( 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 )

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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 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

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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 . 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 .

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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 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 ) .

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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 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 .

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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 - test ( 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 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 .

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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 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 ) .

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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 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 .

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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 and GFP in Nb leaves . The 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 and PBL27 - HA transiently expressed in Nb leaves . Total proteins 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 ) , and microsomal membrane ( 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 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 .

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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 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 .

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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 and MAPKKK5K375M - GFP transiently expressed in Arabidopsis protoplasts .

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


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

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins 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 The S622A mutation strongly reduces phosphorylation by PBL27 . Each of phosphorylation sites was substituted by alanine , and subjected to the 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 . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins were detected by autoradiography .

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 . 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 .

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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 ( Line 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 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 ) .

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

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


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 .

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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 assay .

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


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 .

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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 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 .

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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 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 .

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

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

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 and MKK5 in vitro . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins 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 . The 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.


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 ) .

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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).


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 ) .

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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 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 ) .

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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 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 ) .

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 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

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 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 ) .

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 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 ) .

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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 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 ) .

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 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 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 ) .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O 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 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 the media during differentiation by 3T3 - L1 adipocytes transfected with the indicated si - RNAs . Ponceau stained image provides loading control .

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 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 ±

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

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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 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 ±

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

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 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 ±

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 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 .

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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 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

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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 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 -

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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 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

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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 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 .

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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 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 .

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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 protein in soleus muscle of 8 - 10 week old WT and KO animals 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 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

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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 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 .

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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 ) 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 .

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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 primary hepatocytes 24 h post ‐ infection . Location of parasite is marked with a yellow asterisk . Scale bars , 10 µm .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4 ‐ mCherry ( top ) or IBIS1 ‐ mCherry ( bottom ) were fixed 22 h or 48 h post ‐ infection , respectively , and stained with anti ‐ LC3 to label autophagic membranes . In the right panels , fluorescence is merged , and selected areas of interest are shown enlarged . Scale bars , 10 µm .

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LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes. In the right panels, fluorescence is merged, and selected areas of interest are shown enlarged. Scale bars, 10 µm.


( A ) Quantification of AURKB transcripts in non - IPF lung derived resident - fibroblasts treated with indicated mitogens for 16hrs . * P < 0 . 05 , * * * P < 0 . 0005 , and * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 4 ) .

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(A) Quantification of AURKB transcripts in non-IPF lung derived resident-fibroblasts treated with indicated mitogens for 16hrs. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1-way ANOVA, (n=4).


( B ) Quantification of AURKB gene transcripts in isolated fibroblasts from non - IPF and IPF lung stromal cell cultures . * * P < 0 . 005 , unpaired t - test , ( n = 6 ) .

(B) Quantification of AURKB gene transcripts in isolated fibroblasts from non-IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t-test, (n=6).


( C ) IPF and non - IPF lung sections were immunostained using AURKB antibody ( n = 4 ) . Representative images were obtained at 40X magnification . Scale bar : 40µm . Dotted area : black ; myofibroblastic core , blue ; active fibrotic front . Arrows indicate AURKB positive cells .

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(C) IPF and non-IPF lung sections were immunostained using AURKB antibody (n=4). Representative images were obtained at 40X magnification. Scale bar: 40µm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.


( D ) Immunoblots of Aurkb and Aurka in total lung lysates of normal ( CCSP / - ) and fibrotic ( CCSP / TGFα ) mice fed with Dox for 6wks . Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH . * P < 0 . 05 , unpaired t - test , ( n = 4 ) .

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(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).


( E ) Immunostaining was performed using AURKB antibody in lung sections of control ( CCSP / - ) and TGFα ( CCSP / TGFα ) mice on Dox for 6wks ( n = 6 ) . Representative images were obtained at 20X ( low ; Scale bar : 100µm ) and 63X ( high ; Scale bar : 30µm ) magnification . Dashed box indicates area of the section showing in high magnification .

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(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.


( F ) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice . Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh . * * P < 0 . 005 , unpaired t - test , ( n = 4 - 5 ) .

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(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).


( A ) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( B ) Lung - resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).


( C ) Immunoblot analysis of AURKB and WT1 in the lysates of non - IPF fibroblasts transduced with control or WT1 - adenoviral particles for 72hrs . * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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(C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3).


( D ) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα ( 100ng / mL ) for 16hrs . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Primary lung - resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks . Cell lysates were prepared , and the ChIP assay was performed with anti - WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter - specific PCR primers . Non - immunoprecipitated DNA is represented as input DNA ( product size , 104 bp ) . * * * P < 0 . 0005 , unpaired t - test , ( n = 2 ) .

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(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).


( F ) AURKB - promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing ( OE ) vector . * * * * P < 0 . 00005 , one - way - ANOVA , ( n = 6 ) .

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(F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).


( C ) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB ( green ) and Ki - 67 ( red ) . Merged image shows cells that co - express AURKB and Ki - 67 ( yellow ) .

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(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).


( D ) Quantification of Ki - 67 + and AURKB + double positive cells was performed using five confocal images per mice in control and TGFα mice . Images were obtained at 40X magnification . Scale bar : 50µm . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA . * * * * P < 0 . 00005 , unpaired t - test , ( n = 9 - 11 ) .

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(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).


( F ) Proliferation was measured in primary lung - resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα ( 20ng / mL ) for 24hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( G ) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( H ) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB ( green ) and αSMA ( red ) . Images were obtained at 40X magnification . Scale bar : 50µm . ( n = 4 ) .

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(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).


( I ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in lung - resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h . * * P < 0 . 005 , * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).


( J ) Quantification of BAK , BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

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(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( K ) Quantification of Bak , Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs . * P < 0 . 005 , * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).


( A ) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( B ) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs . * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).


( C ) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody . Images were obtained at 40X magnification . Scale bar : 50µm . The number of PCNA - positive cells and total DAPI - positive cells were quantified using MetaMorph image analysis software . Proliferation is indicated as the percentage of proliferating cells in total DAPI - positive cells . * * * * P < 0 . 00005 , unpaired t - test , ( n = 3 - 4 ) .

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(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).


( D ) Proliferation was measured in primary fibroblasts treated with TGFα ( 20ng / mL ) and indicated doses of barasertib for 48hrs . . * * P < 0 . 005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).


( E ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib . * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).


( B ) Representative images of Masson ' s trichrome - stained lung sections from the vehicle and barasertib treated mice . Images were obtained at 10X magnification . Scale bar : 200μm .

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(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of right lung weight of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).


( D ) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).


( E ) Quantification of Col1α , Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( A ) Lung sections from vehicle and barasertib treated mice were immunostained using Ki - 67 antibody . Representative images were obtained at 20X magnification . Scale bar : 50μm .

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(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.


( B ) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody . Gapdh is used as loading control . * * P < 0 . 005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).


( C ) Quantification of Aurkb , Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib . * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( B ) Quantification of right lung weight of mice treated with vehicle and barasertib . * * P < 0 . 005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( C ) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies . Gapdh is used as loading control . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).


( D ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

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(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( E ) Quantification of lung mechanics in mice treated with vehicle and barasertib . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( B ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 7 - 8 mice / group ) .

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(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).