Figure 2A 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 . 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 . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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.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.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).


Figure 3A 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 . 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 - GFPE 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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Figure 3A 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.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 -GFPE 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.


Figure 4A 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 . 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 . 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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Figure 4A 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.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.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.


Figure 5A 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 . 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 . 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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Figure 5A 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.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.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.


Figure 6A 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 ) . 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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Figure 6A 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).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.


Figure 7A 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 . 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 . E The kinase activity of GST - PBL273xA was analyzed by the in vitro kinase assay using [ 32P ] γ - ATP .

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Figure 7A 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.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.E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.


Figure 8A 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 . 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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Figure 8A 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.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.


Figure 1 ( C - F ) Two months after a single tail vein injection of rAAV , wild - type mice showed similar body weight ( C ) , blood glucose levels ( D ) , serum alanine aminotransferase ( ALT ) ( E ) and aspartate aminotransferase ( AST ) activities ( F ) with the mice injected with PBS . n = 8 - 10 for each group . ( H - I ) Two months after a single injection of rAAV , db / db mice showed similar body weight ( H ) and blood glucose levels ( I ) with the mice injected with PBS . n = 10 for each group . ( J - K ) Serum ALT ( J ) and AST ( K ) of mice in ( H ) . ( M ) The hepatic p - MLKL protein level in normal and necrotic area from mice injected with PBS or rAAV in ( H ) . Ncp , Necroptosis . n = 7 - 8 biological replicates . ( N ) The hepatic mRNA levels of Il - 6 , Il - 1β , Tnf - α and Mcp - 1 involved in necroptosis at normal or necroptotic area from mice injected with PBS or rAAV in ( H ) . n = 7 - 8 biological replicates . ( Q - R ) Six months after a single injection of rAAV , db / db mice showed decreased body weight ( Q ) and similar blood glucose levels ( R ) compared to the mice injected with PBS . n = 8 - 12 . ( S - T ) Serum ALT ( S ) and AST ( T ) activities of mice in ( Q ) . n = 8 - 10 .

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Figure 1(C-F) Two months after a single tail vein injection of rAAV, wild-type mice showed similar body weight (C), blood glucose levels (D), serum alanine aminotransferase (ALT) (E) and aspartate aminotransferase (AST) activities (F) with the mice injected with PBS. n = 8 - 10 for each group.(H-I) Two months after a single injection of rAAV, db/db mice showed similar body weight (H) and blood glucose levels (I) with the mice injected with PBS. n = 10 for each group. (J-K) Serum ALT (J) and AST (K) of mice in (H).(M) The hepatic p-MLKL protein level in normal and necrotic area from mice injected with PBS or rAAV in (H). Ncp, Necroptosis. n = 7 - 8 biological replicates.(N) The hepatic mRNA levels of Il-6, Il-1β, Tnf-α and Mcp-1 involved in necroptosis at normal or necroptotic area from mice injected with PBS or rAAV in (H). n = 7 - 8 biological replicates.(Q-R) Six months after a single injection of rAAV, db/db mice showed decreased body weight (Q) and similar blood glucose levels (R) compared to the mice injected with PBS. n = 8 - 12. (S-T) Serum ALT (S) and AST (T) activities of mice in (Q). n = 8 - 10.


Figure 2Body weight ( B ) were measured 6 weeks later , after injection once a day for 5 consecutive days with vehicle ( Veh ) or streptozotocin ( STZ ) . n = 6 - 19 for each group . blood glucose levels ( C ) were measured 6 weeks later , after injection once a day for 5 consecutive days with vehicle ( Veh ) or streptozotocin ( STZ ) . n = 6 - 19 for each group . Two months after a single injection of rAAV , hyperglycemic mice induced by streptozotocin showed , blood glucose levels ( F ) compared with those mice injected with PBS . n = 9 ( G - H ) Serum ALT ( G ) and AST ( H ) activities of mice in ( D ) . ( J ) The hepatic p - MLKL level of mice in ( D ) . Body weight ( L ) of RHH ( Resistance to HFD - induced Hyperglycemia ) mice fed with HFD for 15 weeks . n = 6 - 12 . blood glucose levels ( Q ) of obese and euglycemic mice in ( L ) after a single injection of PBS or rAAV for 2 months . n = 6 . Serum ALT ( R ) of mice in ( O ) . ( T ) Liver images , H & E staining of liver sections and incidence of hepatic necroptosis for mice in ( O ) . ( U ) The hepatic p - MLKL level of mice in ( O ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O

Figure 2Body weight (B) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.blood glucose levels (C) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.Two months after a single injection of rAAV, hyperglycemic mice induced by streptozotocin showed , blood glucose levels (F) compared with those mice injected with PBS. n = 9 (G-H) Serum ALT (G) and AST (H) activities of mice in (D).(J) The hepatic p-MLKL level of mice in (D).Body weight (L) of RHH (Resistance to HFD-induced Hyperglycemia) mice fed with HFD for 15 weeks. n = 6 - 12.blood glucose levels (Q) of obese and euglycemic mice in (L) after a single injection of PBS or rAAV for 2 months. n = 6. Serum ALT (R) of mice in (O).(T) Liver images, H&E staining of liver sections and incidence of hepatic necroptosis for mice in (O).(U) The hepatic p-MLKL level of mice in (O).


Figure 3Two months after a single injection of PBS or rAAV blood glucose levels ( G ) of hyperglycemic and obese mice in ( B ) were not significantly changed . n = 10 - 11 for each group . ( H - I ) Serum ALT ( H ) and AST ( I ) activities of mice in ( E ) . ( K ) The hepatic p - MLKL protein level in normal or necrotic area from mice injected with PBS or rAAV in ( E ) . ( L ) The hepatic mRNA levels of Il - 6 , Il - 1β , Tnf - α and Mcp - 1 involved in necroptosis at normal or necroptotic area from mice in ( E ) . blood glucose levels ( O ) of hyperglycemic and obese mice after a single injection of PBS or rAAV for 6 months . n = 8 . ( P - Q ) Serum ALT ( P ) and AST ( Q ) activities of mice in ( M ) . ( R ) Liver images , H & E staining and ki67 immunostaining of liver sections and HCC incidence of mice in ( M ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Two months after a single injection of PBS or rAAV blood glucose levels (G) of hyperglycemic and obese mice in (B) were not significantly changed. n = 10 - 11 for each group. (H-I) Serum ALT (H) and AST (I) activities of mice in (E).(K) The hepatic p-MLKL protein level in normal or necrotic area from mice injected with PBS or rAAV in (E).(L) The hepatic mRNA levels of Il-6, Il-1β, Tnf-α and Mcp-1 involved in necroptosis at normal or necroptotic area from mice in (E).blood glucose levels (O) of hyperglycemic and obese mice after a single injection of PBS or rAAV for 6 months. n = 8. (P-Q) Serum ALT (P) and AST (Q) activities of mice in (M).(R) Liver images, H&E staining and ki67 immunostaining of liver sections and HCC incidence of mice in (M).


Figure 4 ( A ) Prednisone treatment markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly ( I : C ) ( pIC ) . n = 3 in duplicate . Prednisone treatment markedly blocked the increase of p - MLKL level ( C , n = 3 in duplicate . ) of macrophages induced by poly ( I : C ) and z - VAD - fmk ( zVAD ) . Scar bar , 100 μm . ( J ) Representative liver images for mice in ( E ) . ( M ) The hepatic p - MLKL level of mice in ( E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O

Figure 4(A) Prednisone treatment markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C) (pIC). n = 3 in duplicate.Prednisone treatment markedly blocked the increase of p-MLKL level (C, n = 3 in duplicate.) of macrophages induced by poly(I:C) and z-VAD-fmk (zVAD). Scar bar, 100 μm.(J) Representative liver images for mice in (E).(M) The hepatic p-MLKL level of mice in (E).


Figure 5 ( A ) The top 15 hepatic genes upregulated in 9 - week - old db / db mice compared with wild type mice . n = 1 , liver samples from 3 mice were mixed together in equal amounts for RNA extraction . Body weight ( D ) of db / db mice after a single injection of rAAV - si - NC / Cyp4a14 / Pebp1 / Tat for 2 months . n = 10 - 12 . rAAV - si - NC was packaged as indicated in Fig . 1A ( I ) Liver images , H & E staining of liver sections and the incidence of death and hepatic necroptosis ( Ncp ) of mice in ( D ) . n = 10 - 14 , NML , Normal . ( K ) The hepatic p - MLKL level of the indicated mice in ( D ) . ( L ) The indicated hepatic mRNA levels of mice in ( K ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O

Figure 5(A) The top 15 hepatic genes upregulated in 9-week-old db/db mice compared with wild type mice. n = 1, liver samples from 3 mice were mixed together in equal amounts for RNA extraction.Body weight (D) of db/db mice after a single injection of rAAV-si-NC/Cyp4a14/Pebp1/Tat for 2 months. n = 10 - 12. rAAV-si-NC was packaged as indicated in Fig. 1A(I) Liver images, H&E staining of liver sections and the incidence of death and hepatic necroptosis (Ncp) of mice in (D). n = 10 - 14, NML, Normal.(K) The hepatic p-MLKL level of the indicated mice in (D).(L) The indicated hepatic mRNA levels of mice in (K).


Figure 6Two months after a single injection of rAAV - si - Pebp1 , HFD - induced hyperglycemic and obese mice showed blood glucose levels ( C ) with the mice injected with rAAV - si - NC . n = 10 - 11 . ( D - E ) Serum ALT ( D ) and AST ( E ) activities from mice in ( B ) . The colored dots indicate the mice with high ALT or AST activity , and the same color dot in ( D ) and ( E ) indicates the same mouse . ( G ) The hepatic p - MLKL level of mice in ( B ) . ( H ) The indicated hepatic mRNA levels of mice in ( B ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O

Figure 6Two months after a single injection of rAAV-si-Pebp1, HFD-induced hyperglycemic and obese mice showed blood glucose levels (C) with the mice injected with rAAV-si-NC. n = 10 - 11. (D-E) Serum ALT (D) and AST (E) activities from mice in (B). The colored dots indicate the mice with high ALT or AST activity, and the same color dot in (D) and (E) indicates the same mouse.(G) The hepatic p-MLKL level of mice in (B).(H) The indicated hepatic mRNA levels of mice in (B).


Figure 7 ( A ) The effect of si - Pebp1 in primary mouse macrophages . n = 3 . ( B ) Knockdown of Pebp1 markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly ( I : C ) . n = 6 . Knockdown of Pebp1 markedly blocked the loss of cell membrane integrity ( C , n = 6 in duplicate or triplicate . ) of mouse macrophages induced by poly ( I : C ) and z - VAD - fmk . ( E ) Knockdown of Pebp1 attenuated the increase of p - Tbk1 level induced by poly ( I : C ) . n = 3 biological replicates . Knockdown of Tbk1 markedly blocked the loss of cell membrane integrity ( H of mouse macrophages induced by poly ( I : C ) and z - VAD - fmk . After a single injection of rAAV - si - Tbk1 for 11 weeks , the db / db mice showed blood glucose levels ( M ) with the mice injected with rAAV - si - NC . n = 11 . ( N - O ) Serum ALT ( N ) and AST ( O ) activities of mice in ( K ) . ( R ) The hepatic p - MLKL level of mice in ( K ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O

Figure 7(A) The effect of si-Pebp1 in primary mouse macrophages. n = 3. (B) Knockdown of Pebp1 markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C). n = 6.Knockdown of Pebp1 markedly blocked the loss of cell membrane integrity (C, n = 6 in duplicate or triplicate.) of mouse macrophages induced by poly(I:C) and z-VAD-fmk.(E) Knockdown of Pebp1 attenuated the increase of p-Tbk1 level induced by poly(I:C). n = 3 biological replicates.Knockdown of Tbk1 markedly blocked the loss of cell membrane integrity (H of mouse macrophages induced by poly(I:C) and z-VAD-fmk.After a single injection of rAAV-si-Tbk1 for 11 weeks, the db/db mice showed blood glucose levels (M) with the mice injected with rAAV-si-NC. n = 11. (N-O) Serum ALT (N) and AST (O) activities of mice in (K).(R) The hepatic p-MLKL level of mice in (K).


Figure 1 ( B ) Non - supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls . PD samples indicated in red . ( D ) qPCR validation of TH levels in SN samples of PD and controls , normalized to beta - actin mRNA . T - test p * = 0 . 025 , Data presented as mean ± SD . n = 18 for CT and 24 for PD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Non-supervised clustering heatmap plot indicates substantial clustering of transcripts from each tissue but not of transcripts from patients compared to controls. PD samples indicated in red.(D) qPCR validation of TH levels in SN samples of PD and controls, normalized to beta-actin mRNA. T-test p*=0.025, Data presented as mean ± SD. n=18 for CT and 24 for PD.


Figure 2 ( D ) Heatmap of circRNA abundance , indicating significant tissue - based SN clustering , PD samples indicated in red .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 2(D) Heatmap of circRNA abundance, indicating significant tissue-based SN clustering, PD samples indicated in red.


Figure 3 ( A ) Total circRNAs detected in the SN , MTG and AMG of both PD and control donors , p * = 0 . 02 p * = 0 . 026 and p * = 0 . 022 for comparison between PD and CT in SN , MTG and AMG . T - test for total SN vs MTG p * = 2 . 27E - 8 and SN vs AMG p * = 5 . 86E - 10 . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . P value was calculated by T - test . ( B ) Global editing levels in the 3 brain regions is based on the Alu editing index , representing the weighted average editing level across all expressed Alu sequences , Wilcoxon , corrected p * = 0 . 038 , 0 . 055 and 0 . 42 for Amygdala , MTG and SN . Wilcoxon test between the Alu editing index of control and PD samples . ( C ) Global RNA editing levels based on the Alu editing index , studied only in Alu elements within circRNA exons and their flanking introns , in the 3 brain regions , for circRNAs corrected p * = 0 . 025 and 0 . 040 for the AMG and MTG and p = 0 . 3 for SN . For flanking introns p * = 0 . 02 and 0 . 040 for AMG and MTG and p = 0 . 34 for SN , Wilcoxon test . n = 8 for Amygdala control , 15 for Amygdala PD , 8 for MTG control and 13 for MTG PD , 10 for SN control and 15 for SN PD . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . ( D ) Negative correlation between total RNA editing and circRNA abundance , correlation = - 0 . 37 , Correlation test p value = 0 . 0015 . ( E ) Adar1 expression levels in all tissues . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . Whiskers mark minimum or maximum values . ( F , G ) Correlations between circRNAs detected in each sample and the age of the control ( green , correlation = 0 . 68 , p - value = 0 . 032 ) and PD donors ( blue , correlation = - 0 . 14 p value = 0 . 62 ) . ( H ) Volcano plot of DE circRNAs in PD vs control tissues , red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test ( DEseq2 analysis ) . CircSLC8A1 is marked is red .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.02 p*=0.026 and p*=0.022 for comparison between PD and CT in SN, MTG and AMG. T-test for total SN vs MTG p*=2.27E-8 and SN vs AMG p*=5.86E-10. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. P value was calculated by T-test.(B) Global editing levels in the 3 brain regions is based on the Alu editing index, representing the weighted average editing level across all expressed Alu sequences, Wilcoxon, corrected p*=0.038, 0.055 and 0.42 for Amygdala, MTG and SN. Wilcoxon test between the Alu editing index of control and PD samples.(C) Global RNA editing levels based on the Alu editing index, studied only in Alu elements within circRNA exons and their flanking introns, in the 3 brain regions, for circRNAs corrected p*= 0.025 and 0.040 for the AMG and MTG and p=0.3 for SN. For flanking introns p*= 0.02 and 0.040 for AMG and MTG and p=0.34 for SN, Wilcoxon test. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.(D) Negative correlation between total RNA editing and circRNA abundance, correlation = -0.37, Correlation test p value = 0.0015.(E) Adar1 expression levels in all tissues. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.(F,G) Correlations between circRNAs detected in each sample and the age of the control (green, correlation = 0.68, p-value = 0.032) and PD donors (blue, correlation = -0.14 p value = 0.62).(H) Volcano plot of DE circRNAs in PD vs control tissues, red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test (DEseq2 analysis). CircSLC8A1 is marked is red.


Figure 4 ( B , C ) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains , T - test p = 0 . 025 , p = 0 . 118 respectively . ( D ) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN , T - test p * = 0 . 025 . n = 18 for CT and 24 for PD , x defines mean values . ( E ) no difference in circSLC8A1 levels in females and males , T - test p = 0 . 583 for control and p = 0 . 262 for PD . n = 15 females and 16 males . . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average . ( F , G ) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control , R2 = 0 . 47 T - test p = 0 . 0017 but not in PD samples , R2 = 0 . 016 , p > 0 . 05 . n = 18 for CT and 23 for PD . ( H ) Increased CircSLC8A1 RNA levels in PQ - exposed SH - SY cells ( normalized to Tubb3 and RPL19 ) while other circRNAs remain unchanged ( I , J ) Immunoblot decline ( western blot and quantification ) of the SLC8A1 protein in PQ - exposed SH - SY cells , with Tubulin as a loading control , T - test p * = 0 . 015 . n = 3 biological replicas for each condition . Data presented as mean ± SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B, C) CircSLC8A1 and linear SLC8A1 levels in the SN of control and PD brains, T-test p=0.025, p= 0.118 respectively.(D) qPCR validations of circSLC8A1 and SLC8A1 mRNA changes in the SN, T-test p*=0.025. n=18 for CT and 24 for PD, x defines mean values.(E) no difference in circSLC8A1 levels in females and males, T-test p=0.583 for control and p=0.262 for PD. n=15 females and 16 males. . The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average.(F, G) CircSLC8A1 and linear SLC8A1 mRNA levels are correlated in control, R2=0.47 T-test p=0.0017 but not in PD samples, R2= 0.016, p>0.05. n=18 for CT and 23 for PD.(H) Increased CircSLC8A1 RNA levels in PQ-exposed SH-SY cells (normalized to Tubb3 and RPL19) while other circRNAs remain unchanged(I,J) Immunoblot decline (western blot and quantification) of the SLC8A1 protein in PQ-exposed SH-SY cells, with Tubulin as a loading control, T-test p*=0.015. n=3 biological replicas for each condition. Data presented as mean ± SD.


Figure 5 ( A ) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH - SY treatment of statins or the PF LRRK2 inhibitor , T - test p * * = 0 . 007 and p * * = 0 . 01 for PF - 06447475 and p * = 0 . 047 and p = * 0 . 0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively ( B , C ) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures , T - test p * = 0 . 027 for Statins and p * = 0 . 011 for PF - 06447475 , n = 3 biological replicas for each condition . Data presented as mean ± SD . ( D ) SLC8A1 immunostaining of statin - treated SH - SY cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O

Figure 5(A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.007 and p**=0.01 for PF-06447475 and p*=0.047 and p=*0.0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively(B,C) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures, T-test p*=0.027 for Statins and p*=0.011 for PF-06447475, n=3 biological replicas for each condition. Data presented as mean ± SD.(D) SLC8A1 immunostaining of statin-treated SH-SY cells.


Figure 6 ( B ) qPCR quantification from nuclear / cytoplasmic fractionations of SH - SY cells , T - test p = 0 . 005 for circSLC8A1 , p = 0 . 009 for NEAT1 and p = 0 . 009 for Rpl19 . n = 3 biological replicas for each condition . ( C ) qPCR quantification of Ago2 - bound RNA , immunoprecipitated ( IP ) / Input values normalized to negative control for a known Ago2 target , circCDR1as and circSLC8A1 , T - test p = 0 . 029 for circSLC8A1 and p = 0 . 001 for circCDR1as , n = 3 biological replicas for each condition . ( G ) relative expression of known miR - 128 targets from SN RNA - seq , p * * = 0 . 0004 for BMI1 , p * = 0 . 011 for SIRT1 and p * = 0 . 005 for AXIN1 . ( H ) relative expression of known miR - 128 targets in SH - SY cells treated with Paraquat , T - test p * = 0 . 03 , p * = 0 . 02 and p * = 0 . 002 for AXIN1 , BM1 and SIRT1 respectively , Data presented as mean ± SD with 4 - 6 biological replicas for each condition .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) qPCR quantification from nuclear/cytoplasmic fractionations of SH-SY cells, T-test p=0.005 for circSLC8A1, p=0.009 for NEAT1 and p=0.009 for Rpl19. n=3 biological replicas for each condition.(C) qPCR quantification of Ago2-bound RNA, immunoprecipitated (IP)/Input values normalized to negative control for a known Ago2 target, circCDR1as and circSLC8A1, T-test p=0.029 for circSLC8A1 and p=0.001 for circCDR1as, n=3 biological replicas for each condition.(G) relative expression of known miR-128 targets from SN RNA-seq, p**=0.0004 for BMI1, p*=0.011 for SIRT1 and p*=0.005 for AXIN1.(H) relative expression of known miR-128 targets in SH-SY cells treated with Paraquat, T-test p*=0.03, p*=0.02 and p*=0.002 for AXIN1, BM1 and SIRT1 respectively, Data presented as mean ± SD with 4-6 biological replicas for each condition.


Figure 2A ) The absolute abundance of centrosomal proteins in whole cell lysates , prepared from asynchronous growing cells , was determined using SRM mass spectrometry combined with stable isotope dilution . Copy numbers per cell were measured in three independent experiments . Because not all peptides could be detected in all experiments , histograms show average values determined in those two measurements in which most peptides could be measured reliably ; Results presented are means ± SEM . RPE - 1 ( non - transformed ) cells were compared with various tumor - derived ( U2OS , HeLa , KE37 ) or transformed cells ( HEK293T ) . Note the different scale for γ - tubulin on the y - axis . B ) Representative image showing centrosome preparation stained with antibodies to NEDD1 ( green ) and CP110 ( red ) . Bar : 10 μm . C ) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution . Histogram a priori represents relative copy numbers per centrosome ( Results presented are means ± SEM . ) . To predict protein abundance in absolute numbers ( average number of copies / centrosome ) , data were normalized to 1340 γ - tubulin molecules , i . e . the average number of γ - tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements ( Figs 4 and 5 ) . Bars for Sas - 6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50 % of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins . Note the different scale for γ - tubulin on the y - axis .

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Figure 2A) The absolute abundance of centrosomal proteins in whole cell lysates, prepared from asynchronous growing cells, was determined using SRM mass spectrometry combined with stable isotope dilution. Copy numbers per cell were measured in three independent experiments. Because not all peptides could be detected in all experiments, histograms show average values determined in those two measurements in which most peptides could be measured reliably; Results presented are means ± SEM. RPE-1 (non-transformed) cells were compared with various tumor-derived (U2OS, HeLa, KE37) or transformed cells (HEK293T). Note the different scale for γ-tubulin on the y-axis.B) Representative image showing centrosome preparation stained with antibodies to NEDD1 (green) and CP110 (red). Bar: 10 μm.C) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution. Histogram a priori represents relative copy numbers per centrosome (Results presented are means ± SEM.). To predict protein abundance in absolute numbers (average number of copies/centrosome), data were normalized to 1340 γ-tubulin molecules, i.e. the average number of γ-tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements (Figs 4 and 5). Bars for Sas-6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50% of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins. Note the different scale for γ-tubulin on the y-axis.


Figure 3Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method . The iBAQ values were normalized with the trendline as obtained from the SRM measurements ( Fig EV1 , panel E ) and γ - tubulin was normalized to 1340 copies / centrosome . A subset of centrosomal proteins is shown as described in ( Azimzadeh et al , 2012 ) . Black bars represent the centrosome subset and red bars represent the values obtained by SRM . Results presented are means ± SEM .

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Figure 3Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method. The iBAQ values were normalized with the trendline as obtained from the SRM measurements (Fig EV1, panel E) and γ-tubulin was normalized to 1340 copies/centrosome. A subset of centrosomal proteins is shown as described in (Azimzadeh et al, 2012). Black bars represent the centrosome subset and red bars represent the values obtained by SRM. Results presented are means ± SEM.


Figure 4A ) Fluorescence analysis of RPE - 1 cells expressing endogenously tagged γ - tubulin - EGFP . Cells were synchronized in G2 , using RO - 3306 , to allow for a clear distinction of the two centrosomes present at this cell cycle stage . Panels illustrate sum projections of the stacks from an original picture ( left ) , a whole cell mask ( middle ) and a centrosome mask ( right ) ; both masks are based on intensity thresholding . Bar : 10 μm . B ) EGFP - tagged viral like particles ( GFP - VLP2 / 6 ) are used as a reference for quantification of γ - tubulin - EGFP . The histogram shows the distribution of the total fluorescence intensity associated with single GFP - VLP2 / 6 ; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2 / 6 particle ( Charpilienne et al , 2001 ) . The inset shows a sum projection of EGFP - VLPs fluorescence image . Bar : 10 μm . C ) Scatter plots show the numbers of γ - tubulin molecules per centrosome determined by calibrations using either SRM measurements ( n = 46 cells ) or EGFP - VLP2 / 6 fluorescence ( n = 40 cells ) . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm.B) EGFP-tagged viral like particles (GFP-VLP2/6) are used as a reference for quantification of γ-tubulin-EGFP. The histogram shows the distribution of the total fluorescence intensity associated with single GFP-VLP2/6; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2/6 particle (Charpilienne et al, 2001). The inset shows a sum projection of EGFP-VLPs fluorescence image. Bar: 10 μm.C) Scatter plots show the numbers of γ-tubulin molecules per centrosome determined by calibrations using either SRM measurements (n=46 cells) or EGFP-VLP2/6 fluorescence (n=40 cells). The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot.


Figure 5To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing ( endogenous ) γ - tubulin - EGFP ( A ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of γ - tubulin ( A ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : γ - tubulin : 45 ; in late S / G2 phase : γ - tubulin : 45 ; in late G2 phase : γ - tubulin : 48 ; in early mitosis : γ - tubulin : 31 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) . To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing Sas - 6 ( B ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : Sas - 6 : 43 ; in late S / G2 phase : Sas - 6 : 46 ; in late G2 phase : Sas - 6 : 48 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) . To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing STIL ( C ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of STIL ( C ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : STIL : 79 ; in late S / G2 phase : STIL : 68 ; in late G2 phase : STIL : 68 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing (endogenous) γ-tubulin-EGFP (A) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of γ-tubulin (A) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: γ-tubulin: 45; in late S/G2 phase: γ-tubulin: 45; in late G2 phase: γ-tubulin: 48; in early mitosis: γ-tubulin: 31. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing Sas-6 (B) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: Sas-6: 43; in late S/G2 phase: Sas-6: 46; in late G2 phase: Sas-6: 48. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing STIL (C) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of STIL (C) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: STIL: 79; in late S/G2 phase: STIL: 68; in late G2 phase: STIL: 68. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


Figure 1 ( A ) Heatmap of genes in the livers of HFD - feeding induced NAFL mice and WD / CCl4 - treatment induced NASH mice . n = 5 . The color gradient represents log2 ( Fold change ) . ( B ) Relative mRNA levels of KLF10 in the livers from NAFL and NASH mice . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t test ( C ) Protein levels of KLF10 examined by western blots ( left ) and its expression was normalized to the β - actin ( right ) . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t test ( D ) Representative immunohistochemistry of KLF10 protein expression in the liver sections from NAFL and NASH mice . The bar chart showed the fold change of KLF10 positive cells per field ( NASH vs . NAFL ) . n = 5 . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t test ( E ) Correlations between KLF10 and NAS , inflammation score , fibrosis scores , and hepatic triglyceride contents ( TG ) from NAFL mice ( n = 7 ) and NASH mice ( n = 8 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Spearman ' s correlation ( E ( F ) H & E staining , Sirius red staining , and KLF10 immunofluorescence staining of liver sections from NAFL and NASH patients . n = 3 . ( G ) Gene expression analysis in liver tissues from NAFL and NASH patients using GEO datasets ( GSE48452 and GSE61260 ) from NCBI database . In the dataset GSE48452 , n = 17 for NAFL patients and n = 15 for NASH patients . In the dataset GSE61260 , n = 21 for NAFL patients and n = 24 for NASH patients . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t test G ) . ( H ) Spearman correlations between KLF10 expression and NAS in liver tissues from NAFL and NASH patients using GEO datasets ( GSE48452 ) . Left panel , n = 32 ( including 17 NAFL patients and 15 NASH patients ) . Right panel , n = 21 ( including 6 NAFL and 15 NASH patients ) . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Spearman ' s correlation H ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Heatmap of genes in the livers of HFD-feeding induced NAFL mice and WD/CCl4-treatment induced NASH mice. n=5. The color gradient represents log2(Fold change).(B) Relative mRNA levels of KLF10 in the livers from NAFL and NASH mice. n=5. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test(C) Protein levels of KLF10 examined by western blots (left) and its expression was normalized to the β-actin (right). n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test(D) Representative immunohistochemistry of KLF10 protein expression in the liver sections from NAFL and NASH mice. The bar chart showed the fold change of KLF10 positive cells per field (NASH vs. NAFL). n=5. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test(E) Correlations between KLF10 and NAS, inflammation score, fibrosis scores, and hepatic triglyceride contents (TG) from NAFL mice (n=7) and NASH mice (n=8). Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Spearman's correlation (E(F) H&E staining, Sirius red staining, and KLF10 immunofluorescence staining of liver sections from NAFL and NASH patients. n=3.(G) Gene expression analysis in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452 and GSE61260) from NCBI database. In the dataset GSE48452, n=17 for NAFL patients and n=15 for NASH patients. In the dataset GSE61260, n=21 for NAFL patients and n=24 for NASH patients. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test G).(H) Spearman correlations between KLF10 expression and NAS in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452). Left panel, n=32 (including 17 NAFL patients and 15 NASH patients). Right panel, n=21 (including 6 NAFL and 15 NASH patients). Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Spearman's correlation H).


Figure 28 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( B ) Protein levels of KLF10 in the livers ( left ) and its expression was normalized to the β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( C ) H & E staining of liver sections . 8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( D ) CD68 immunofluorescence staining of liver sections ( left ) and its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( E ) Body weight ( BW ) , liver / body weight ratio , serum AST and ALT levels , and hepatic TG contents of two groups of mice . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( F ) Sirius red staining of liver sections ( left ) and its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( G ) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( H ) Protein levels of TNF - α in the livers ( left ) and its expression was normalized to the β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( J ) Protein levels of KLF10 in the livers ( left ) and its expression was normalized to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( K ) H & E staining of liver sections . 8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( L ) CD68 immunofluorescence staining of liver sections ( left ) and its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( M ) Body weight ( BW ) , liver / body weight ratio , serum AST and ALT levels , and hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( N ) Sirius red staining of liver sections ( left ) and its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( O ) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test8 - week - old male C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , and then kept on HFD - feeding for 12 weeks . n = 5 per group . ( P ) Protein levels of TNF - α in the livers ( left ) and its expression was normalized to the β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 28-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Protein levels of KLF10 in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) H&E staining of liver sections.8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (D) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents of two groups of mice. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (F) Sirius red staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (G) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (H) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (J) Protein levels of KLF10 in the livers (left) and its expression was normalized to β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (K) H&E staining of liver sections.8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (L) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (M) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (N) Sirius red staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (O) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (P) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


Figure 3 ( A ) Heatmap of genes in mouse primary hepatocytes transfected with lentivirus vectors expressing KLF10 ( KLF10OE ) or empty vector ( Ctrl ) , P ＜ 0 . 001 . n = 5 per group . The color gradient represents log2 ( Fold change ) . ( B ) Relative mRNA ( n = 5 ) and protein ( n = 3 ) levels of zDHHC7 in MPHs expressing KLF10 or empty vector ( Ctrl ) . The protein level of KLF10 or zDHHC7 was normalized to β - actin . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test ( B ( C - D ) Spearman correlations between KLF10 and zDHHC7 in liver tissues from NASH and NAFL patients using GEO datasets ( GSE48452 and GSE61260 ) . In the dataset GSE61260 ( C ) , n = 83 ( including 38 healthy controls , 21 NAFL patients and 24 NASH patients ) . In the dataset GSE48452 ( D ) , n = 73 ( including 41 healthy controls , 17 NAFL patients and 15 NASH patients ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Spearman ' s correlation ( C , D ) . ( E ) Relative mRNA levels of DHHCs family in HepG2 cells overexpressing KLF10 or empty vector ( ctrl ) . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test ( F ) Protein levels of zDHHC7 in the livers of HFD mice or WD / CCl4 mice The protein level of zDHHC7 was normalized to the β - actin . n = 5 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test ( H ) The transcriptional activity of wild - type or mutant zDHHC7 promoter in HepG2 cells . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test ( I ) Chromatin immunoprecipitation analysis of KLF10 binding activity at the zDHHC7 promoter . Soluble chromatin was prepared from the livers of WD / CCl4 treatment - induced NASH mice and immunoprecipitated with antibodies against KLF10 or against IgG . The final DNA extractions were amplified using pairs of primers that cover the regions of zDHHC7 and GAPDH gene promoters . ( J ) Quantitative real - time RCR results of ChIP assays . IgG , immunoglobulin G . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test J ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Heatmap of genes in mouse primary hepatocytes transfected with lentivirus vectors expressing KLF10 (KLF10OE) or empty vector (Ctrl), P＜0.001. n=5 per group. The color gradient represents log2(Fold change).(B) Relative mRNA (n=5) and protein (n=3) levels of zDHHC7 in MPHs expressing KLF10 or empty vector (Ctrl). The protein level of KLF10 or zDHHC7 was normalized to β-actin. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test (B(C-D) Spearman correlations between KLF10 and zDHHC7 in liver tissues from NASH and NAFL patients using GEO datasets (GSE48452 and GSE61260). In the dataset GSE61260 (C), n=83 (including 38 healthy controls, 21 NAFL patients and 24 NASH patients). In the dataset GSE48452 (D), n=73 (including 41 healthy controls, 17 NAFL patients and 15 NASH patients). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Spearman's correlation (C, D).(E) Relative mRNA levels of DHHCs family in HepG2 cells overexpressing KLF10 or empty vector (ctrl). n=5. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test(F) Protein levels of zDHHC7 in the livers of HFD mice or WD/CCl4 mice The protein level of zDHHC7 was normalized to the β-actin. n=5. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test(H) The transcriptional activity of wild-type or mutant zDHHC7 promoter in HepG2 cells. n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test(I) Chromatin immunoprecipitation analysis of KLF10 binding activity at the zDHHC7 promoter. Soluble chromatin was prepared from the livers of WD/CCl4 treatment-induced NASH mice and immunoprecipitated with antibodies against KLF10 or against IgG. The final DNA extractions were amplified using pairs of primers that cover the regions of zDHHC7 and GAPDH gene promoters. (J) Quantitative real-time RCR results of ChIP assays. IgG, immunoglobulin G. n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test J).


Figure 4The liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( A ) Representative immunofluorescence staining of CD36 and β - catenin . The arrows represent the colocalization of CD36 and β - catenin . The liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( B ) Quantitative results of CD36 colocalization rate . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test ( BThe liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( C ) Expression of CD36 in whole livers and plasma membrane fractions . Protein levels were normalized to the β - actin or ATP1a1 , respectively . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testThe liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( D ) Protein levels of palmitoylated CD36 in mouse livers . ( E ) Quantitative results of CD36 colocalization rate . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test E , The liver tissues were collected from HFD - fed mice expressing KLF10 or control ( F ) Representative immunofluorescence staining of CD36 and β - catenin . The arrows represent the colocalization of CD36 and β - catenin . The liver tissues were collected from HFD - fed mice expressing KLF10 or control ( G ) Quantitative results of CD36 colocalization rate . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testThe liver tissues were collected from HFD - fed mice expressing KLF10 or control ( H ) Expression of CD36 in whole livers and plasma membrane fractions . Protein levels were normalized to the β - actin or ATP1a1 , respectively . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testThe liver tissues were collected from HFD - fed mice expressing KLF10 or control ( I ) Protein levels of palmitoylated CD36 in mouse livers . ( J ) Quantitative results of CD36 colocalization rate . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t test J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (A) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin.The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (B) Quantitative results of CD36 colocalization rate. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test (BThe liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (C) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t testThe liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (D) Protein levels of palmitoylated CD36 in mouse livers. (E) Quantitative results of CD36 colocalization rate. n=3. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test E,The liver tissues were collected from HFD-fed mice expressing KLF10 or control (F) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin.The liver tissues were collected from HFD-fed mice expressing KLF10 or control (G) Quantitative results of CD36 colocalization rate. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t testThe liver tissues were collected from HFD-fed mice expressing KLF10 or control (H) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t testThe liver tissues were collected from HFD-fed mice expressing KLF10 or control (I) Protein levels of palmitoylated CD36 in mouse livers. (J) Quantitative results of CD36 colocalization rate. n=3. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test J).