( A , B ) NIH3T3 stably expressing GFP ‐ Gal3 either empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , were fixed at indicated time points subjected to immunocytochemistry for Lamp1 DAPI ( blue ) ( A ) . number of GFP ‐ Gal3 or Lamp1 per was quantified using G ‐ ( see also Supplementary Figure S4A B ) . Then , of GFP ‐ Gal3 ‐ positive Lamp1 was determined ( B ) . data represent means ± s . d . At least 70 were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on online supplementary information page .

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(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( C - E ) HeLa transfected with tfGal3 either One ‐ STrEP ‐ FLAG ‐ tagged Atg4BC74A ( Atg4B mutant ) or empty vector ( control ) were observed by confocal microscopy after treatment as shown in Figure 2A B . number of GFP ± RFP + or GFP + RFP + per was quantified using G ‐ ( D ) . Then , of GFP + RFP + tfGal3 was calculated ( E ) . data represent means ± s . d . At least 30 were counted ( n = 3 ) . Bar : 10 μm .

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(C-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm.


( F - H ) NIH3T3 stably expressing empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , were cultured in presence or absence of both 10 μg / ml E64d Pepstatin A , fixed at indicated time points , subjected to immunocytochemistry for Gal3 ( green ) DAPI ( blue ) ( F ) . number of endogenous Gal3 per control ( G ) or Atg4B ‐ mutant ( H ) was quantified by G ‐ . data represent means ± s . d . At least 50 were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on online supplementary information page .

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(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( A - F ) HeLa stably expressing GFP ‐ LC3 were transfected with mStrawberry ‐ Gal3 , treated with 1000 μM LLOMe for 1 h . Then , were fixed observed by confocal microscopy ( A ) . same specimens were further examined by transmission electron microscopy ( B - F ) . Green : LC3 ; magenta : Gal3 ; blue : DAPI .

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(A-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.


( G - I ) NIH3T3 stably expressing CFP ‐ Gal3 YFP ‐ LC3 , either empty vector ( control ) ( G , H ) or mStrawberry ‐ Atg4BC74A ( I ) were treated with 1000 μM LLOMe for 2 h , fixed , observed by confocal microscopy . electron were taken in same sample field as transmission electron microscope . Green : LC3 ; blue : Gal3 DAPI ; black arrow : single ; arrow : autophagosome ; arrowhead : ER .

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(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.


( A ) Plasmacreatinine ureanitrogen in Atg5F / F Atg5F / F ; KAPmice treated with vehicle or UA + OA . values displayed represent means ± s . e . Statistically significant differences ( * P0 . 05 ) are indicated . F / F : Atg5F / Fmice ; F / F ; KAP : Atg5F / F ; KAPmice .

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(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.


( B , C ) PAS ‐ stained renal cortical region of Atg5F / F Atg5F / F ; KAP mice treated with vehicle or UA + OA ( n = 4 - 7 ) . Bars : 40 μm ( B ) . Kidney injury score of Atg5F / F Atg5F / F ; KAP mice treated with vehicle or UA + OA ( C ) .

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(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).


( D ) Kidney cortexes from Atg5F / F Atg5F / F ; KAP mice treated with vehicle or UA + OA were subjected to immunohistocheminal of Lamp1 LC3 . Green : LC3 ; magenta : Lamp1 ; blue : DAPI . F / F : Atg5F / F mice ; F / F ; KAP : Atg5F / F ; KAP mice . Bar : 5 μm .

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(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.


( E ) Electron of kidney proximal tubules in Atg5 F / F Atg5F / F ; KAP mice treated with UA + OA . Yellow arrowhead : autophagosome ; asterisk : lysosome ; Mt : mitochondrion . Bar : 1 μm .

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(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.


Immunofluorescence microscopy reveals highly similar mostly localization of SMARCB1 wild type R377C mutant , visualized by anti - HA ( green ) . Phalloidin DAPI was used to visualize actin cytoskeleton nucleus , respectively . A novel MS - microscopy approach was used to further define molecular context of . This identified possible chromosomal , endosomal localization of . possible endosomal localization is in agreement with anti - HA immunofluorescence microscopy results . ( key : scale bar for immunofluorescence is 10 µm , color gradient on MS - microscopy indicates localization scores calculated by MS - microscopy tool .

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Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool.


proliferation in HCT116 CRC shows growth advantage conferred by SMARCB1 R377C mutation compared with wild type SMARCB1 or vector control . error bars designate standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control. The error bars designate the standard deviation (SD)


Immunofluorescence microscopy of STK38L wild type R105W mutant display no clear difference in localization . Both wild type R105W mutant display , plasma localization . However , MS - microscopy suggests a more prominent endosomal localization with R105W mutant . ( key : scale bar for immunofluorescence is 10 µm , color gradient on MS - microscopy indicates localization scores calculated by MS - Microscopy tool .

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Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.


proliferation in HCT116 CRC shows growth advantage conferred by expression of wild type STK38L , whereas R105W mutant only slightly increased number of colonies . error bars designate standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD)


Each mitochondrial is colored according to ( A ) its fold change calculated by standard differential expression using limma package ( Ritchie et al , 2015 ; Phipson et al , 2016 ) ; ( B ) its CNV value ( 5 with highest CNV value 5 with lowest CNV values are highlighted annotated in boxes ) ; ( C ) absolute difference between two

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Each mitochondrial protein is colored according to (A) its fold change calculated by standard differential expression using the limma package (Ritchie et al, 2015; Phipson et al, 2016); (B) its CNV value (5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes); and (C) the absolute difference between the two values


of abundance distribution for ( A ) , ( B ) mitochondrial , ( C ) extracellular across 5 groups in C . elegans ( Walther et al , 2015 ) . Each group was compared against a pool reference sample using SILAC ( Ong et al , 2002 ) . Distribution of fold changes for different time points are shown as density plots as as boxplots ( inset ) ; below boxplots , asterisks mark time points that show significantly different distribution of fold changes compared to first time point ( Mann - Whitney p < 0 . 01 )

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Analysis of the abundance distribution for (A) cytoplasmic, (B) mitochondrial, and (C) extracellular proteins across 5 age groups in C.elegans (Walther et al, 2015). Each age group was compared against a pool reference sample using SILAC (Ong et al, 2002). Distribution of protein fold changes for the different time points are shown as density plots and as as boxplots (inset); below the boxplots, asterisks mark the time points that show significantly different distribution of fold changes compared to the first time point (Mann-Whitney test p<0.01)


Abundance variation of members for ( D ) three mitochondrial complexes ( respiratory I , mitochondrial ribosome mitochondrial import / export ) . For each , abundance variation of each member ( colored in grey ) over 5 groups is reported with Δ SILAC ( difference between experimental SILAC of a time point SILAC at day 1 , left ) CNV value ( right ) . For each group , box plot ( colored as corresponding time - point in panels A - B - C ) represents variance between abundances of members . Significant changes ( Mann - Whitney p < 0 . 05 ) are marked with a red

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Abundance variation of complex members for (D) three mitochondrial complexes (respiratory complex I, mitochondrial ribosome and mitochondrial import/export proteins). For each complex, the abundance variation of each member (colored in grey) over the 5 age groups is reported with the Δ SILAC ratio (difference between the experimental SILAC ratio of a time point and the SILAC ratio at day 1, left) and the CNV value (right). For each age group, the box plot (colored as the corresponding time-point in the panels A-B-C) represents the variance between the abundances of the complex members. Significant changes (Mann-Whitney test p<0.05) are marked with a red star


( E ) Variation of mitochondrial proteome over lifespan of C . elegans ( day 1 day 22 ) . Respiratory chain I mitochondrial ribosomal are highlighted in green purple , respectively ( selected , with extreme CNV values , are indicated )

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(E) Variation of the mitochondrial proteome over the lifespan of C.elegans (day 1 and day 22). Respiratory chain complex I and mitochondrial ribosomal proteins are highlighted in green and purple, respectively (selected proteins, with extreme CNV values, are indicated)


( F ) Variation of extracellular proteome abundance over lifespan of C . elegans ( day 1 day 22 ) . Four have been selected ( highlighted with a colored circle : purple for NAS - 34 , green for Y45F10C . 4 , blue for Vitellogenin - 1 , black for Y45F10C . 2 ) for their slower or accelerated increase of abundance compared to of extracellular . variation of selected ( highlighted with same colors ) throughout worm lifespan is represented in middle box , with average of extracellular proteome colored in yellow . Related to Figure EV5 Dataset

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(F) Variation of the extracellular proteome abundance over the lifespan of C.elegans (day 1 and day 22). Four proteins have been selected (highlighted with a colored circle: purple for NAS-34, green for Y45F10C.4, blue for Vitellogenin-1, black for Y45F10C.2) for their slower or accelerated increase of abundance compared to the rest of extracellular proteins. The variation of the selected proteins (highlighted with the same colors) throughout the worm lifespan is represented in the middle box, with the average of the extracellular proteome colored in yellow. Related to Figure EV5 and Dataset EV6


B MAPKKK5 - C interacts with PBL27 in yeast two - hybrid experiments . growth of yeast colonies on plates ( - ULWH ) lacking uracil ( U ) , leucine ( L ) , tryptophan ( W ) , histidine ( H ) with 2 mM 3 - aminotriazole ( 3 - AT ) indicates a positive interaction .

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B MAPKKK5-C interacts with PBL27 in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 2 mM 3-aminotriazole (3-AT) indicates a positive interaction.


C Chitin - induced MAPK activation was analyzed by immunoblots with α - pMAPK .

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C Chitin-induced MAPK activation was analyzed by immunoblots with α-pMAPK.


D Complementation of chitin - induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5 - FLAG . MAPK activation was analyzed by immunoblots with α - pMAPK .

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D Complementation of chitin-induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5-FLAG. MAPK activation was analyzed by immunoblots with α-pMAPK.


E Flg22 - induced MAPK activation was analyzed by immunoblots with α - pMAPK . Experiments ( B ) - ( E ) were performed three times with similar results .

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E Flg22-induced MAPK activation was analyzed by immunoblots with α-pMAPK. Experiments (B) - (E) were performed three times with similar results.


A Chitin - induced callose deposition in mapkkk5 mutants . Seedlings were analyzed at 18 hours after treatment with 10 μM chitin . Representative pictures from biological replicates are presented . Data are means ± SD from three independent biological replicates , where each biological replicate consists of two technical replicates . asterisks indicate statistically significant differences from WT controls by Student ' s t - ( P < 0 . 05 ) .

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A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


B Leaf discs were treated with 10 μM ( GlcNAc ) 7 in a solution containing 500 μM L - 012 10 μg / ml horseradish peroxidase . ROS production was quantified using a luminescence microplate reader . Data are means ± SD calculated using three biological replicates , where each biological replicate consisted of two technical replicates .

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B Leaf discs were treated with 10 μM (GlcNAc)7 in a solution containing 500 μM L-012 and 10 μg/ml horseradish peroxidase. ROS production was quantified using a luminescence microplate reader. Data are means ±SD calculated using three biological replicates, where each biological replicate consisted of two technical replicates.


C mapkkk5 mutations reduce resistance to Alternaria brassiciicola . Lesions sizes were measured 6 days after inoculation . Values are mean ± S . E . , n ≥ 92 . Asterisks indicate significant difference from wild - type controls by Student ' s t - ( P < 0 . 01 ) .

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C mapkkk5 mutations reduce resistance to Alternaria brassiciicola. Lesions sizes were measured 6 days after inoculation. Values are mean ±S.E., n ≥92. Asterisks indicate significant difference from wild-type controls by Student's t-test (P < 0.01).


D of chitin - induced transcriptional reprogramming in mapkkk5 - 1 . Total was extracted from seedlings treated with mock or 40 μM chitin for 3 hours analyzed by - seq . Genes that are significantly induced or suppressed by chitin in wild type were selected ( q - value < 0 . 05 ; 12 , 992 genes ) . log2 fold changes of selected genes compared to mock in wild type mapkkk5 - 1 were plotted . Yellow blue dots indicate genes that show reduced induction or suppression ( 506 genes ) or enhanced induction or suppression ( 151 genes ) in mapkkk5 - 1 compared to wildtype , respectively . slope of linear regression ( red ) indicates that overall transcriptional response is weakened in mapkkk5 - 1 compared to wild type . As a comparison , y = x ( black ) is shown . For more details , see Methods . E A showing MAPKKK5 - dependent genes . Genes showing reduced induction or suppression in mapkkk5 - 1 compared to wild type were selected as described in Methods ( 339 genes ) . log2 fold changes of selected genes compared to mock were subjected to hierarchical clustering . Yellow indicates positive values , blue indicates negative values black indicates zero : see color scale .

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D Analysis of chitin-induced transcriptional reprogramming in mapkkk5-1. Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA-seq. Genes that are significantly induced or suppressed by chitin in the wild type were selected (q-value < 0.05; 12,992 genes). The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5-1 were plotted. Yellow and blue dots indicate genes that show reduced induction or suppression (506 genes) or enhanced induction or suppression (151 genes) in mapkkk5-1 compared to the wildtype, respectively. The slope of the linear regression line (red) indicates that the overall transcriptional response is weakened in mapkkk5-1 compared to the wild type. As a comparison, the line y=x (black) is shown. For more details, see the Methods.E A heatmap showing MAPKKK5-dependent genes. Genes showing reduced induction or suppression in mapkkk5-1 compared to the wild type were selected as described in the Methods (339 genes). The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis. Yellow indicates positive values, blue indicates negative values and black indicates zero: see the color scale.


F Expression patterns of representative MAPKKK5 - dependent genes . qRT - PCR of defense - related genes in 8 - day - old seedlings exposed to 40μM ( GlcNAc ) 7 for 3h . Data are shown as average of three independent biological replicates ± SD . asterisks indicate statistically significant differences from WT controls by Student ' s t - ( P < 0 . 05 ) .

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F Expression patterns of representative MAPKKK5-dependent genes. qRT-PCR analysis of defense-related genes in 8-day-old seedlings exposed to 40μM (GlcNAc)7 for 3h. Data are shown as the average of three independent biological replicates ± SD. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


A MAPKKK5 - GFP was detected in fraction . total ( T ) , soluble ( S ) , microsomal ( M ) fractions purified from Nb leaves expressing MAPKKK5 - GFP were used for immunoblots with antibodies against GFP , PM - localized aquaporin PIP1 ascorbate peroxidase EAP1 .

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A The MAPKKK5-GFP protein was detected in the membrane fraction. The total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5-GFP were used for immunoblots with antibodies against GFP, PM-localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1.


B Subcellular localization of MAPKKK5 - GFP GFP in Nb leaves . results were photographed at 38 hpi . Scale bar = 50µm .

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B Subcellular localization of MAPKKK5-GFP and GFP in Nb leaves. The results were photographed at 38 hpi. Scale bar=50µm.


C Detection of MAPKKK5 - GFP PBL27 - HA transiently expressed in Nb leaves . Total were purified from Nb leaves at 38 hpi .

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C Detection of MAPKKK5-GFP and PBL27-HA transiently expressed in Nb leaves. Total proteins were purified from Nb leaves at 38 hpi.


D Localization of MAPKKK5K375M - GFP was analyzed by using total ( T ) , soluble ( S ) , microsomal ( M ) fractions purified from Nb leaves expressing MAPKKK5 K375M -

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D Localization of MAPKKK5K375M-GFP was analyzed by using total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5 K375M -GFP


E Co - immunoprecipitation assays show that MAPKKK5K375M - GFP forms with PBL27 - HA in Nb leaves . microsomal fractions purified from Nb leaves co - expressing MAPKKK5K375M - GFP PBL27 - HA at 48 hpi were used for co - immunoprecipitation with α - GFP .

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E Co-immunoprecipitation assays show that MAPKKK5K375M-GFP forms the complex with PBL27-HA in Nb leaves. The microsomal fractions purified from Nb leaves co-expressing MAPKKK5K375M-GFP and PBL27-HA at 48 hpi were used for co-immunoprecipitation with α-GFP.


F Visualization of interaction between PBL27 MAPKKK5K375M by bimolecular complementation ( BiFC ) in Nb leaves . results were photographed at 38 hpi . Venus indicates interaction between PBL27 MAPKKK5K375M . Scale bar = 50µm .

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F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation (BiFC) analysis in Nb leaves. The results were photographed at 38 hpi. Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M. Scale bar=50µm.


A Detection of MAPKKK5 - GFP MAPKKK5K375M - GFP transiently expressed in Arabidopsis protoplasts .

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A Detection of MAPKKK5-GFP and MAPKKK5K375M-GFP transiently expressed in Arabidopsis protoplasts.


B Expression of MAPKKK5 - GFP reduces level of PBL27 . CBB , Coomassie brilliant blue .

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B Expression of MAPKKK5-GFP reduces the level of PBL27. CBB, Coomassie brilliant blue.


C Arabidopsisprotoplasts expressing PBL27 - HA MAPKKK5 - GFP were treated with 30 μM MG132 for 3 h subjected to immunoblots with α - HA α - GFP .

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C Arabidopsisprotoplasts expressing PBL27-HA and MAPKKK5-GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α-HA and α-GFP.


D co - immunoprecipitation shows that PBL27 forms with MAPKKK5 in Arabidopsis protoplasts . Total purified from protoplasts co - expressing PBL27 MAPKKK5 were subjected to immunoprecipitation with α - HA .

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D The co-immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts. Total proteins purified from protoplasts co-expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α-HA.


E Visualization of interaction between PBL27 MAPKKK5K375M by BiFC in Arabidopsis protoplasts . FLS - associated RLCK BIK1 was also used for this . Venus indicates interaction between PBL27 MAPKKK5K375M . Scale bar = 10µm .

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E Visualization of the interaction between PBL27 and MAPKKK5K375M by BiFC analysis in Arabidopsis protoplasts. FLS-associated RLCK BIK1 was also used for this assay. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M. Scale bar=10µm.


F disassociation of PBL27 - MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts . Protoplasts were co - transfected with MAPKKK5K375M - GFP PBL27 - HA , stimulated with or without 10μM chitin or 1 μM flg22 for 10 min subjected to co - immunoprecipitation . Experiments ( A ) - ( F ) were performed three times with similar results .

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F The disassociation of PBL27-MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts. Protoplasts were co-transfected with MAPKKK5K375M-GFP and PBL27-HA, stimulated with or without 10μM chitin or 1 μM flg22 for 10 min and subjected to the co-immunoprecipitation assay. Experiments (A) - (F) were performed three times with similar results.


A PBL27 phosphorylates MAPKKK5 - C in vitro . in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , phosphorylated were detected by autoradiography . CBB , Coomassie brilliant blue .

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A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue.


B BIK1 does not phosphorylate MAPKKK5 - C in vitro . RBOHD was used as a positive control . Asterisk indicates artificial bands .

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B BIK1 does not phosphorylate MAPKKK5-C in vitro. RBOHD was used as a positive control. Asterisk indicates artificial bands.


D S622A mutation strongly reduces phosphorylation by PBL27 . Each of phosphorylation sites was substituted by alanine , subjected to in vitro phosphorylation assays .

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D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays.


E PBL27 does not phosphorylate MAPKKK5 - C6xAin vitro . in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , phosphorylated were detected by autoradiography .

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E PBL27 does not phosphorylate MAPKKK5-C6xAin vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.


F PBL27 interacts with MAPKKK5 - C6xA in yeast two - hybrid experiments . growth of yeast colonies on plates ( - ULWH ) lacking uracil ( U ) , leucine ( L ) , tryptophan ( W ) , histidine ( H ) with 10 mM 3 - AT indicated a positive interaction .

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F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.


A Chitin - induced MAPK activation in a mapkkk5 mutant ( 1 ) expressing MAPKKK56xA . MAPK activity was detected using immunoblots with α - pMAPK .

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A Chitin-induced MAPK activation in a mapkkk5 mutant (Line 1) expressing MAPKKK56xA. MAPK activity was detected using immunoblots with α-pMAPK.


B Chitin - induced callose deposition in MAPKKK56xA ( 1 ) . Seedlings were analyzed at 18 hours after treatment with 10 μM chitin . Data are means ± SD from three independent biological replicates , where each biological replicate consists of two technical replicates . asterisks indicate statistically significant differences from WT controls by Student ' s t - ( P < 0 . 05 ) .

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B Chitin-induced callose deposition in the MAPKKK56xA plant (Line 1). Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


C Detection of MAPKKK5K375M - GFP MAPKKK5K375M , 6xA - GFP transiently expressed in Arabidopsis protoplasts .

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C Detection of MAPKKK5K375M -GFP and MAPKKK5K375M,6xA-GFP transiently expressed in Arabidopsis protoplasts.


D Visualization of interaction between PBL27 MAPKKK5K375M , 6xA by BiFC in Arabidopsis protoplasts . Venus indicates interaction between PBL27 MAPKKK5K375M , 6xA . Scale bar = 10µm .

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D Visualization of interaction between PBL27 and MAPKKK5K375M, 6xA by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M, 6xA. Scale bar=10µm.


A PBL27 does not interact with MAPKKK5 - KD : C in a yeast two - hybrid .

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A PBL27 does not interact with MAPKKK5-KD:C in a yeast two-hybrid assay.


B Addition of CERK1 to an in vitro kinase enhances phosphorylation of MAPKKK5 - KDK375M : C by PBL27 . Asterisks indicate artificial bands derived from CERK1 : IC .

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B Addition of CERK1 to an in vitro kinase assay enhances phosphorylation of MAPKKK5-KDK375M:C by PBL27. Asterisks indicate artificial bands derived from CERK1:IC.


C CERK1 does not phosphorylate C - terminal domain of MAPKKK5 in vitro . in vitro kinase was performed with recombinant of GST - PBL27 , MAPKKK5 - C intracellular kinase domain of CERK1 ( GST - CERK1 : IC ) . loading control was shown by with Coomassie Brilliant Blue . in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , phosphorylated were detected by autoradiography . Asterisks indicate artificial bands .

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C CERK1 does not phosphorylate the C-terminal domain of MAPKKK5 in vitro. The in vitro kinase assay was performed with recombinant proteins of GST-PBL27, MAPKKK5-C and the intracellular kinase domain of CERK1 (GST-CERK1:IC). The protein loading control was shown by staining with Coomassie Brilliant Blue. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. Asterisks indicate artificial bands.


D alanine substitution mutations at residues conserved in activation loop of reduces phosphorylation of PBL27 by CERK1 : IC . in vitro kinase was carried out with recombinant of GST - PBL27K112E , GST - PBL27K112E , 3xA CERK1 - IC .

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D The alanine substitution mutations at the residues conserved in the activation loop of reduces the phosphorylation of PBL27 by CERK1:IC. The in vitro kinase assay was carried out with recombinant proteins of GST-PBL27K112E , GST-PBL27K112E, 3xA and CERK1-IC.


E kinase activity of GST - PBL273xA was analyzed by in vitro kinase using [ 32P ] γ - ATP .

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E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.


A Visualization of interaction between MAPKKK5K375M MKK1 / MKK2 / MKK4 / MKK5 by BiFC in Arabidopsis protoplasts . Venus indicates interaction . Scale bar = 10µm .

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A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction. Scale bar=10µm.


B MAPKKK5 - KD phosphorylates MKK4 MKK5 in vitro . in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , phosphorylated were detected by autoradiography .

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B MAPKKK5-KD phosphorylates MKK4 and MKK5 in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.


C MAPKKK5 - KD does not phosphorylate a T224A / S230A mutant of MKK4 or a T215A / S221A mutant of MKK5 . above experiments were performed three times with similar results .

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C MAPKKK5-KD does not phosphorylate a T224A/S230A mutant of MKK4 or a T215A/S221A mutant of MKK5. The above experiments were performed three times with similar results.


weights of WT Rcan1 - KO mice fed normal chow diet ( NC ) for 20 starting at 8 - 10 of ( , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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Body weights of WT and Rcan1-KO mice fed normal chow diet (NC) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


weights of WT Rcan1 - KO mice fed a high - fat diet ( HFD ) for 20 starting at 8 - 10 of ( , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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Body weights of WT and Rcan1-KO mice fed a high-fat diet (HFD) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


C Food consumption per gram of gained ( g / g ∆ BW ) over first 15 on NC or HFD , starting at 8 of ( , n = 9 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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C Food consumption per gram of body weight gained (g/g ∆BW) over the first 15 weeks on NC or HFD, starting at 8 weeks of age (male, n=9 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Cholesterol remaining in feces of WT Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( , n = 4 over 3 days ) . Cholesterol content of NC HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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Cholesterol remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


triglyceride remaining in feces of WT Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( , n = 4 over 3 days ) . triglyceride content of NC HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). triglyceride content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI measures of total , lean mass , fat mass prior to after 6 on HFD ( , n = 5 ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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

MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI image of WT KO following 8 on a HFD . Yellow delineates visceral WAT . Pink delineates subcutaneous WAT . graphed on right . Data information : values shown are mean ± * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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

MRI image of WT and KO males following 8 weeks on a HFD. Yellow delineates visceral WAT. Pink delineates subcutaneous WAT. Quantification graphed on the right. Data information: values shown are mean ±SEM * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Representative H & E of gWAT from WT KO after 25 on NC or HFD . Scale bar = 100 μm . K of cross - sectional ( CSA ) of adipocytes in K ( n = 3 , 3 / each , 75 - 100 adipocytes ) . Scale bar = 100 μm . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

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

Representative H&E staining of gWAT from WT and KO males after 25 weeks on NC or HFD. Scale bar = 100 μm. K Quantification of cross-sectional area (CSA) of adipocytes in K (n=3 animals, 3 images/each, 75-100 adipocytes). Scale bar = 100 μm. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


L Transcript for inflammatory markers Monocyte chemoattractant - 1 ( Mcp - 1 ) , macrophage antigen F4 / 80 ( F480 ) , necrosis factor - alpha ( Tnfα ) , necrosis factor - beta ( Tnfβ ) in gWAT of WT KO after 25 on NC or HFD ( , n = 4 - 5 ) . Transcript were normalized to 18S Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A B , which used multiple t - ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transcript levels for inflammatory markers Monocyte chemoattractant protein-1 (Mcp-1), macrophage antigen F4/80 (F480), tumor necrosis factor-alpha (Tnfα), and tumor necrosis factor-beta (Tnfβ) in gWAT of WT and KO after 25 weeks on NC or HFD (males, n=4-5). Transcript levels were normalized to 18S Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


A , B Oil Red O of livers from WT KO after 25 on NC or HFD , with of relative stain ( n = 3 , 3 each ) . Scale bar = 100 μm . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


C Transcript for genes involved in lipid metabolism gluconeogenesis in livers of WT KO mice after 25 on NC or HFD . Stearoyl - Coenzyme A desaturase ( Scd1 ) , fatty acid transporter ( CD36 ) , glycerol - 3 - phosphate acyltransferase ( Gpat ) , carnitine palmitoyltransferase I ( Cpt1 ) , peroxisome proliferator - activated gamma ( PPARγ ) , pyruvate dehydrogenase acetyl - transferase ( Pdk4 ) , PPARγ coactivator 1 alpha ( PGC1α ) , diacylglycerol O - acyltransferase ( Dgat ) , facilitated glucose transporter ( Glut4 ) . ( , n = 4 - 5 ) . Transcript were normalized to 18S . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

O O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


D - F Serum of free fatty ( FAA ) , insulin , leptin in WT KO after 25 on NC or HFD ( , n = 8 - 10 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Glucose tolerance ( GTT ) after 25 on NC or HFD . GTT was performed following an overnight fast Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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

Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Insulin tolerance ( ITT ) after 25 on NC or HFD . , ITT following a three hour fast ( , n = 6 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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

Insulin tolerance test (ITT) after 25 weeks on NC or HFD. , ITT following a three hour fast (males, n=6). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Oxygen consumption ( VO2 ) of 10 - 12 week old WT KO mice on normal chow were measured using CLAM metabolic cage system ( , n = 6 ) . Bar graphs show average value of metabolic measurements made within 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


carbon dioxide production ( VCO2 ) of 10 - 12 week old WT KO mice on normal chow were measured using CLAM metabolic cage system ( , n = 6 ) . Bar graphs show average value of metabolic measurements made within 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


respiratory exchange VCO2 / VO2 ( RER ) of 10 - 12 week old WT KO mice on normal chow were measured using CLAM metabolic cage system ( , n = 6 ) . Bar graphs show average value of metabolic measurements made within 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


physical activity of 10 - 12 week old WT KO mice on normal chow were measured using CLAM metabolic cage system ( , n = 6 ) . Bar graphs show average value of metabolic measurements made within 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


Transcript for Rcan1 . 1 Rcan1 . 4 during differentiation of 3T3 - L1 adipocytes , measured at 0 , 1 , 2 , 3 , 4 , 6 24 hours following addition MDI as well as at 3 , 7 10 days as indicated ( n = 3 ) . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - , unpaired , vs 0 time point . Data information : Transcript were normalized to 18S . values shown are mean ±

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

Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


B Western blot for RCAN1 . 1 , RCAN1 . 4 tubulin ( TUB ) in whole extracts of 3T3 - L1 adipocytes transfected with a control si - or ones targeting either Rcan1 . 4 or Rcan1 . 1 ( 20 μg per lane ) .

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

B Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).


C Representative of 3T3 - L1 adipocytes transfected with indicated si - RNAs prior to differentiation stained with Oil Red O on day 10 . Scale bar = 100 μm .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-TISSUE O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.


D Western blot for adiponectin ( ADPOQ ) released into media during differentiation by 3T3 - L1 adipocytes transfected with indicated si - RNAs . Ponceau stained image provides loading control .

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

D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.


E Transcript for Pgc1 - α , in 3T3 - L1 adipocytes transfected with indicated si - RNAs assayed over 10 - days of differentiation ( n = 3 ) . Data information : Transcript were normalized to 18S . values shown are mean ±

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

E Transcript levels for Pgc1-α, in 3T3-L1 adipocytes transfected with the indicated si-RNAs and assayed over 10-days of differentiation (n=3). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


Representative bright - field showing lipid accumulation at 10 - days differentiation of Ing - svf treated with indicated siRNA . Scale bar = 50 μm .

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

Representative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.


G Transcript for Adrenoceptor beta 3 ( Adrb3 ) , uncoupling 1 ( Ucp1 ) , PPARγ coactivator 1 alpha ( PGC1α ) , deiodinase type II ( Dio2 ) Ing - svf cultures transfected with control siRNA ( C ) or one targeting Rcan1 ( KD ) then maintained in growth media ( G ) , or differentiated for ten - days ( D ) with without addition of 100 mM isoproterionol for 6 hours prior to harvesting ( D + ISO ) ( n = 2 in triplicate ) . * = p < 0 . 05 ; * * = p < 0 . 01 , two - way ANOVA with multiple comparisons . Data information : Transcript were normalized to 18S . values shown are mean ±

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


H , I Transcript for indicated panel of genes relevant to adipocyte differentiation in Platelet - derived growth factor receptor alpha - positive ( PDGFRα + ) lineage - negative ( Lin - ) stromal vascular isolated by FACS sorting from sWAT of WT ( black bars ) KO ( red bars ) . In H , isolated were maintained in growth media . In I , cultures were treated with MDI then differentiated for 10 days . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - , unpaired , WT vs . KO . Data information : Transcript were normalized to 18S . values shown are mean ±

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

H, I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet-derived growth factor receptor alpha-positive (PDGFRα+) and lineage-negative (Lin-) stromal vascular cells isolated by FACS sorting from sWAT of WT (black bars) and KO (red bars). In H, isolated cells were maintained in growth media. In I, cultures were treated with MDI then differentiated for 10 days. * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, WT vs. KO. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


A Representative H & E histological sections of sWAT from WT KO mice probed with anti - UCP1 antibodies . Mice were either maintained at 24 ° C ( RT ) or housed at 6 ° C for 5 days . Scale bar = 200 μm .

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

A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm.


Transcript of Pgc1α Ucp1 in sWAT of WT KO mice after 24 hours at RT or 4 ° C ( n = Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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

Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n= Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


C Transcript of Ucp1 , Pgc1α , Adrb3 , retinoblastoma 1 ( Rb1 ) , zinc finger 423 ( Zfp423 ) , PR domain containing 16 ( Prdm16 ) were quantified in sWAT of WT KO mice after 25 on a HFD ( n = 4 - 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ; unpaired student ' s t -

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

C Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. ; unpaired student's t-test


D Transcript of Pgc1α - 1b Pgc1α - 1a isoforms in sWAT of WT KO 8 week old maintained at 24 ° C ( RT ) or housed at 4 ° C for 24 hours ( n = 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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D Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


E . Western blot for RCAN1 in various skeletal muscle groups . Each lane was loaded with 20 μg of total from plantaris ( PL ) , soleus ( SOL ) , extensor digitorum longus ( EDL ) , tibialis anterior ( TA ) , red gastrocnemius ( RG ) , gastrocnemius ( WG ) , vastus lateralis ( WV ) , heart ventricle ( HT ) , atria , diaphragm ( Dia ) , Tong . lane containing brain extract was loaded with 10 μg of . Antibody specificity was validated by running 20 μg of from heart of an Rcan1 - KO ( HT - KO ) . asterisks * indicate location of putative proteolytic fragments of RCAN1 . 1 .

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E. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1.


F Transcript of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT KO mice housed at RT . ( n = 4 - 5 each gender ) . G Transcript of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT KO housed at RT . ( n = 5 ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisons F , ; unpaired student ' s t - in G .

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F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender). G Transcript levels of Sln in skeletal muscle (tibialis anterior ) of 8-10 week old male WT and KO animals housed at RT. (n=5).Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons F, ; unpaired student's t-test in G.


H Representative western blot showing SLN in soleus muscle of 8 - 10 week old WT KO housed at RT . GAPDH was used as a loading control .

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H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control.


J Activity of Sln - Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin ( CnA * ) Rcan1 . Luciferase activity was normalized to beta - galactosidase activity from a co - transfected vector . ( n = 3 , assayed in duplicate ) . Data information : Transcript were normalized to 18S . values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


A Correlation of Rcan1 expression in liver gonadal adipose tissue with HOMA - IR , using mice from over 100 strains from hybrid mouse diversity panel . Regression ( red ) , r = biweight midcorrelation , p = p - value .

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A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value.


B , C Correlation of Rcan1 expression in gonadal adipose liver with , plasma insulin plasma triglycerides , in a data set comparing backgrounds susceptible ( BTBR background ) or resistant ( C57BL / 6 background ) to diabetes when carrying leptinob / ob ( ob ) mutation . Measures are reported as of mean log10 ( ml ) . Regression ( black ) , r = linear regression , p = p - value .

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B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value.


A ) Live confocal imaging of liver stage P . berghei expressing UIS4 ‐ mCherry GFP reveals different morphologies of LS ‐ TVN : elongated clusters ( left ) , vesicles in host cytoplasm ( center ) , a thin tubule protruding from PVM ( right ) . Live imaging was performed 20 h after infection of hepatoma . Features are marked with arrowheads .

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A) Live confocal imaging of liver stage P. berghei expressing UIS4‐mCherry and cytoplasmic GFP reveals different morphologies of the LS‐TVN: elongated membrane clusters (left), vesicles in the host cell cytoplasm (center), and a thin tubule protruding from the PVM (right). Live imaging was performed 20 h after infection of hepatoma cells. Features are marked with white arrowheads.


B ) UIS4 ‐ IBIS1 ‐ positive tubules rapidly extend from retract to PVM . Confocal time ‐ lapse imaging of a UIS4 ‐ mCherry ‐ expressing at 22 h ( top ) an IBIS1 ‐ mCherry ‐ expressing at 26 h ( bottom ) after infection of hepatoma . Tubules are indicated with a yellow arrow , time is shown in minutes seconds .

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B) The UIS4‐ and IBIS1‐positive tubules rapidly extend from and retract to the PVM. Confocal time‐lapse imaging of a UIS4‐mCherry‐expressing parasite at 22 h (top) and an IBIS1‐mCherry‐expressing parasite at 26 h (bottom) after infection of hepatoma cells. Tubules are indicated with a yellow arrow, and the time is shown in minutes and seconds.


C ) Imaging of IBIS1 ‐ mCherry ‐ expressing P . berghei reveals similar features of LS ‐ TVN , i . e . elongated cluster ( left ) , vesicles in host cytoplasm ( center ) , a tubule ( right ) . Infected were fixed 26 h post ‐ infection imaged by epifluorescence microscopy . Features are indicated with a yellow arrowhead . Scale bars , 10 µm .

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C) Imaging of IBIS1‐mCherry‐expressing P. berghei reveals similar features of the LS‐TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26 h post‐infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10 µm.


D ) of distinct UIS4 ‐ IBIS1 ‐ positive membranous structures in fixed infected hepatoma at three different time points : 6 h after infection ( period of sporozoite transformation into exo ‐ erythrocytic forms ) , 24 h after infection ( onset of replication growth ) , 48 h after infection ( mature liver stages ) . Values are shown as mean ( ± SD ) from three separate wells ( n = 100 per well ) of one experiment are representative of two experimental repetitions .

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D) Quantification of the distinct UIS4‐ and IBIS1‐positive membranous structures in fixed infected hepatoma cells at three different time points: 6 h after infection (the period of sporozoite transformation into exo‐erythrocytic forms), 24 h after infection (onset of parasite replication and growth), and 48 h after infection (mature liver stages). Values are shown as mean (± SD) from three separate wells (n = 100 per well) of one experiment and are representative of two experimental repetitions.


Hepatoma infected with Plasmodium berghei ANKA were fixed stained with anti - UIS4 anti - HSP70 A ) Some UIS4 ‐ positive vesicles are tethered to PVM as ' beads ‐ ona ‐ string ' . Infected fixed 24 h post ‐ infection .

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Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4 and anti-HSP70 A) Some UIS4‐positive vesicles are tethered to the PVM as 'beads‐ona‐string'. Infected cell fixed 24 h post‐infection.


B ) UIS4 ‐ mCherry features we detect in live imaging of UIS4 ‐ mCherry ‐ expressing P . berghei , i . e . elongated clusters ( left ) , vesicles ( center ) tubules ( right ) are also apparent in fixed wildtype stained with anti ‐ UIS4 antibody . Infected 24 h post ‐ infection . Scale bars , 10 µm .

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

B) The UIS4‐mCherry features we detect in live imaging of UIS4‐mCherry‐expressing P. berghei, i.e. elongated membrane clusters (left), vesicles (center) and tubules (right) are also apparent in fixed wildtype parasites stained with anti‐UIS4 antibody. Infected cells 24 h post‐infection. Scale bars, 10 µm.


Hepatoma infected with Plasmodium berghei ANKA were fixed stained with anti - UIS4 . C ) Often LS ‐ TVN is a composite of vesicles , tubules clusters . Infected 22 h post ‐ infection .

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

Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4. C) Often the LS‐TVN is a composite of vesicles, tubules and membrane clusters. Infected cell 22 h post‐infection.


D ) LS ‐ TVN detectable in hepatocytes infected with UIS4 ‐ mCherry ‐ expressing P . berghei also consists of elongated clusters ( yellow arrowhead ) , vesicles ( arrow ) tubules ( yellow arrow ) . Infected 18 h post ‐ infection .

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

D) The LS‐TVN detectable in primary hepatocytes infected with UIS4‐mCherry‐expressing P. berghei also consists of elongated membrane clusters (yellow arrowhead), vesicles (white arrow) and tubules (yellow arrow). Infected cell 18 h post‐infection.


E ) Dynamic tubules ( yellow arrow ) are detectable with UIS4 ‐ mCherry in infected hepatocytes 24 h post ‐ infection . Location of is marked with a yellow asterisk . Scale bars , 10 µm .

O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

E) Dynamic tubules (yellow arrow) are detectable with UIS4‐mCherry in infected primary hepatocytes 24 h post‐infection. Location of parasite is marked with a yellow asterisk. Scale bars, 10 µm.


A ) Confocal microscopy show a tubule ( upper panel ) labeled with UIS4 IBIS1 ( arrowheads ) , an elongated cluster ( middle panel ) in which both are present , vesicles ( bottom panel ) that are often labeled with both UIS4 IBIS1 ( yellow arrow ) frequently labeled with only one ( yellow arrowheads ) .

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A) Confocal microscopy images show a tubule (upper panel) labeled with UIS4 and IBIS1 (white arrowheads), an elongated membrane cluster (middle panel) in which both proteins are present, and vesicles (bottom panel) that are often labeled with both UIS4 and IBIS1 (yellow arrow) and frequently labeled with only one protein (yellow arrowheads).


B ) Epifluorescence microscopy of IBIS1 UIS4 reveals that while most tubules are labeled with both as above ( arrowhead ) , some are labeled exclusively with UIS4 ( upper panel ) or IBIS1 ( bottom panel ) . Single ‐ labeled tubules were highlighted with arrows their frequency in infected with tubules is indicated in ( ± SD ) , which was calculated from two separate wells 30 h post ‐ infection ( n > 150 infected per well ) .

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

B) Epifluorescence microscopy of IBIS1 and UIS4 reveals that while most tubules are labeled with both proteins as above (white arrowhead), some are labeled exclusively with UIS4 (upper panel) or IBIS1 (bottom panel). Single‐labeled tubules were highlighted with white arrows and their frequency in infected cells with tubules is indicated in percent (± SD), which was calculated from two separate wells 30 h post‐infection (n > 150 infected cells per well).


A ‐ C ) Time ‐ lapse imaging of hepatoma infected with UIS4 ‐ mCherry ‐ expressing Plasmodium berghei was used to determine velocities of UIS4 ‐ positive features , e . g . , a tubule ( A ) , elongated cluster ( B ) vesicle ( C ) were tracked over time . Measured speeds of dynamic features indicated with arrows are depicted graphically to right . Scale bars , 10 µm .

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

A‐C) Time‐lapse imaging of hepatoma cells infected with UIS4‐mCherry‐expressing Plasmodium berghei was used to determine the velocities of the UIS4‐positive features, e.g., a tubule (A), elongated membrane cluster (B) and vesicle (C) were tracked over time. Measured speeds of the dynamic features indicated with white arrows are depicted graphically to the right. Scale bars, 10 µm.


D ) velocities of several UIS4 ‐ or IBIS1 ‐ positive features [ tubules ( left ) , elongated clusters ( center ) vesicles ( right ) ] were measured over time , peak velocity of each was plotted . Plotted data was derived from multiple imaged infected : For IBIS1 ‐ positive tubules data is derived from three infected for UIS4 ‐ positive tubules from 13 infected . Elongated cluster speeds were analyzed in at least five infected for both UIS4 IBIS1 , vesicle speeds were analyzed in at least 12 different for both markers .

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

D) The velocities of several UIS4‐ or IBIS1‐positive features [tubules (left), elongated membrane clusters (center) and vesicles (right)] were measured over time, and the peak velocity of each was plotted. Plotted data was derived from multiple imaged infected cells: For IBIS1‐positive tubules data is derived from three infected cells and for UIS4‐positive tubules from 13 infected cells. Elongated membrane cluster speeds were analyzed in at least five infected cells for both UIS4 and IBIS1, and vesicle speeds were analyzed in at least 12 different cells for both protein markers.


B ) infected with UIS4 ‐ mCherry ‐ expressing were treated with nocodazole for 1 . 5 h ( top ) or cytochalasin D for 30 min ( bottom ) imaged by confocal microscopy . Imaging occurred 29 24 h post ‐ infection , respectively . UIS4 ‐ positive tubules , static ( top ) dynamic ( bottom ) , are indicated with arrowheads . Displayed are representative of five imaged from at least two different experiments .

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B) Cells infected with UIS4‐mCherry‐expressing parasites were treated with nocodazole for 1.5 h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post‐infection, respectively. UIS4‐positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments.


C ) No colocalization between host microtubules UIS4 ‐ positive tubules is detectable . Infected were fixed 25 h post ‐ infection stained with antibodies against UIS4 Tubulin . Scale bars , 10 µm .

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C) No colocalization between host microtubules and UIS4‐positive tubules is detectable. Infected cells were fixed 25 h post‐infection and stained with antibodies against UIS4 and Tubulin. Scale bars, 10 µm.


A B ) bleach was applied to elongated clusters of LS ‐ TVN approximately 27 h post ‐ infection . UIS4 ‐ mCherry signal typically did not recover in bleached ( A ) , although it was found to recover in some elongated clusters ( B ) .

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

A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).


C ) When bleach was applied to PVM at 25 h post ‐ infection , signal recovered rapidly in bleached region . Upon application of additional bleach pulses in same , bleached ( region # 1 measured graphically ) repeatedly recovered , remaining PVM ‐ associated UIS4 ‐ mCherry signal depleted ( region # 2 ) although LS ‐ TVN ‐ associated signal remained ( region # 3 ) . When a region of LS ‐ TVN was subsequently bleached ( yellow arrow , region # 4 ) , no recovery was observed . times are shown in minutes seconds . Scale bars , 10 µm .

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C) When the bleach was applied to the PVM at 25 h post‐infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM‐associated UIS4‐mCherry signal depleted (region #2) although the LS‐TVN‐associated signal remained (region #3). When a region of the LS‐TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10 µm.


A B ) A phase ‐ contrast image containing infected of interest ( A ) was used for comparison to EM grid after fixation preparation of for ( B ) . Green outlining of in phase ‐ contrast image was used as a tool for comparison alignment . insets defined in are shown in right panels merged with image of UIS4 ‐ mCherry signal acquired immediately prior to fixation . Scale bars , 10 µm .

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

A and B) A phase‐contrast image containing the infected cell of interest (A) was used for comparison to the EM grid after fixation and preparation of the cells for TEM (B). Green outlining of the cells in the phase‐contrast image was used as a tool for comparison and alignment. The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4‐mCherry signal acquired immediately prior to fixation. Scale bars, 10 µm.


C ) Detection of UIS4 ‐ mCherry signal by widefield microscopy in of interest immediately prior to fixation . location of is marked with an asterisk , UIS4 ‐ positive features are highlighted with arrowheads .

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C) Detection of the UIS4‐mCherry signal by widefield fluorescence microscopy in the cell of interest immediately prior to fixation. The location of the parasite is marked with an asterisk, and UIS4‐positive features are highlighted with arrowheads.


D ) Thirty ‐ six sections of this sample were prepared imaged ( Movies S4 S5 ) . Features corresponding to UIS4 ‐ positive tubule ( arrowhead ) were found in sections 10 - 16 to UIS4 ‐ positive signal adjacent to PVM ( yellow arrowhead ) were found in Sections 16 - 29 . Sections 11 ( top ) 20 ( bottom ) are displayed to highlight these two features . Scale bars , 1 µm .

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

D) Thirty‐six TEM sections of this sample were prepared and imaged (Movies S4 and S5). Features corresponding to the UIS4‐positive tubule (white arrowhead) were found in sections 10-16 and to the UIS4‐positive signal adjacent to the PVM (yellow arrowhead) were found in Sections 16-29. Sections 11 (top) and 20 (bottom) are displayed to highlight these two features. Scale bars, 1 µm.


A ) Hepatoma infected with UIS4 − mCherry − expressing Plasmodium berghei were fixed 22 h post − infection stained with antibodies against host markers of distinct organelles : anti − EEA1 to label early endosomes , anti − CD63 anti − LAMP1 to label late endosomes lysosomes , anti − Calnexin to label endoplasmic reticulum , anti − Golgin97 to label Golgi . In samples , either anti − mCherry or anti − UIS4 was used to amplify UIS4 − mCherry signal . Imaging was carried out by epifluorescence microscopy . In right panels , is merged , selected regions with areas of interest are shown enlarged . At least 20 infected possessing LS − TVN features were visually examined for each condition , representative of UIS4 − positive elongated clusters ( left ) UIS4 − positive tubules ( right , indicated with yellow arrows ) are displayed . Scale bars , 10 µm .

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

A) Hepatoma cells infected with UIS4−mCherry−expressing Plasmodium berghei parasites were fixed 22 h post−infection and stained with antibodies against host cell protein markers of distinct organelles: anti−EEA1 to label early endosomes, anti−CD63 and anti−LAMP1 to label late endosomes and lysosomes, anti−Calnexin to label the endoplasmic reticulum, and anti−Golgin97 to label the Golgi. In all samples, either anti−mCherry or anti−UIS4 was used to amplify the UIS4−mCherry signal. Imaging was carried out by epifluorescence microscopy. In the right panels, fluorescence is merged, and selected regions with areas of interest are shown enlarged. At least 20 infected cells possessing LS−TVN features were visually examined for each condition, and representative images of UIS4−positive elongated membrane clusters (left) and UIS4−positive tubules (right, and indicated with yellow arrows) are displayed. Scale bars, 10 µm.


B ) Profiling of red ( UIS4 ) green ( CD63 , left or LAMP ‐ 1 , right ) signals along indicated within insets from in ( A ) above .

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

B) Profiling of the red (UIS4) and green (CD63, left or LAMP‐1, right) signals along the indicated line within the insets from the images in (A) above.