kinases in vitro. The kinase reactions were set up by mixing the desired GST fusions of 17K, 17K3A, 17K5A, HvGRIK1, and HvSnRK1 kinase domain (KD), with the reaction products analyzed using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. P, phosphorylated 17K bands; arrowhead, unphosphorylated 17K.


Confirmation of expression of SnRK1K139R - YFP or SnRK1 - YFP fusion each in two transgenic lines , SnRK1K139R - YFP - L13 - L22 or SnRK1 - YFP - L4 - L17 , by immunoblotting . samples analyzed here or in ( B ) ( C ) were prepared from BYDV - infected barley at 21 dpi .

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

Confirmation of the expression of SnRK1K139R-YFP or SnRK1-YFP fusion protein each in two transgenic lines, SnRK1K139R-YFP-L13 and -L22 or SnRK1-YFP-L4 and -L17, by immunoblotting. The protein samples analyzed here or in (B) and (C) were all prepared from BYDV-infected barley plants at 21 dpi.


Phosphorylation of BYDV 17K was increased in SnRK1 - YFP - L4 overexpressing SnRK1 - YFP but reduced in SnRK1K139R - YFP - L13 ectopically expressing SnRK1K139R - YFP compared with WT control . P , phosphorylated 17K bands .

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

Phosphorylation of BYDV 17K was increased in SnRK1-YFP-L4 plants overexpressing SnRK1-YFP but reduced in SnRK1K139R-YFP-L13 ectopically expressing SnRK1K139R-YFP compared with WT control. P, phosphorylated 17K bands.


Quantitative comparisons of height ( D ) , maximum root ( E ) among set of BYDV - infected barley materials shown in ( C ) ( using samples collected at 21 dpi ) .

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

Quantitative comparisons of plant height (D), maximum root length (E) among the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).


( H ) of viral CP gene in set of BYDV - infected barley materials shown in ( C ) ( using samples collected at 21 dpi ) .

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protein (H) levels of viral CP gene in the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).


of GFP siRNA detected by sRNA blotting for panel of tobacco materials shown in ( A ) , with arrowhead indicating main band of GFP siRNA .

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Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in (A), with arrowhead indicating the main band of GFP siRNA.


of PEBV CP corrected expression of 17K , 17K5A or 17K5D in panel of tobacco shown in ( D ) , revealed using immunoblotting with anti - PEBV CP or anti - 17K antibody .

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

Protein levels of PEBV CP and corrected expression of 17K, 17K5A or 17K5D in the panel of tobacco plants shown in (D), revealed using immunoblotting with anti-PEBV CP or anti-17K antibody.


17K5D , but not BYDV 17K , interacted with HvSDN1 in LC ( B ) assays . Expression of 17K 17K5D in LC assays were verified by immunoblotting using anti - 17K antibody .

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

17K5D, but not BYDV 17K, interacted with HvSDN1 in LC (B) assays. Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti-17K antibody.


C 17K5D , but not 17K or 17K5A , showed binding to BYDV vsiRNA . GST fusions of 17K , 17K5D , 17K5A , as well as free GST , were used in EMSA assays with biotin - labeled vsiRNA899 or vsiRNA899 duplex as probes .

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

C 17K5D, but not 17K or 17K5A, showed binding to BYDV vsiRNA. The GST fusions of 17K, 17K5D, and 17K5A, as well as free GST, were used in the RNA EMSA assays with biotin-labeled vsiRNA899 or vsiRNA899 duplex as probes.


Cleavage of BYDV vsiRNA899 by HvSDN1 ( His - HvSDN1 ) , with AtSDN1 ( His - AtSDN1 ) serving as a positive control . Asterisk denotes cleavage products . GST fusions of 17K 17K5D did not show vsiRNA cleavage activity .

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

Cleavage of BYDV vsiRNA899 by HvSDN1 (His-HvSDN1), with AtSDN1 (His-AtSDN1) serving as a positive control. Asterisk denotes cleavage products. The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity.


Inhibition of HvSDN1 - catalyzed vsiRNA cleavage by GST - 17K5D but not GST - 17K . Free GST served as a negative control . Asterisk denotes cleavage products .

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

Inhibition of HvSDN1-catalyzed vsiRNA cleavage by GST-17K5D but not GST-17K. Free GST protein served as a negative control. Asterisk denotes cleavage products.


three mutants of 17K5D ( 17K5Dm4 , 17K5Dm2a , 17K5Dm2b ) retained ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 ( D ) . Neither GST nor GST - 17K showed vsiRNA899 binding .

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

The three mutants of 17K5D (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) retained the ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 (D). Neither GST nor GST-17K showed vsiRNA899 binding.


Quantitative of HvSDN1 cleavage activities , with recorded using Typhoon FLA9500 ( F G ) Compared with free GST or GST - 17K , GST fusions of 17K5D or three derivative mutants ( 17K5Dm4 , 17K5Dm2a , 17K5Dm2b ) inhibited HvSDN1 - catalzyed cleavage of fluorogenic vsiRNA899 , which was revealed by a fluorospot ( F ) , relative comparison of values ( G )

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

Quantitative analysis of HvSDN1 cleavage activities, with the fluorescence recorded using Typhoon FLA9500 (F and G) Compared with free GST or GST-17K, the GST fusions of 17K5D or three derivative mutants (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) inhibited HvSDN1-catalzyed cleavage of fluorogenic vsiRNA899, which was revealed by a fluorospot assay (F), relative comparison of fluorescence values (G)


Co - IP assays revealing presence of vsiRNA - containing HvAGO1 - HvSDN1 - P17K in BYDV - infected WT , SnRK1K139R - YFP - L13 , SnRK1 - YFP - L4 , with mock - inoculated SnRK1 - YFP - L4 individuals as controls . immunoprecipitates , prepared using anti - HvAGO1 antibody , were analyzed for presence of HvSDN1 ( by immunoblotting with anti - HvSDN1 antibody ) , P17K ( asterisk ) 17K ( arrowhead ) ( using Phos - tag SDS - PAGE coupled immunoblotting ) , BYDV vsiRNA ( by sRNA blotting ) . Compared with WT control , HvAGO1 immunoprecipitates derived from SnRK1 - YFP - L4 contained higher of P17K vsiRNA but less amount of HvSDN1 , whereas reverse was observed for SnRK1K139R - YFP - L13 .

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

Co-IP assays revealing the presence of vsiRNA-containing HvAGO1-HvSDN1-P17K complex in BYDV-infected WT, SnRK1K139R-YFP-L13, and SnRK1-YFP-L4 plants, with mock-inoculated SnRK1-YFP-L4 individuals as controls. The immunoprecipitates, prepared using anti-HvAGO1 antibody, were analyzed for the presence of HvSDN1 (by immunoblotting with anti-HvSDN1 antibody), P17K (asterisk) and 17K (arrowhead) (using Phos-tag SDS-PAGE coupled immunoblotting), and BYDV vsiRNA (by sRNA blotting). Compared with WT control, the HvAGO1 immunoprecipitates derived from SnRK1-YFP-L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1, whereas the reverse was observed for SnRK1K139R-YFP-L13.


Co - IP assays demonstrating that interaction between HvAGO1 HvSDN1 was weakened by P17K . immunoprecipitates were prepared using anti - HvSDN1 antibody from panel of shown in ( B ) . Relative to WT control , HvSDN1 immunoprecipitates derived from SnRK1 - YFP - L4 contained much less HvAGO1 , whereas opposite was observed for SnRK1K139R - YFP - L13 .

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

Co-IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K. The immunoprecipitates were prepared using anti-HvSDN1 antibody from the panel of plants shown in (B). Relative to WT control, the HvSDN1 immunoprecipitates derived from SnRK1-YFP-L4 contained much less HvAGO1, whereas the opposite was observed for SnRK1K139R-YFP-L13.


17K5D compromised interaction between HvSDN1 PAZ domain of HvAGO1 . LUC signal resulted from interaction between HvSDN1 - HvAGO1 PAZ domain was significantly decreased in presence of 17K5D but not that of 17K .

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

17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1. The LUC signal resulted from the interaction between HvSDN1-HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K.


Verification of 17K5D expression in transgenic lines 17K5D - L3 - L7 by immunoblotting with anti - 17K antibody .

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

Verification of 17K5D expression in the transgenic lines 17K5D-L3 and -L7 by immunoblotting with anti-17K antibody.


B - D Comparison of BYDV disease symptoms including growth ( B ) , height ( C ) , maximum root ( D ) between two transgenic lines WT control at 21 dpi .

O O O O O O O O O O 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-D Comparison of BYDV disease symptoms including plant growth (B), plant height (C), and maximum root length (D) between two transgenic lines and WT control at 21 dpi.


( F ) of viral CP gene in BYDV - infected samples shown in ( B ) .

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

protein (F) levels of viral CP gene in the BYDV-infected samples shown in (B).


BYDV vsiRNA abundance in set of samples shown in ( B ) , as detected by sRNA blotting at 14 dpi .

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

BYDV vsiRNA abundance in the set of samples shown in (B), as detected by sRNA blotting at 14 dpi.


Relative to TaSDN1 - DD , TaSDN1 was decreased in TaSDN1 - Dd TaSDN1 - dd mutants , with a stronger reduction found for TaSDN1 - dd . reactive TaSDN1 detected in TaSDN1 - dd was caused by un - mutated TaSDN1 - A - B homoeologs .

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

Relative to TaSDN1-DD line, TaSDN1 protein was decreased in TaSDN1-Dd and TaSDN1-dd mutants, with a stronger reduction found for TaSDN1-dd. The reactive TaSDN1 protein detected in TaSDN1-dd was caused by the un-mutated TaSDN1-A and -B homoeologs.


( M ) of BYDV CP gene in TaSDN1 - DD , TaSDN1 - Dd TaSDN1 - dd at 21 dpi .

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

protein (M) levels of BYDV CP gene in TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi.


BYDV vsiRNA abundance in TaSDN1 - DD , TaSDN1 - Dd , TaSDN1 - dd detected by sRNA blotting at 21 dpi of BYDV .

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

BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.


graphs depict recovery of CAR1 - mcherry ( left ) tPH - CynA - KikGR ( right ) . Mean ± is shown for n = 2 .

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The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.


Time - lapse confocal of tPH - CynA - KikGR expressing AX3 showing docking events at back of a migrating Dictyostelium . Front back of are shown .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.


Time - lapse confocal of AX3 co - expressing PH - Crac - GFP ( green ) another established PI ( 3 , 4 ) P2 biosensor tPH - TAPP1 - RFP ( red ) . Box shows vesicle .

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

Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.


Time - lapse confocal of tPH - CynA - KikGR ( green ) expressing wild - type AX3 . FM4 - 64 ( red ) was added at time 0 during live imaging . Front back of are shown . Box shows vesicle .

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

Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.


Time - lapse confocal of wild - type AX3 expressing tPH - CynA - KikGR ( green ) PH - crac ( red ) before after PI3K inhibition by 5 µM Wortmannin .

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Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.


Confocal of tPH - CynA - KikGR ( green ) PH - Crac ( red ) expressing wild - type Dictyostelium AX3 ( left ) PI3K1 - 2 - Dictyostelium ( right ) treated with 5 µM Latrunculin A .

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

Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.


Time - lapse confocal showing dynamic localization of PI ( 3 , 4 ) P2 . Nocodazole was added at time 0 during live imaging . of vesicle number , vesicle number / min , vesicle lifetime ( s ) from experiment in E . T - was carried out by GraphPad Prism , * * P ≤ 0 . 01 versus After Nocodazole group ; mean ± ( n = 10 ) .

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

Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).


( L ) Representative confocal of tPH - CynA - KikGR in wild - type Dictyostelium AX2 or RacH − treated with 5 μM latrunculin A Scale bar : 5 μm . ( M ) of to cytosol of tPH - CynA - KikGR in wild - type Dictyostelium AX2 RacH − . T - was carried out by GraphPad Prism , * * * * P ≤ 0 . 0001 versus AX2 group ; mean ± ( n = 33 ) .

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

(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).


Time - lapse confocal of tPH - CynA - KikGR expressing Dictyostelium AX3 . macropinosome at front of was photoconverted at time 0 . arrow points to PI ( 3 , 4 ) P2 localization at back of . front back are shown , box showing photoconverted region .

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

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.


B , C MCF - 7 expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for indicated times , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( B ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to growth media - treated GFP - vector which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( C ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in C ,

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

B, C MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (B). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (C). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in C,


D , E MCF - 7 expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were cultured in growth media or EBSS for indicated times , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( D ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to growth media - treated NT shRNA which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( E ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in E .

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

D, E MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (D). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (E). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in E.


F , G MCF - 7 expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for 4 hours , then fixed immunostained with p62 antibodies , co - stained with DAPI phalloidin ( F ) . Data represent number of p62 + relative to ( µm2 ) ( n = 3 experiments , > 50 / experiment ) ( G ) . Data information : Data is presented as mean ± SD . insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in F p values determined by one - way ANOVA with Tukey post hoc in G ,

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

F, G MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F p values determined by one-way ANOVA with Tukey post hoc test in G,


H , I MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( H ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector which were assigned an arbitrary value of 1 ( I ) ( n = 3 experiments ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in , I ,

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

H, I MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (I) (n=3 experiments). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in , I,


J , K MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then fixed immunostained with p62 antibodies , co - stained with DAPI phalloidin ( J ) . Data represent number of p62 + relative to ( µm2 ) ( n = 3 experiments , > 50 / experiment ) ( K ) . Data information : Data is presented as mean ± SD . insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in J . p values determined by one - way ANOVA with Tukey post hoc in K ,

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

J, K MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (J). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (K). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in J. p values determined by one-way ANOVA with Tukey post hoc test in K,


L , M MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 2 µM BYL719 or DMSO as a vehicle control for indicated times . were lysed immunblotted with LC3B , pAKTS473 , AKT ( pan ) , pS6KT389 or S6K antibodies GAPDH antibodies as a loading control ( L ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( M ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in M ,

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

L, M MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times. Cells were lysed and immunblotted with LC3B, pAKTS473, AKT(pan), pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control (L). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (M). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in M,


N , O MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours . were lysed immunblotted with LC3B antibodies GAPDH antibodies as a loading control ( N ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( O ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc in O ,

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

N, O MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (N). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (O). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in O,


A , B MCF - 7 expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were fixed immunostained with recombinant GST - 2xFYVE LAMP2 antibodies , co - stained with DAPI ( A ) . Data represent proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 40 / experiment ) ( B ) . Arrows indicate co - localization between 2xFYVE LAMP2 . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in A , p values determined by one - way ANOVA with Tukey post hoc in

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

A, B MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >40 cells/experiment) (B). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA with Tukey post hoc test in B


C MCF - 7 were treated with 0 . 02 % ( w / v ) saponin for 30 seconds , then fixed immunostained with INPP4B either LAMP1 or CD63 antibodies , co - stained with DAPI . Arrows indicate co - localization between INPP4B CD63 . Data information : insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in C ,

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

C MCF-7 cells were treated with 0.02% (w/v) saponin for 30 seconds, then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies, and co-stained with DAPI. Arrows indicate co-localization between INPP4B and CD63. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,


D , E MCF - 7 co - expressing GFP - INPP4B or GFP - vector , Hrs or NT shRNA , were fixed immunostained with recombinant GST - 2xFYVE LAMP2 antibodies , co - stained with DAPI ( D ) . Data represent proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 / experiment ) ( E ) . Arrows indicate co - localization between 2xFYVE LAMP2 . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in D , p values determined by one - way ANOVA with Tukey post hoc

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

D, E MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (D). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (E). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA with Tukey post hoc test in


F , G MCF - 7 co - expressing GFP - INPP4B or GFP - vector , Hrs or NT shRNA , were fixed immunostained with recombinant GST - 2xFYVE CD63 antibodies , co - stained with DAPI ( F ) . Data represent proportion of 2xFYVE + late endosomes ( n = 3 experiments , > 30 / experiment ) ( G ) . Arrows indicate co - localization between 2xFYVE LAMP2 . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in F . p values determined by one - way ANOVA with Tukey post hoc in G ,

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and CD63 antibodies, and co-stained with DAPI (F). Data represent the proportion of 2xFYVE+ late endosomes (n=3 experiments, >30 cells/experiment) (G). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F. p values determined by one-way ANOVA with Tukey post hoc test in G,


H , I MCF - 7 co - expressing GFP - INPP4B or GFP - vector , Hrs or NT shRNA , were lysed immunoblotted with LC3B antibodies , GAPDH antibodies as a loading control ( H ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to GFP - vector ; NT shRNA which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( I ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in I .

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

H, I MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to GFP-vector;NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (I). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in I.


A , B MCF - 7 expressing GFP - INPP4B or GFP - vector were fixed immunostained with LAMP1 or LAMP2 antibodies , co - stained with DAPI phalloidin ( A ) . Data represent number of LAMP1 + or LAMP2 + relative to ( µm2 ) ( n = 3 experiments , > 50 / experiment ) ( B ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in A , p values determined by two - unpaired t in B ,

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

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained with LAMP1 or LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP1+ or LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (B). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in A, p values determined by two-tailed unpaired t test in B,


C , D Snapshots of Magic Red cathepsin B substrate Hoechst 33342 captured in live MCF - 7 expressing GFP - INPP4B or GFP - vector ( C ) . Data represent number of Magic Red cathepsin B + per ( n = 3 experiments , > 50 / experiment ) ( D ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in C , p values determined by two - unpaired t in D ,

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

C, D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF-7 cells expressing GFP-INPP4B or GFP-vector (C). Data represent the number of Magic Red cathepsin B+ puncta per cell (n=3 experiments, >50 cells/experiment) (D). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in C, p values determined by two-tailed unpaired t test in D,


E , F MCF - 7 expressing NT or INPP4B # 1 shRNA were cultured in growth media or EBSS for indicated times , then fixed immunostained with LAMP1 antibodies co - stained with DAPI phalloidin ( E ) . Data represent number of LAMP1 + relative to ( µm2 ) ( n = 3 experiments , > 30 per experiment ) ( F ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in p values determined by one - way ANOVA with Tukey post hoc in

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

E, F MCF-7 cells expressing NT or INPP4B #1 shRNA were cultured in growth media or EBSS for the indicated times, then fixed and immunostained with LAMP1 antibodies and co-stained with DAPI and phalloidin (E). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >30 cells per experiment) (F). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in p values determined by one-way ANOVA with Tukey post hoc test in F


G , H MCF - 7 expressing GFP - INPP4B or GFP - vector were transfected with NT , PIK3CA # 1 or PIK3CA # 2 siRNA . After 24 hours , were fixed immunostained with LAMP1 antibodies , co - stained with DAPI phalloidin ( G ) . Data represent number of LAMP1 + relative to ( µm2 ) ( n = 3 experiments , > 40 per experiment ) ( H ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in G . p values determined by one - way ANOVA in H .

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

G, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, PIK3CA #1 or PIK3CA #2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (G). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (H). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in G. p values determined by one-way ANOVA in H.


A , B Snapshots of LAMP1 - GFP , Magic Red cathepsin B substrate Hoechst 33342 captured in live HeLa transfected with INPP4B or NT siRNA ( A ) . Data represent number of LAMP1 + / Magic Red Cathepsin B + LAMP1 + / Magic Red Cathepsin B − lysosomes ( terminal storage lysosomes ) per ( n = 3 experiments , > 50 / experiment ) ( B ) . Yellow arrows indicate LAMP1 + / MR Cathepsin B + lysosomes , arrows indicate terminal storage lysosomes . Data information : Data is presented as mean ± SD . insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in A , p values determined by two - way ANOVA with Holm - Sidak post - hoc in

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

A, B Snapshots of LAMP1-GFP, Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA (A). Data represent the number of LAMP1+/Magic Red Cathepsin B+ and LAMP1+/Magic Red Cathepsin B− lysosomes (terminal storage lysosomes) per cell (n=3 experiments, >50 cells/experiment) (B).Yellow arrows indicate LAMP1+/MR Cathepsin B+ lysosomes, and white arrows indicate terminal storage lysosomes. Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by two-way ANOVA with Holm-Sidak post-hoc test in B


C Timelapse snapshots of LAMP1 - mCherry signals from MCF - 7 expressing GFP - INPP4B or GFP - vector captured by spinning disk microscopy . Maximum projections of 3 z - planes taken 0 . 27 µm apart are shown . Arrows indicate lysosome reformation . Data information : insets at lower right of each image are higher power regions of boxed areas . Scale bar is 10 µm in C .

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

C Timelapse snapshots of LAMP1-mCherry signals from MCF-7 cells expressing GFP-INPP4B or GFP-vector captured by spinning disk microscopy. Maximum intensity projections of 3 z-planes taken 0.27 µm apart are shown. Arrows indicate lysosome reformation. Data information: The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C.


E Data represent number of LAMP1 + reformation events per per minute ( n = 12 GFP - vector , n = 15 GFP - INPP4B ) . Data information : Data is presented as mean ± SD . p values determined by two - unpaired t in E .

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

E Data represent the number of LAMP1+ reformation events per cell per minute (n=12 GFP-vector cells, n=15 GFP-INPP4B cells). Data information: Data is presented as mean ± SD. p values determined by two-tailed unpaired t test in E.


A , B MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 for 2 hours . were fixed immunostained with recombinant GST - 2xFYVE LAMP2 antibodies , co - stained with DAPI ( A ) . Data represent proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 / experiment ) ( B ) . Arrows show co - localization between 2xFYVE LAMP2 . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in A p values determined by one - way ANOVA in

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

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours. Cells were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (B). Arrows show co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in B


D - F MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then YM201636 was washed out for 1 , 2 or 4 hours . were fixed immunostained with LAMP1 antibodies , co - stained with DAPI phalloidin ( D ) . Data represent number of LAMP1 + relative to ( µm2 ) ( E ) , lysosome fold change relative to YM201636 - treated condition for each ( F ) ( n = 3 experiments , > 30 per experiment ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in D . p values determined by one - way ANOVA in E , or by two - unpaired t of under curve in F .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then YM201636 was washed out for 1, 2 or 4 hours. Cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (D). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (E), and the lysosome fold change relative to the YM201636-treated condition for each cell line (F) (n=3 experiments, >30 cells per experiment). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D. p values determined by one-way ANOVA in E, or by two-tailed unpaired t test of the area under the curve in F.


G , H MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 1 , 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours . were lysed immunoblotted with LC3B antibodies , GAPDH antibodies as a loading control ( G ) . Data represent relative LC3B - II normalized to GAPDH , expressed relative to DMSO - treated GFP - vector which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( H ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in H ,

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1, 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (G). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (H). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in H,


A , B MCF - 7 expressing GFP - INPP4B or GFP - vector were transfected with NT , SNX1 or SNX2 siRNA . After 24 hours , were fixed immunostained with LAMP2 antibodies , co - stained with DAPI phalloidin ( A ) . Data represent number of LAMP2 + relative to ( µm2 ) ( n = 3 experiments , > 40 per experiment ) ( B ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in A , p values determined by one - way ANOVA in with Tukey post hoc in

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

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, SNX1 or SNX2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA in with Tukey post hoc test in B


C Timelapse snapshots of LAMP1 - mCherry GFP - SNX2 signals from MCF - 7 captured by spinning disk microscopy . Maximum projections of 2 z - planes taken 0 . 27 µm apart are shown . Data information : insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in C ,

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

C Timelapse snapshots of LAMP1-mCherry and GFP-SNX2 signals from MCF-7 cells captured by spinning disk microscopy. Maximum intensity projections of 2 z-planes taken 0.27 µm apart are shown. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,


D , E MCF - 7 expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . were fixed immunostained with SNX2 LAMP1 antibodies , co - stained with DAPI ( D ) . Data represent proportion of SNX2 + LAMP2 ( n = 3 experiments , > 40 / experiment ) ( E ) . Arrows show co - localization between SNX2 LAMP2 . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in D , p values determined by one - way ANOVA in with Tukey post hoc in E ,

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

D, E MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2 and LAMP1 antibodies, and co-stained with DAPI (D). Data represent the proportion of SNX2+ LAMP2 puncta (n=3 experiments, >40 cells/experiment) (E). Arrows show co-localization between SNX2 and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA in with Tukey post hoc test in E,


F HeLa were transfected with GFP - SNX2 . After 24 hours , were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . were fixed subjected to immuno - electron microscopy using GFP ( 10 nm ) antibodies . Arrows show GFP - SNX2 localization to lysosome . Data information : Scale bar is 100 nm in F .

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

F HeLa cells were transfected with GFP-SNX2. After 24 hours, cells were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and subjected to immuno-electron microscopy analysis using GFP (10 nm) antibodies. Arrows show GFP-SNX2 localization to lysosome. Data information: Scale bar is 100 nm in F.


G HeLa were transfected with GFP - PIKfyve . After 24 hours , were were treated with 5 µM YM201636 for 2 hours , then YM201636 was washed out for 1 hour . were fixed immunostained with SNX2 , GFP LAMP2 antibodies , imaged using super resolution microscopy . Arrows show co - localization between GFP - PIKfyve , SNX2 LAMP2 . Data information : insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm

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

G HeLa cells were transfected with GFP-PIKfyve. After 24 hours, cells were were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2, GFP and LAMP2 antibodies, and imaged using super resolution microscopy. Arrows show co-localization between GFP-PIKfyve, SNX2 and LAMP2. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in


H MCF - 7 expressing LAMP1 - mCherry were transfected with NT or SNX2 siRNA . After 24 hours , were imaged by spinning disk microscopy for 20 minutes . Representative timelapse snapshots of LAMP1 - mCherry from a maximum projection of 3 z - planes taken 0 . 27 µm apart are shown . Arrow indicates lysosome fission . I Data represent number of LAMP1 + reformation events per per minute ( n = 21 NT siRNA , n = 27 SNX2 siRNA ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in H , p values determined by two - unpaired t in I .

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

H MCF-7 cells expressing LAMP1-mCherry were transfected with NT or SNX2 siRNA. After 24 hours, cells were imaged by spinning disk microscopy for 20 minutes. Representative timelapse snapshots of LAMP1-mCherry from a maximum projection of 3 z-planes taken 0.27 µm apart are shown. Arrow indicates lysosome fission. I Data represent the number of LAMP1+ reformation events per cell per minute (n=21 NT siRNA cells, n=27 SNX2 siRNA cells). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in H, p values determined by two-tailed unpaired t test in I.


A , B HeLa transfected with INPP4B or NT siRNA were treated for 1 hour with 5 µg / mL puromycin . were fixed immunostained with ubiquitin antibodies , co - stained with DAPI phalloidin ( A ) . Data represent of with ubiquitinated ( n = 3 experiments , > 200 / experiment ) ( B ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in A p values determined by one - way ANOVA in with Tukey post hoc in

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

A, B HeLa cells transfected with INPP4B or NT siRNA were treated for 1 hour with 5 µg/mL puromycin. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (A). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in with Tukey post hoc test in B


C , D HeLa were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then 5 µg / mL puromycin for 1 hour . were fixed immunostained with ubiquitin antibodies , co - stained with DAPI phalloidin ( C ) . Data represent of with ubiquitinated ( n = 3 experiments , > 200 / experiment ) ( D ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in C , p values determined by one - way ANOVA in with Tukey post hoc in D ,

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

C, D HeLa cells were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then 5 µg/mL puromycin for 1 hour. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (C). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (D). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C, p values determined by one-way ANOVA in with Tukey post hoc test in D,


E , F MCF - 7 expressing GFP - INPP4B or GFP - vector were treated for 4 hours with 10 µg / mL puromycin , 5 µm MG132 either 5 µM YM201636 or DMSO as a vehicle control . were fixed immunostained with ubiquitin antibodies , co - stained with DAPI phalloidin ( E ) . Data represent of with ubiquitinated ( n = 3 experiments , > 200 / experiment ) ( F ) . Data information : Data is presented as mean ± SD . insets at lower right or bottom of each image are higher power regions of boxed areas . Scale bar is 10 µm in E . p values determined by one - way ANOVA in with Tukey post hoc

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

E, F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated for 4 hours with 10 µg/mL puromycin, 5 µm MG132 and either 5 µM YM201636 or DMSO as a vehicle control. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (E). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (F). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in E. p values determined by one-way ANOVA in with Tukey post hoc test in