Figure 4Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( A ) Spironolactone reduces ERBB4 levels . T - 47D cells were stimulated with 10 ng / ml EGFld , 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated . Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284 . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( B ) Quantification of band intensities for phospho - ERBB4 levels ( n = 4 per condition ) shown in ( A ) using ImageJ . Phosphorylation levels are normalized to protein levels of ERBB4 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0356 for p - ERBB4 ( Y1056 ) , and * * , p = 0 . 0079 for p - ERBB4 ( Y1284 ) . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( D ) Spironolactone reduces phospho - Erbb4 levels in Nrg1 - tg mice . Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis . Lysates were probed with indicated antibodies . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( E ) Quantification of band intensities for phospho - Erbb4 and phospho - Limk1 levels shown in ( D ) using ImageJ . Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0330 for p - Erbb4 , and p = 0 . 0201 for p - Limk1 . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( F ) Canrenone ( applied as 10 µM ) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex ( PFC ) . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of canrenone . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( G ) Spironolactone ( applied as 10 µM ) significantly increases frequencies ( n = 12 ; p = 0 . 0454 ) and amplitudes ( n = 12 , p = 0 . 0478 ) of sIPSCs in pyramidal neurons of PFC . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of spironolactone . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( H ) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0286 ) . Spironolactone ( 5 µM ) was applied in the presence of canrenone ( 10 µM ) . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib . Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( I ) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0124 ) . Lapatinib was applied in 5 µM . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib .

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 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 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 O O O O O O O O O O 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 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 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

Figure 4Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(B) Quantification of band intensities for phospho-ERBB4 levels (n=4 per condition) shown in (A) using ImageJ. Phosphorylation levels are normalized to protein levels of ERBB4. Data shown as mean, error bars represent SD; t-test, with *, p=0.0356 for p-ERBB4 (Y1056), and **, p=0.0079 for p-ERBB4 (Y1284).Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(D) Spironolactone reduces phospho-Erbb4 levels in Nrg1-tg mice. Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis. Lysates were probed with indicated antibodies.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (E) Quantification of band intensities for phospho-Erbb4 and phospho-Limk1 levels shown in (D) using ImageJ. Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1. Data shown as mean, error bars represent SD; t-test, with *, p=0.0330 for p-Erbb4, and p=0.0201 for p-Limk1.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (F) Canrenone (applied as 10 µM) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex (PFC). Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of canrenone.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(G) Spironolactone (applied as 10 µM) significantly increases frequencies (n=12; p=0.0454) and amplitudes (n=12, p=0.0478) of sIPSCs in pyramidal neurons of PFC. Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of spironolactone.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(H) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0286). Spironolactone (5 µM) was applied in the presence of canrenone (10 µM).For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(I) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0124). Lapatinib was applied in 5 µM. For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.


Figure 5Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( B ) Nrg1 - tg mice travelled longer distances in the open field arena ( effect of genotype F ( 1 , 44 ) = 10 . 53 ; p = 0 . 0022 ; 2 - way ANOVA ) . Bonferroni post - hoc analysis revealed a significant genotype - dependent difference between vehicle - treated groups ( p = 0 . 0044 ) but not in spironolactone - treated groups ( p = 0 . 3783 ) . Genotype differences were abolished upon spironolactone treatment . Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( C ) When vehicle treated animal were analyzed in 1 min intervals , transgenic mice showed an increased activity throughout the entire test ( effect of genotype F ( 1 , 22 ) = 9 . 27 ; p = 0 . 0060 ; 2 - way ANOVA ) , most prominent in intervals 2 , 7 , and 8 ( p = 0 . 0427 , p = 0 . 0385 and p = 0 . 000036 , respectively , Bonferroni post - hoc test ) . Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( D ) There was no significant difference between the genotypes when treated with spironolactone ( effect of genotype F ( 1 , 22 ) = 2 . 19 ; p = 0 . 1535 ; 2 - way ANOVA ) . However , the interaction of genotype and treatment was significant ( F ( 9 , 198 ) = 1 . 94 ; p = 0 . 0481 ; 2 - way ANOVA ) . Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( E ) Nrg1 - tg mice treated with spironolactone spent more time in the light compartment during the light - dark test ( interaction gene × treatment F ( 1 , 41 ) = 4 . 90 ; p = 0 . 0324 ; 2 - way ANOVA , and p = 0 . 0219 , Bonferroni post - test ) . Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( F ) In the Y - maze test , transgenic mice performed less alterations ( effect of genotype F ( 1 , 44 ) = 11 . 50 ; p = 0 . 0015 ; 2 - way ANOVA ) . The Bonferroni test confirmed this phenotype in vehicle - treated groups ( p = 0 . 0011 ) , but not in spironolactone - treated animals ( p = 0 . 5950 ) . Spironolactone treatment had a significant effect on the number of alterations ( F ( 1 , 44 ) = 4 . 12 ; p = 0 . 0484 ; 2 - way ANOVA ) . Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( G ) Spironolactone treatment significantly enhanced PPI in Nrg1 - tg mice ( effect of treatment F ( 1 , 21 ) = 5 . 07 ; p = 0 . 0325 ; 2 - way repeated measures ANOVA ) . Data are shown as mean , error bars represent SEM . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 , Bonferroni test following 2 - way ANOVA ; Spiro , spironolactone ; Veh , vehicle . n = 12 per genotype and treatment with an exception of ( E ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 10 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) and ( G ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 12 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) .

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 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 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 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 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

Figure 5Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(B) Nrg1-tg mice travelled longer distances in the open field arena (effect of genotype F (1,44) =10.53; p=0.0022; 2-way ANOVA). Bonferroni post-hoc analysis revealed a significant genotype-dependent difference between vehicle-treated groups (p=0.0044) but not in spironolactone-treated groups (p=0.3783). Genotype differences were abolished upon spironolactone treatment.Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(C) When vehicle treated animal were analyzed in 1 min intervals, transgenic mice showed an increased activity throughout the entire test (effect of genotype F (1,22) =9.27; p=0.0060; 2-way ANOVA), most prominent in intervals 2, 7, and 8 (p=0.0427, p=0.0385 and p=0.000036, respectively, Bonferroni post-hoc test).Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(D) There was no significant difference between the genotypes when treated with spironolactone (effect of genotype F (1,22) =2.19; p=0.1535; 2-way ANOVA). However, the interaction of genotype and treatment was significant (F (9,198) =1.94; p=0.0481; 2-way ANOVA).Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test).Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice. (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA).Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).


Figure 1B . Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs . C . qPCR validations of neutrophil genes . n = 7 DFUs and n = 3 Skin D3 wounds . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) . Data presented as mean ± SD .

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Figure 1B. Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs.C. qPCR validations of neutrophil genes. n=7 DFUs and n=3 Skin D3 wounds. **P < 0.01 (two-tailed unpaired Student's t-test). Data presented as mean ± SD.


Figure 2A . Representative images of human neutrophils treated with the FOXM1 inhibitor , FDI - 6 . Vehicle ( DMSO ) served as a control . Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red . Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . Data presented as mean ± SD . ( Scale bar : 100µm ) . B . Quantification of ROS levels in human neutrophils treated with FDI - 6 or in combination with N - acetylcysteine ( NAC ) . Phorbol 12 - myristate 13 - acetate ( PMA ) treatment served as a positive control . Data presented as mean ± SD . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . C . Quantification of NET formation in human neutrophils treated with FDI - 6 or in combination with NAC . PMA treatment served as a positive control . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . Data presented as mean ± SD . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) .

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 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

Figure 2A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).


Figure 3A . Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response .

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Figure 3A. Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response.


Figure 4A . Representative images of wounded skin after topical treatment with either vehicle ( IgG isotype control ) or ⍺ - TREM1 activator at 0 , 2 , 4 , 6 , and 8 days after wounding . Percent of wound area at each time following vehicle or ⍺ - TREM1 activator treatment relative to the original wound area . Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺ - TREM1 treated wounds were performed with Fiji software . Data presented as mean ± SD . * * P < 0 . 01 , and * * * * P < 0 . 0001 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . B . Representative pictures of vehicle ( IgG isotype control ) and ⍺ - TREM1 treated diabetic wounds at day 4 show basal keratin marker K5 , and neutrophil marker Ly6G , FOXM1 and citH3 . Treatment of wounds with ⍺ - TREM1 resulted in increased FOXM1 + and decreased citH3 + neutrophils compared to vehicle treated diabetic wounds . ( Scale bar : 50µm ) . Quantification of mean fluorescence intensity was performed with Fiji software . n = 7 diabetic vehicle wounds and 6 diabetic ⍺ - TREM1 wounds . Data presented as mean ± SD . * P < 0 . 05 , * P < 0 . 05 , and * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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 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

Figure 4A. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1 treated diabetic wounds at day 4 show basal keratin marker K5, and neutrophil marker Ly6G, FOXM1 and citH3. Treatment of wounds with ⍺-TREM1 resulted in increased FOXM1+ and decreased citH3+ neutrophils compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic vehicle wounds and 6 diabetic ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).


Figure 5A . qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs ( n = 5 healing and n = 6 nonhealing ) . Data presented as mean ± SD . * P < 0 . 05 . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) . B . Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing ( n = 4 ) and nonhealing ( n = 4 ) is shown in the graph . Data presented as mean ± SEM . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) . C . Representative images of cit - H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs , which was confirmed by corresponding quantification from healing ( n = 3 ) and nonhealing ( n = 3 ) , shown in the graph . Data presented as mean ± SD . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) .

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 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

Figure 5A. qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs (n = 5 healing and n= 6 nonhealing). Data presented as mean ± SD. *P < 0.05. **P < 0.01 (two-tailed unpaired Student's t-test).B. Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing (n=4) and nonhealing (n=4) is shown in the graph. Data presented as mean ± SEM. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).C. Representative images of cit-H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs, which was confirmed by corresponding quantification from healing (n=3) and nonhealing (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).


Figure 1 ( G ) GTT or ITT in 17 - week - old wild - type or JFKTG mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) . ( H ) H & E and Oil Red O staining of liver sections from wild - type or JFKTG mice fed on ND or HFD . Scale bar , 100 μm .

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Figure 1(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.


Figure 2 ( D ) The body weight of male wild - type or JfkKO mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) . ( G ) H & E and Oil Red O staining of liver sections from wild - type or JfkKO mice fed on HFD . Scale bar , 100 μm . ( H ) Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector or JFK via tail vein . Seven days after injection , total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) . H & E and Oil Red O staining of liver sections are shown . Scale bar , 100 μm .

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Figure 2(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.


Figure 3 ( A ) HepG2 cells were stably transfected with vector or FLAG - JFK . Cellular extracts were immunopurified with anti - FLAG immunoafﬁnity resin and eluted with FLAG peptides . The eluates were resolved on SDS - PAGE and silver - stained . The proteins bands were retrieved and analyzed by mass spectrometry . ( B ) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins .

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Figure 3(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.


Figure 4 ( E ) HepG2 cells were transfected with FLAG - JFK and treated with 50 μg / ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis . Quantitation was done by densitometry and expressed as signals of ING5 / β - actin . ( H ) Bacterially - expressed GST - JFK or JFK mutants was incubated with in vitro transcribed / translated ING5 for in vitro ubiquitination assays .

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Figure 4(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.


Figure 5 ( D ) ChIP - seq track at the representative promoter of target genes . The red rectangles indicate the peak regions / binding sites of ING5 on the target genes promoter .

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Figure 5(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.


Figure 6 ( C ) HepG2 cells were treated with control siRNAs or siRNAs against JFK or / and ING5 , or transfected with vector , JFK , or / and ING5 . Cellular extracts were prepared for western blotting analysis with the indicated antibodies . ( D ) HepG2 cells were transfected with vector or JFK , AMPKα1 , or / and ING5 , or treated with control siRNAs or siRNAs against JFK , AMPKα1 , or / and ING5 , Cellular extracts were prepared for AMPK activity assays by ELISA . Error bars represent mean ± SD for triplicate experiments ( * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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Figure 6(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).


Figure 7 ( D ) The measurement of AMPK activity and the rate of fatty acid β - oxidation of wild - type or JfkKO mice . Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA . Mitochondria were isolated from MEFs or liver tissues , and the rate of fatty acid β - oxidation was quantified by spectrophotometric detection for ferricyanide - trapped reduced products . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) . Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector control , shIng5 , or shAmpkα1 via tail vein . Seven days after injection , cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content ( E ) . Asterisk ( * ) represents significant comparison to wild - type mice , octothorpe ( # ) represents significant comparison to JfkKO mice . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , # p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) .

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Figure 7(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).


Figure 1 ( A ) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n ( number of animals per group ) = 6 . ( B ) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n ( number of animals per group ) = 5 . ( C ) Tumor volume in SCIDmice treated or not with LPS n ( number of animals per group ) = 5 . ( D ) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n ( number of animals per group ) = 6 . ( E ) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n ( number of animals per group ) ≥ 4 . NT , control ; DT , diphtheria toxin treated animals to deplete DCs ; LPS , mice treated with LPS . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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Figure 1(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


Figure 2 ( A ) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel . ( B ) Detection of blood vessels by immunohistochemistry using anti - CD31 antibody ( brown staining ) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in ( A ) . Nuclei are stained with hematoxylin . Two representative sections are shown , magnification 20X . Left panel scale bars , 500m ; right panel scale bars 100 m . Quantification of vessel mean dimensions and area coverage by vessels is also shown . Three sections from three tumors have been analyzed . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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Figure 2(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


Figure 3 ( A , B ) Absolute numbers of IFN - γ + and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 ( 25g / mouse ) to reduce the ingress of lymphocytes in the efferent lymphatics . ( C ) Tumor volume in mice treated or not with LPS and / or FTY720 according to the scheme . ( D ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with FTY720 . n ( number of animals per group ) = 4 . Error bars depict SEM . FTY : FTY720 . Statistical significance was determined with a two - tail t test . NT , untreated .

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Figure 3(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.


Figure 4 ( A ) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti - CD62L antibody to inhibit NK cel ingress in the lymph node from blood . Where indicated mice were challenged with LPS . ( B ) Absolute numbers of IFN - γ + NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti - CD62L treatment . ( C ) ( left panel ) Absolute numbers of endogenous and adoptively transferred ( CFSE + ) NK cells in the draining and contralateral lymph node 5 hours after LPS administration ; ( right panel ) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration . ( D ) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration . Lymph node cells were stained with anti - CD3 , anti - DX5 and anti - IFN - γ antibodies and the analysis performed on gated CD3 - DX5high cells . One representative of 4 independent experiments is shown . ( E ) Tumor volume in mice treated or not with LPS , where indicated mice received anti - CD62L antibody 12h before LPS treatment . ( F ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with the anti - CD62L antibody .

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Figure 4(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.


Figure 5 ( A ) Representative image of an NK cell ( red ) interacting with four DCs ( green ) . ( B , C , D ) Plot of the confinement ratio ( CR , black squares ) , the normalized NK speed Vthr ( ti ) ( red circles ) and the digitalized NK - DC distance , DistD ( ti ) ( green triangles ) as a function of time for the NK cell reported in the image . The B , C and D panels correspond respectively to the interaction with the DC1 , DC2 and DC4 displayed in ( A ) . For reference also the normalized distance Dist ( ti ) is reported in the plots ( blue down triangles ) . The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots . The green up - triangles indicate the parameter TonToff defined as TonToff = - 1 if Dist ( ti ) < d = 25 µm and TonToff = - 1 if Dist ( ti ) d and indicate the putative interactions according to a more simplified algorithm based on the DC - NK cell distance alone .

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Figure 5(A) Representative image of an NK cell (red) interacting with four DCs (green). (B, C, D) Plot of the confinement ratio (CR, black squares), the normalized NK speed Vthr(ti) (red circles) and the digitalized NK-DC distance, DistD(ti) (green triangles) as a function of time for the NK cell reported in the image. The B, C and D panels correspond respectively to the interaction with the DC1, DC2 and DC4 displayed in (A). For reference also the normalized distance Dist(ti) is reported in the plots (blue down triangles). The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots. The green up-triangles indicate the parameter TonToff defined as TonToff = -1 if Dist(ti) < d = 25 µm and TonToff = -1 if Dist(ti) d and indicate the putative interactions according to a more simplified algorithm based on the DC-NK cell distance alone.


Figure 6 ( A ) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope . The sequence of images shows the interaction between an NK cell ( red ) and a DC ( green ) . In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back . In the right lower panel the stable contact DC - NK cell is clearly visible . ( B ) Time duration of the DC - NK contacts in steady state ( - LPS ) and inflammatory ( + LPS ) conditions . Bars indicate the mean of the distribution . The percent of cells showing long interaction times ( ≥ 15 min ) are shown . ( C ) NK cell 3D velocities . 3D velocities of NK cells taking contacts with DCs before ( black ) and 5 h ( green ) after LPS treatment .

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Figure 6(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.


Figure 7 ( A ) Differential interference contrast images ( left panels ) and confocal microscopy analysis of IL - 18 ( right panels ) in a DC / NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells ( NT ) . Representative images from 3 experiments are shown , magnification 63X . Scale bars , 4 . 36 m . ( B ) IFN - γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs ( untreated ) or DCs treated O / N with LPS ( LPS ) . Where indicated IL - 18 was added to the cultures . ( C ) Cell - contact dependent activation of NK cells by DCs depends on IL - 18 . Unstimulated or LPS - activated DCs and NK cells were cocultured in the same wells ( DC + NK + LPS ) or separated by a porous membrane ( ( DC + LPS ) / NK ) . Where indicated IL - 18 was added to the transwells ( ( DCs + LPS ) / ( NK + IL - 18 ) ) . NK cells alone were also cultured in the presence of the three selected cytokines ; IL - 2 and IFN - β in the upper chamber and NK cells and IL - 18 in the lower chamber . IFN - γ in the supernatant was then measured by ELISA after 8 hours of coculture . ( B , C ) . n ( number of independent experiments ) = 3 . Error bars depict SEM .

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Figure 7(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.


Figure 1C Western blot analysis of ACE2 in Caco - 2 cells treated with vitamin ( Vit ) compounds ( VitB1 , 500 µM ; VitB6 , 500 µM ; VitB12 , 50 nM ; VitC , 5 mM ; VitD3 , 25 µM ; VitK1 , 0 . 5 µM ) for 24 hrs . Data are representative of three biological replicates . D Western blot analysis of ACE2 in HEK293T , 2fTGH , Caco - 2 and A549 cells treated with VitC at indicated concentrations for 24 hrs . Data are representative of three biological replicates . G Western blot analysis of ACE2 , IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs . Data are representative of three biological replicates . H Fluorescence microscopy of the SARS - CoV - 2 GFP / ΔN or VSV - GFP viruses in Caco - 2 - N cells pretreated with VitC ( 5 mM and 10 mM ) for 24 hrs , and then infected with SARS - CoV - 2 GFP / ΔN ( MOI = 0 . 1 ) or VSV - GFP ( MOI = 0 . 1 ) viruses for 24 hrs . Data are representative of three biological replicates . Scale bar : 100 µm .

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Figure 1C Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. Data are representative of three biological replicates.D Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.G Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.H Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Data are representative of three biological replicates. Scale bar: 100 µm.


Figure 2A RT - qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC ( 5 mM ) as indicated . Data are shown as mean and s . d . of three biological replicates ( n = 3 ) . N . S , not significant ( two - tailed unpaired Student ' s t - test ) . F IP - IB analysis of ubiquitination types of Myc - ACE2 in HEK293T cells cotransfected with Myc - ACE2 and different types of HA - Ub , and then treated with VitC ( 5 mM ) for 12 hrs . The red dashed contour represents the group of K48 - linked ubiquitination we focused on studying here . Data are representative of three biological replicates . H IP - IB analysis of K48 - Ub of endogenous ACE2 in 2fTGH cells treated with VitC ( 2 . 5 mM and 5 mM ) for 12 hrs . Data are representative of three biological replicates .

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Figure 2A RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant (two-tailed unpaired Student's t-test).F IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.H IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.


Figure 3C IP - IB analysis of the interaction between Flag - USP50 and Myc - ACE2 in HEK293T cells cotransfected with these two constructs . Data are representative of three biological replicates . G Western blot analysis of ACE2 in HEK293T cells transfected with Flag - USP50 and then treated with CHX ( 50 μM ) as indicated . Data are representative of three biological replicates . K Fluorescence microscopy of the VSV virus with a SARS - CoV - 2 - S gene and a GFP gene ( VSV - Spike ) in Usp50 + / + and Usp50 - / - HEK293T cells pretreated with or without VitC ( 5 mM ) for 12 hrs , followed by infection with the VSV - Spike virus ( MOI = 0 . 1 ) for 24 hrs . Scale bar : 100 µm . The average intensity each unit area was shown as mean and s . d . of three - unit areas ( n = 3 ) . N . S , not significant , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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Figure 3C IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. Data are representative of three biological replicates.G Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.K Fluorescence microscopy of the VSV virus with a SARS-CoV-2-S gene and a GFP gene (VSV-Spike) in Usp50+/+ and Usp50-/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with the VSV-Spike virus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. The average intensity each unit area was shown as mean and s.d. of three-unit areas (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).


Figure 4D IP - IB analysis of ubiquitination types of Myc - ACE2 in HEK293T cells cotransfected with Myc - ACE2 , Flag - USP50 and different types of HA - Ub . The red dashed contour represents the group of K48 - linked ubiquitination we focused on studying here . Data are representative of three biological replicates . E IP - IB analysis of K48 - Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl ( - ) or shUSP50 ( # 1 , # 2 ) . Data are representative of three biological replicates .

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Figure 4D IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.E IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). Data are representative of three biological replicates.


Figure 5B IP - IB analysis of Myc - ACE2 K48 - linked ubiquitination in HEK293T cells cotransfected with Myc - ACE2 ( WT or K788R ) and HA - K48 , together with shCtrl or shUSP50 . Data are representative of three biological replicates . C Western blot analysis of Myc - ACE2 in HEK293T cells transfected with Myc - ACE2 ( WT or K788R ) and then treated with CHX ( 50 μM ) as indicated . Data are representative of three biological replicates . D IP - IB analysis of K48 - Ub of Myc - ACE2 in HEK293T cells transfected with Myc - ACE2 ( WT or K788R ) and then treated with VitC ( 5 mM ) for 12 hrs , by a specific anti - K48 - Ub antibody . Data are representative of three biological replicates . G ACE2 - KO Caco - 2 cells transfected with ACE2 ( WT or K788R ) were treated with VitC ( 5 mM ) for 12 hrs , and then were infected with SARS - CoV - 2 GFP / ΔN ( MOI = 0 . 1 ) for 2 hrs . RT - qPCR was used to analyze SARS - CoV - 2 RNA levels . Data are shown as mean and s . d . of three biological replicates ( n = 3 ) . N . S , not significant , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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Figure 5B IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. Data are representative of three biological replicates.C Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.D IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. Data are representative of three biological replicates.G ACE2-KO Caco-2 cells transfected with ACE2 (WT or K788R) were treated with VitC (5 mM) for 12 hrs, and then were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).


Figure 6A IP - IB analysis of the interaction between Flag - USP50 and Myc - ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC ( 2 . 5 mM and 5 mM ) for 12 hrs . Data are representative of three biological replicates . D Flag - USP50 proteins were obtained as ( C ) . Flag - USP50 and r - hACE2 proteins were mixed , together with increasing amounts of VitC or with VitC - Na + ( 20 mM ) . HCl is a pH control ( pH = 4 ) . After 2 hrs incubation , ACE2 proteins were analyzed by immunoblotting by a specific anti - ACE2 antibody . Data are representative of three biological replicates .

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Figure 6A IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.D Flag-USP50 proteins were obtained as (C). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na+ (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three biological replicates.


Figure 7D Western blot analysis of hACE2 levels in lung tissues of hACE2 mice ( n = 6 ) administrated with VitC as ( B ) . Data are representative of three biological replicates . G RT - qPCR analysis of the SARS - CoV - 2 - Spike mRNA levels in lung , kidney , liver and spleen tissues of hACE2 mice treated with VitC and the VSV - Spike viruses as ( F ) . All graphs show the mean ± SEM for five individual mice ( n = 5 ) . * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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Figure 7D Western blot analysis of hACE2 levels in lung tissues of hACE2 mice (n = 6) administrated with VitC as (B). Data are representative of three biological replicates.G RT-qPCR analysis of the SARS-CoV-2-Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and the VSV-Spike viruses as (F). All graphs show the mean ± SEM for five individual mice (n = 5). ***p < 0.001 (two-tailed unpaired Student's t-test).


Figure 1 ( A - C ) qPCR and western blot analysis indicated an upregulation of hepatic ZBTB22 in diabetic db / db mice , obese ob / ob mice and high - fat diet for 12 weeks mice ( n = 6 mice ) . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D ) qPCR and western blot analysis showed an increase of hepatic ZBTB22 in normal C57 mice after overnight fasting , and a recovery of ZBTB22 expression after refeeding ( n = 6 mice ) . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( E ) qPCR and immunofluorescence analysis displayed an enhanced expression of ZBTB22 in MPHs after FSK treatment for 24h and a reduction of ZBTB22 in MPHs after insulin treatment for 15min ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 1(A-C) qPCR and western blot analysis indicated an upregulation of hepatic ZBTB22 in diabetic db/db mice, obese ob/ob mice and high-fat diet for 12 weeks mice (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.(D) qPCR and western blot analysis showed an increase of hepatic ZBTB22 in normal C57 mice after overnight fasting, and a recovery of ZBTB22 expression after refeeding (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.(E) qPCR and immunofluorescence analysis displayed an enhanced expression of ZBTB22 in MPHs after FSK treatment for 24h and a reduction of ZBTB22 in MPHs after insulin treatment for 15min (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.


Figure 2 ( A - B ) qPCR analysis the change of genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in primary hepatocytes after ZBTB22 overexpression ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D - E ) ZBTB22 overexpression increase lipid deposition in primary hepatocytes , as shown by the enhanced lipid TOX probes and TG content ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( F - G ) qPCR analysis the change of genes involved in gluconeogenesis , lipogenesis , and fatty acid oxidation in primary hepatocytes after ZBTB22 knockdown ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( I - J ) ZBTB22 knockdown suppress lipid deposition in primary hepatocytes , as shown by the weakened lipid TOX probes and TG content ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( K ) qPCR analysis of mRNA levels of Pgc1α , PCK1 in primary hepatocytes infected with Ad - shCtrl or Ad - shZBTB22 with or without forskolin treatment ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 2(A-B) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in primary hepatocytes after ZBTB22 overexpression (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D-E) ZBTB22 overexpression increase lipid deposition in primary hepatocytes, as shown by the enhanced lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(F-G) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in primary hepatocytes after ZBTB22 knockdown (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(I-J) ZBTB22 knockdown suppress lipid deposition in primary hepatocytes, as shown by the weakened lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(K) qPCR analysis of mRNA levels of Pgc1α, PCK1 in primary hepatocytes infected with Ad-shCtrl or Ad-shZBTB22 with or without forskolin treatment (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 3ZBTB22 deletion effectively decrease the fat mass of mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D ) ZBTB22 deletion enhanced the expression of genes involved in thermogenesis and fatty acid oxidation in brown adipose tissue from mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( E ) ZBTB22 deletion increased the heat production , CO2 generation and O2 consumption in mice fed on HFD ( n = 3 mice ) .

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Figure 3ZBTB22 deletion effectively decrease the fat mass of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) ZBTB22 deletion enhanced the expression of genes involved in thermogenesis and fatty acid oxidation in brown adipose tissue from mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(E) ZBTB22 deletion increased the heat production, CO2 generation and O2 consumption in mice fed on HFD (n=3 mice).


Figure 4ZBTB22 deletion effectively decrease the fasting blood glucose of mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D ) ZBTB22 deletion significantly changed hepatic expression of genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( E ) ZBTB22 deletion significantly improved the phosphorylation of AKT and GSK3β in the liver of mice fed on HFD . ( I ) Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance in ZBTB22 deficiency mice fed on HFD ( n = 6 mice ) . ( J ) Hepatic ZBTB22 overexpression decreased the insulin - induced phosphorylation of AKT and GSK3β in the liver of ZBTB22 deficiency mice fed on HFD . ( K ) qPCR analysis of hepatic genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in ZBTB22 deficiency mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 4ZBTB22 deletion effectively decrease the fasting blood glucose of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) ZBTB22 deletion significantly changed hepatic expression of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(E) ZBTB22 deletion significantly improved the phosphorylation of AKT and GSK3β in the liver of mice fed on HFD.(I) Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance in ZBTB22 deficiency mice fed on HFD (n=6 mice).(J) Hepatic ZBTB22 overexpression decreased the insulin-induced phosphorylation of AKT and GSK3β in the liver of ZBTB22 deficiency mice fed on HFD.(K) qPCR analysis of hepatic genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in ZBTB22 deficiency mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 5 ( A - B ) Hepatic ZBTB22 silencing decreases the expression of gene involved in gluconeogenesis , lipogenesis , and fatty acid oxidation in db / db mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D ) Hepatic ZBTB22 silencing improved the glucose tolerance , insulin sensitivity and pyruvate tolerance in db / db mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( J - K ) Hepatic ZBTB22 silencing decreases serum inflammatory cytokines levels and hepatic inflammatory genes expression ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 5(A-B) Hepatic ZBTB22 silencing decreases the expression of gene involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) Hepatic ZBTB22 silencing improved the glucose tolerance, insulin sensitivity and pyruvate tolerance in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(J-K) Hepatic ZBTB22 silencing decreases serum inflammatory cytokines levels and hepatic inflammatory genes expression (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 6Hepatic ZBTB22 overexpression increases the expression of genes involved in gluconeogenesis in normal C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( C ) Hepatic ZBTB22 overexpression significantly induced glucose intolerance and insulin resistance in normal C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( D ) Hepatic ZBTB22 overexpression significantly decreased insulin - induced phosphorylation of hepatic AKT and GSK3β in normal C57 mice . Hepatic ZBTB22 overexpression increase serum inflammatory cytokines levels Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . Hepatic ZBTB22 overexpression increase hepatic expression of inflammatory genes ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 6Hepatic ZBTB22 overexpression increases the expression of genes involved in gluconeogenesis in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(C) Hepatic ZBTB22 overexpression significantly induced glucose intolerance and insulin resistance in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(D) Hepatic ZBTB22 overexpression significantly decreased insulin-induced phosphorylation of hepatic AKT and GSK3β in normal C57 mice.Hepatic ZBTB22 overexpression increase serum inflammatory cytokines levels Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.Hepatic ZBTB22 overexpression increase hepatic expression of inflammatory genes (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 7Western Blot analysis showed that ZBTB22 - mediated induced the up - regulated expression of PEPCK1 in Ad - ZBTB22 infected mice primary hepatocytes and livers ( C ) Chip and Chip - qPCR displayed increase occupancy of ZBTB22 on the promoter region of PCK1 ( n = 3 biological replicates ) . PCK1 silencing , as well as the stimulatory effects of ZBTB22 on SREBP1C expression ( J ) ( n = 4 biological replicates ) and cellular glucose output ( K ) ( n = 4 biological replicates ) Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 7Western Blot analysis showed that ZBTB22-mediated induced the up-regulated expression of PEPCK1 in Ad-ZBTB22 infected mice primary hepatocytes and livers (C)Chip and Chip-qPCR displayed increase occupancy of ZBTB22 on the promoter region of PCK1 (n=3 biological replicates).PCK1 silencing , as well as the stimulatory effects of ZBTB22 on SREBP1C expression (J) (n=4 biological replicates) and cellular glucose output (K) (n=4 biological replicates) Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 8Hepatic ZBTB22 overexpression failed to change glucose tolerance , insulin sensitivity in PCK1 - silencing C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . ( G ) Hepatic ZBTB22 overexpression failed to change serum and hepatic TG and TC levels in PCK1 - silencing C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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Figure 8Hepatic ZBTB22 overexpression failed to change glucose tolerance, insulin sensitivity in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.(G) Hepatic ZBTB22 overexpression failed to change serum and hepatic TG and TC levels in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Figure 1B , ITC experiment shows the CBS - pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one - to - one stoichiometry . D , Structure of CBS - pair domain of CNNM2 ( PDB 4IY0 ) with the residues DPFYE indicated . F , ITC data of CBS - pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding . G , Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics . Addition of the wild - type CBS - pair domain but not the D426A mutant inhibits phosphatase activity .

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Figure 1B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry.D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated.F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding.G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.


Figure 2A , Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue , cysteine 101 . Recombinant PRL2 was treated with 3 - O - methylfluorescein phosphate ( OMFP ) and analyzed by SDS - PAGE with the Phos - tag reagent . Phosphocysteine is hydrolyzed by boiling . B , Detection of phosphorylated by electrospray mass spectrometry . OMFP - treatment increased the mass of the intact protein by 79 . 99 amu but had no effect on the C101A mutant . C , 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2 . The broad peak is TCEP in the buffer . D , Cysteine phosphorylated PRL2 does not bind to CNNM3 . Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull - down by the CBS - pair domain of CNNM3 as a GST fusion . Only unphosphorylated PRL2 was observed in the pull - down lanes while phosphorylated PRL2 was in the flow - through lanes . E , ITC measurement of the affinity of PRL2 for the CNNM3 GST - CBS - pair domain . Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200 - fold . F , The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS - pair domain .

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Figure 2A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.


Figure 3A , Mutations in the CBS - pair loop of CNNM4 prevent binding to PRL3 . Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation ( IP ) and immunoblotting ( IB ) with the indicated antibodies . B C , Wild - type CNNM4 - dependent magnesium efflux is inhibited by co - transfection by PRL3 but the efflux by the CNNM4 D485A mutant , which is unable to bind PRL3 , is resistant to inhibition . HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2 + removed from the medium at the indicated time point ( arrowhead ) . The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s . e . m . at 5 min plotted in panel C . P - values were calculated using one way ANOVA followed by Bonferroni ' s multiple comparison test . n . s . , not significant . B C , Wild - type CNNM4 - dependent magnesium efflux is inhibited by co - transfection by PRL3 but the efflux by the CNNM4 D485A mutant , which is unable to bind PRL3 , is resistant to inhibition . HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2 + removed from the medium at the indicated time point ( arrowhead ) . The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s . e . m . at 5 min plotted in panel C . P - values were calculated using one way ANOVA followed by Bonferroni ' s multiple comparison test . n . s . , not significant .

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Figure 3A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies.B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.


Figure 4A , The CBS - pair domain ( yellow ) forms a dimer and binds two molecules of PRL2 via an extended loop . The positions of the PRL2 C - terminal CAAX prenylation motif [ 42 ] and CNNM3 N - terminus suggest that the membrane would sit above the complex . B , Contacts between the CNNM3 CBS2 loop and the PRL2 active site . C , Stereoview of the 2FO - FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 . D , Detail of complex with reduced PRL2 , showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101 . E , Model of phosphocysteine preventing CNNM binding . Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding . Residues are numbered according to the PRL1 structure ( PDB 1XM2 ) .

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Figure 4A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex.B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site.C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101.E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).


Figure 5A , PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine . Phos - tag analysis of cell lysates shows close to 50 % of transiently expressed FLAG - PRL1 , - 2 , and - 3 are phosphorylated . The phosphorylated PRL bands are marked by an asterisk . B , Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine ( C104 ) but not the adjacent cysteine ( C49 ) in the active site . C , Co - immunoprecipitation of Myc - PRL by FLAG - CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4 . D , Endogenous PRLs in HeLa cells are cysteine phosphorylated . E , Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium .

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Figure 5A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk.B, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site.C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4.D, Endogenous PRLs in HeLa cells are cysteine phosphorylated.E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.