Figure 2Relative mRNA expression of the Slc2a glucose transporter ( GLUT ) family members ( 1 - 13 ) in cardiac ECs derived from Vegfb + / + ( n = 4 ) and Vegfb - / - ( n = 4 ) mice . Data represent normalized PLIER values ( mean ± SEM ) from microarray analysis performed on n = 4 + 4 RNA samples ( one RNA sample / mouse ) . mRNA expression levels of the SLC2A transporter ( GLUT ) family members ( 1 - 14 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . mRNA expression levels of VEGFB , SLC2A1 , NRP1 and FLT1 ( VEGFR1 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . 2 - NBD - glucose uptake in response to 2 h treatment with VEGF - B186 ( B186 ) in HUVECs exhibiting siRNA - mediated knock - down of FLT1 , NRP1 , SCL2A1 , KDR ( VEGFR2 ) or control siRNA . Data presented as mean ± SEM from three individual experiments . Statistical evaluation using one - way ANOVA and Sidak ´ s multiple comparisons test , p - value : * < 0 . 05 ( comparisons made to respective untreated control ) . Representative images of GLUT1 ( left panels ) and CD31 ( right panels ) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts . Scale bar , 100 μm . mRNA expression analysis from enriched cardiac endothelial cells ( open columns , n = 4 mice ) or whole heart ( closed columns , n = 3 mice ) . Data presented as mean ± StDev relative to Rpl19 expression . Enrichment factor is calculated as mean cardiac EC / mean whole heart and presented in the box .

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 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 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 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

Figure 2Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse).mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm.mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.


Figure 3Immunofluorescence labeling of GLUT1 ( white ) , early endosomes ( EEA1 , green ) and nuclei ( blue ) in HUVECs treated with VEGF - B186 for 10 min , 30 min or 2 h . Scale bar , 10 µm . GLUT1 protein expression in HUVECs treated with VEGF - B186 for 15 min or 2 h . Data presented as mean ± StDev of 3 - 4 independent experiments relative to β - Actin expression . Statistical evaluation using t - test revealed no significant changes . Western blots shown in right panel . Sucrose gradient ( 15 - 50 % sucrose ) fractions of HUVEC cellular lysates after 2 h VEGF - B186 stimulation were analyzed with Western blots shown in Appendix Fig S2 . Different cellular sub - compartments were identified by accumulation of specific protein markers : EEA1 for early endosomes , LIMPII for late endosomes / lysosomes , flotillin for plasma membrane / lipid rafts and calnexin for endoplasmic reticulum ( ER ) ( left panels ) . Amount and sub - cellular distribution of VEGFR1 , NRP1 and GLUT1 protein in control or VEGF - B186 stimulated HUVECs shown in panels to the right . Data represent % protein relative to total protein of interest measured from a representative experiment . Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF - B186 stimulation in different sucrose fractions ( upper panel ) . Data from two independent experiments are shown . Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF - B186 stimulation . Changes in GLUT1 content in response to VEGF - B treatment are shown in the table ( bottom panel ) .

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 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 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

Figure 3Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm.GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel.Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).


Figure 4Representative confocal ( grey ) and epifluorescence ( blue ) images of filipin staining in HUVECs in response to 2 h VEGF - B186 stimulation ( left panels ) . Cellular cholesterol content measured by quantification of fluorescent filipin staining . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 . Scale bars , 10 µm . Measurement of cholesterol content ( B ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Measurement of glucose uptake ( C ) and FA uptake ( D ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Colorimetric quantification of non - esterified cholesterol extracted from HUVECs stimulated with VEGF - B186 for 20 min . Data are presented as mean ± SEM of three individual experiments . Statistical evaluation using t - test , p - value : * < 0 . 05 . Uptake of a fluorescent cholesterol tracer ( TopFluor ) in HUVECs after VEGF - B186 stimulation for 20 min or 2 h . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 , * * * < 0 . 001 ( compared to respective untreated control ) . Quantification of the major cholesteryl ester ( CE ) species in HUVECs after VEGF - B186 stimulation for 20 min , analyzed by lipid mass spectrometry . Data presented as mean ± StDev of an experiment performed in triplicates .

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 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 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 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

Figure 4Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.Measurement of cholesterol content (B) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Measurement of glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.


Figure 5Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 1 h or after 15 min treatment with cholesterol - extracting methyl - beta - cyclodextrin ( MbCD ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to untreated control ) . Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 15 min alone or supplemented with soluble methyl - beta - cyclodextrin complexed with cholesterol ( 5 , 10 or 20 μg / ml cholesterol ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to control w / o cholesterol ) .

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 O 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 5Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).


Figure 6mRNA expression analysis of lipoprotein receptors in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . glucose uptake ( C ) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Cellular cholesterol content of ECs freshly isolated from Ldlr + / + and Ldlr - / - mice stimulated for 15 min with VEGF - B186 . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to Ldlr + / + control ) . Cellular cholesterol content ( left panel ) and glucose uptake ( right panel ) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . p - value : # < 0 . 05 , # # # < 0 . 001 ( compared to simvastatin control ) . Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF - B167 or VEGF - B186 stimulation . Data presented as mean ± SEM from three independent experiments . Western blot shown below . Statistical evaluation using t - test , p - value : * < 0 . 05 ( compared to control ) . Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non - permeabilized cells ( white , left panels ) , Dil - LDL cell surface binding ( evaluated at 4 ° C , middle panels ) and Dil - LDL uptake ( evaluated at 37 ° C , right panels ) in HUVECs in response to VEGF - B186 stimulation for 10 min . Scale bar , 10 µm .

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 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 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 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 O O O O

Figure 6mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.


Figure 7Quantification of non - esterified cholesterol content by means of filipin staining in the blood vessel compartment ( podocalyxin + ) in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - mice ( n = 7 ) , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 ( compared to Vegfb + / + mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - ( n = 7 ) mice , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 ( compared to Vegfb + / + mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 25 - week old male CD1 Flt1 + / lacz ( n = 20 ) mice , normalized to wildtype CD1 ( Flt1 + / + ) mice ( n = 16 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 12 to 16 - week old male C57BL / 6 wildtype ( Vegfb + / + ) ( n = 4 ) , BKS . C57BL / 6 db / + Vegfb + / + ( n = 8 ) , db / db Vegfb + / - ( n = 10 ) and db / db Vegfb - / - ( n = 4 ) mice , normalized to db / db Vegfb + / + ( n = 6 ) mice . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to db / db Vegfb + / + mice ) . Left panel : Quantification of non - esterified cholesterol content by means of filipin staining in hearts of C57BL / 6 male mice after 25 weeks on HFD , long - term treated with isotype control ( n = 17 ) or VEGF - B blocking antibody ( 2H10 , n = 17 ) initiated after 5 weeks on HFD , normalized to age - matched mice on chow diet ( n = 10 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Right panel : Body weights ( mean ± SEM ) of HFD ( n = 17 + 17 ) and chow fed littermate mice ( n = 10 ) . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to HFD control mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody ( n = 5 ) compared to control antibody treated mice ( n = 5 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 .

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

Figure 7Quantification of non-esterified cholesterol content by means of filipin staining in the blood vessel compartment (podocalyxin+) in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- mice (n=7), normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01 (compared to Vegfb+/+ mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- (n=7) mice, normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01 (compared to Vegfb+/+ mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 25-week old male CD1 Flt1+/lacz (n=20) mice, normalized to wildtype CD1 (Flt1+/+) mice (n=16). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using t-test, p-value: ** <0.01.Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 12 to 16-week old male C57BL/6 wildtype (Vegfb+/+) (n=4), BKS.C57BL/6 db/+ Vegfb+/+ (n=8), db/db Vegfb+/- (n=10) and db/db Vegfb-/- (n=4) mice, normalized to db/db Vegfb+/+ (n=6) mice. Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to db/db Vegfb+/+ mice).Left panel: Quantification of non-esterified cholesterol content by means of filipin staining in hearts of C57BL/6 male mice after 25 weeks on HFD, long-term treated with isotype control (n=17) or VEGF-B blocking antibody (2H10, n=17) initiated after 5 weeks on HFD, normalized to age-matched mice on chow diet (n=10). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Right panel: Body weights (mean ± SEM) of HFD (n=17+17) and chow fed littermate mice (n=10). Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to HFD control mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody (n=5) compared to control antibody treated mice (n=5). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values.Statistical evaluation using t-test, p-value: ** <0.01.


Figure 1 ( A ) The hair coats of Sirt7 − / − and Sirt7 + / + mice were clipped on P60 in the mid second telogen phase . Images were captured at P60 , P120 and P150 . ( B ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 − / − and Sirt7 + / + mice . n = 6 mice / genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( C ) H & E staining of the skin in unshaved Sirt7 − / − and Sirt7 + / + mice on P90 and P130 . ( D ) Representative immunofluorescence images showing Ki67 and p - cadherin ( Pcad , labeling HG cells ) expression in Sirt7 − / − and Sirt7 + / + HFs on P74 and P90 . The HF boundary and dermal papilla ( DP ) is denoted by the dotted and dashed lines , respectively . ( E ) Percent Ki67 positive cells in each hair germ in D ; n = 3 mice and 10 HGs were counted in each mouse . ( F ) Telogen duration in Sirt7 − / − and Sirt7 + / + mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( G ) The hair coats of Sirt7 - TG and WT mice were clipped on P45 and images were captured on P56 and P90 . ( H ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( I ) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7 - TG and WT HFs at P60 and P65 . The HF boundary and DP is noted by the dotted and dashed lines , respectively . ( J ) The percentage of Ki67 positive cells in each HG ; n = 3 mice and 10 HGs were counted in each mouse . ( K ) H & E staining of skin tissues in unshaved Sirt7 - TG and WT mice on P65 . ( L ) Telogen duration in Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

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 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 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 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 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 O 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 B-PANEL_START 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

Figure 1(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150.(B) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130.(D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively.(E) Percent Ki67 positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse.(F) Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90.(H) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(I) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP is noted by the dotted and dashed lines, respectively.(J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse.(K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65.(L) Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


Figure 2 ( A - B ) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation - induced hair cycle in WT mice . P56 , P69 , P74 and P84 indicate the postnatal day in the second hair cycle . Pd , post depilation . Dashed line indicates bulge ( Bu ) of a hair follicle and solid line indicates the Ki - 67 positive staining region . ( C ) The hair coats of Sirt7f / f and Sirt7f / f ; K15 - Cre mice were clipped at P60 and images were captured on P60 , P125 and P150 . ( D ) The postnatal day of hair coat recovery by 25 % , 50 % 75 % and 100 % in shaved Sirt7f / f and Sirt7f / f ; K15 - Cre mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( E ) H & E staining of the skin in Sirt7f / f and Sirt7f / f ; K15 - Cre mice at P100 . ( F ) Representative immunofluorescence images showing Ki67 / Pcad expression ( upper ) and Cd34 / Ki67 expression ( lower ) in Sirt7fl / fl and Sirt7f / f ; K15 - Cre HFs on P56 , P74 and P85 . The white dashed line indicates bulge of hair follicle . ( G ) The percentage of Ki67 - positive cells per HG ; n = 3 mice and 10 HGs were counted in each mouse . ( H ) Ki67 - positive cells per bulge ; n = 3 mice and 10 bulges were counted in each mouse .

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 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 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 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

Figure 2(A-B) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74 and P84 indicate the postnatal day in the second hair cycle. Pd, post depilation. Dashed line indicates bulge (Bu) of a hair follicle and solid line indicates the Ki-67 positive staining region.(C) The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60 and images were captured on P60, P125 and P150.(D) The postnatal day of hair coat recovery by 25%, 50% 75% and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100.(F) Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74 and P85. The white dashed line indicates bulge of hair follicle.(G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse.(H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse.


Figure 3 ( A ) Representative colonies formed by Sirt7 + / + , Sirt7 - / - and Sirt7 - TG bulge SCs that were puriﬁed from telogen - phase HFs and allowed to grow for 2 weeks . ( B - C ) Quantiﬁcations of colony number and size in ( A ) ; n = 3 mice per genotype . Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice ( D ) proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice Nfatc1 protein levels were quantified using Image J Plus 6 . 0 ( E , n = 3 mice for each group ) . ( F ) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs . ( G ) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells . ( H ) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments ( n = 3 ) . The relative luciferase activity was determined by dual luciferase reporter assay . ( I - J ) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f / f , Sirt7f / f ; K15 - Cre and Sirt7 - TG mice at the indicted time points . The black dashed line indicates the bulge ( Bu ) of hair follicle . ( K - L ) qPCR analysis of Nfatc1 - targeted genes in HFSCs colonies cultured from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice , n = 3 for each genotypes .

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 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 B-PANEL_START 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 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 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) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks. (B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype. Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group).(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.(G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells.(H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay.(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.


Figure 4 ( A - B ) Immunoblot analysis of the interactions between ectopic HA - SIRT7 and FLAG - NFATc1in HEK293T cells . ( C ) Co - immunoprecipitation ( CoIP ) between endogenous NFATc1 and SIRT7 in HEK293T cells . ( D ) Immunoblots showing endogenous NFATc1 pull - down by GST - SIRT7 in HEK293T cells . ( E ) Immunoblot analysis of HA - SIRT7 WT and various mutants in the anti His - NFATc1 immunoprecipitates . ∆ N indicates N - terminal deletion ; ∆ C indicates C - terminal deletion ; AC indicates N - terminal and C - terminal deletion . ( F ) Immunoblot analysis of HA - SIRT7 levels in truncated FLAG - NFATc1 immunoprecipitates . N indicates the N - terminal domain ; C indicates C - terminal domain . ( G - H ) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells . ( I - J ) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments . Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vitro ( K ) Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vivo ( L ) . Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) and acetylation levels under the indicated treatments . Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) and acetylation levels under the indicated treatments . ( O ) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants , noting that K612R represents deacetylation while K612Q hyperacetylation .

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

Figure 4(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.(E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion.(F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain.(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K)Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vivo (L).Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.


Figure 5 ( A ) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide ( CHX ) , with or without SIRT7 overexpression . ( B ) Quantification of NFATc1 levels in ( A ) from 3 independent experiments . ( C ) Immunoblots of Nfatc1 protein levels in Sirt7 + / + and Sirt7 - / - keratinocytes . ( D ) Quantification of Nfatc1 levels in ( C ) from 3 independent experiments . ( E - F ) Immunoblots ( E ) and related quantitative data ( F , n = 3 ) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA - SIRT7 or a SIRT7 enzyme - dead mutant ( H187Y ) . ( G - H ) Immunoblots ( G ) and quantitative data ( H , n = 3 ) showing NFATc1 levels in HEK293T cells transfected with NFATc1 - CA , with or without ectopic SIRT7 , treated with MG132 . ( I - J ) Immunoblots ( I ) and related quantitative data ( J , n = 3 ) showing NFATc1 - WT and NFATc1 - K612R protein turnover in HEK293T cells . Immunoblots showing the interaction ( by co - immunoprecipitation ) between ectopic NFATc1 / PA28γ in HEK293T cells ( K ) ( L ) after manipulating SIRT7 levels . Immunoblots showing the interaction ( by co - immunoprecipitation ) between endogenous NFATc1 / PA28γ in HaCaT cells ( L ) after manipulating SIRT7 levels . ( M - N ) Immunoblot ( M ) and quantitative data ( N , n = 3 ) analysis of NFATc1 levels upon overexpression of HA - SIRT7 with or without PA28γ oligo siRNAs treatment . ( O ) Co - immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA - SIRT7 or an enzyme - dead mutant ( H187Y ) .

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 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 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 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 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 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

Figure 5(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression. (B) Quantification of NFATc1 levels in (A) from 3 independent experiments. (C) Immunoblots of Nfatc1 protein levels in Sirt7+/+ and Sirt7-/- keratinocytes. (D) Quantification of Nfatc1 levels in (C) from 3 independent experiments. (E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) (L) after manipulating SIRT7 levels.Immunoblots showing the interaction (by co-immunoprecipitation) between endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.(M-N) Immunoblot (M) and quantitative data (N, n=3) analysis of NFATc1 levels upon overexpression of HA-SIRT7 with or without PA28γ oligo siRNAs treatment.(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).


Figure 6 ( A ) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7 + / + and Sirt7 - / - mice with or without Cyclosporin A ( CsA ) treatment . The black dashed line indicates bulge ( Bu ) of hair follicle . ( B ) Quantitative analysis of Nfatc1 levels in ( A , n = 10 hair follicles ) using ImageJ plus 6 . 0 . ( C ) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7 + / + and Sirt7 - / - HFs with or without CsA treatment . ( D ) The percentage of Ki67 - positive cells per HG in ( C , n = 10 hair follicles ) . ( E ) H & E staining of the skin in the indicated treatment groups at P100 . ( F ) Representative images showing haircoat recovery after the indicated treatments . The dashed circle indicates the shaved area ; the dotted line indicates the hair growth boundary .

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

Figure 6(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle. (B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0. (C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).(E) H&E staining of the skin in the indicated treatment groups at P100.(F) Representative images showing haircoat recovery after the indicated treatments. The dashed circle indicates the shaved area; the dotted line indicates the hair growth boundary.


Figure 7 ( A ) Immunoblot analysis of Sirt7 expression in young ( 3M ) and aged ( 18M ) HFSCs . # 1 , # 2 , # 3 represented individual mice . Below , quantification of Sirt7 protein levels ; n = 3 mice for each group ; the Y - axis represents the relative band intensity ( measured with Image J ® ) normalized to the signal in young mice . ( B - D ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young ( 3 months ) and old ( 18 months ) mice ( B ) , and the relative levels of Sirt7 and Nfatc1 - positive cells in the bulge ( C - D ) . The white / black dashed line indicates bulge ( Bu ) of hair follicle . ( E ) Images were captured of the hair coats of Sirt7 + / + and Sirt7 - / - mice at 15 months . The yellow dashed lines indicate hair thinning or hair loss . ( F ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15 - month - old Sirt7 + / + and Sirt7 - / - mice . The white / black dashed line indicates bulge ( Bu ) of hair follicle . ( G ) Quantification of Nfatc1 - positive cells per bulge in ( F , n = 10 hair follicles ) . ( H ) Images captured of the skin in the plucked regions ( blue dotted lines ) of WT ( 3 months ) , aged WT ( 20 months ) and aged Sirt7 - TG mice ( 20 months ) , 14 days after depilation . Pigmentation of the skin indicates hair regrowth . n = 3 mice per genotype . ( I ) Hair re - growth scores were analyzed for young WT , old WT and old Sirt7 - TG mice in ( H ) , n = 3 mice for each group . ( J ) The hair coat recovery score ( described in methods ) after the back hair of old WT and old Sirt7 - TG mice was shaved . n = 3 mice per genotype . ( K ) Images captured of the hair coats of WT and Sirt7 - TG mice at 28 months - of - age . The yellow dashed lines indicate the hair thinning or hair loss regions .

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

Figure 7(A) Immunoblot analysis of Sirt7 expression in young (3M) and aged (18M) HFSCs. #1, #2, #3 represented individual mice. Below, quantification of Sirt7 protein levels; n=3 mice for each group; the Y-axis represents the relative band intensity (measured with Image J®) normalized to the signal in young mice.(B-D) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young (3 months) and old (18 months) mice (B), and the relative levels of Sirt7 and Nfatc1-positive cells in the bulge (C-D). The white/black dashed line indicates bulge (Bu) of hair follicle.(E) Images were captured of the hair coats of Sirt7+/+ and Sirt7-/- mice at 15 months. The yellow dashed lines indicate hair thinning or hair loss.(F) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15-month-old Sirt7+/+ and Sirt7-/- mice. The white/black dashed line indicates bulge (Bu) of hair follicle.(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).(H) Images captured of the skin in the plucked regions (blue dotted lines) of WT (3 months), aged WT (20 months) and aged Sirt7-TG mice (20 months), 14 days after depilation. Pigmentation of the skin indicates hair regrowth. n = 3 mice per genotype.(I) Hair re-growth scores were analyzed for young WT, old WT and old Sirt7-TG mice in (H), n=3 mice for each group.(J) The hair coat recovery score (described in methods) after the back hair of old WT and old Sirt7-TG mice was shaved. n = 3 mice per genotype.(K) Images captured of the hair coats of WT and Sirt7-TG mice at 28 months-of-age. The yellow dashed lines indicate the hair thinning or hair loss regions.


Figure 1 ( A - J ) Confocal images of different z sections ( 0 . 2 µm of thickness ) of a neuron transfected with GFP - LC3 ( A - D ) and all sections are flattened ( E ) . Higher magnification images of the area outlined in white are shown in the inserts ( A - D ) . Different sections of images of transfected neuron with GFP - LC3 ( green ) merged with DAPI ( blue ) . Scale bar , 20 µm . ( K - O ) Three - dimensional reconstruction from serial sections using IMARIS software , cell body and process ( K ) and higher magnification of the zoomed area outlined in white ( K - I ) . Reconstructed 3D image of the same neuron using IMARIS , which evaluates AV localization in the reconstructed 3D image ( M - O ) . Enlarged areas of neuron revealed the presence of AV in soma and neurites ( N - O ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).


Figure 2 ( B ) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV - infected microglia supernatant for 3 or 24 hr . Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV - infected microglia supernatant ( Sup ) exposure for 24 hr . * * * P < 0 . 001 , * P < 0 . 05 for n = 6 experiments . All values are mean ± SEM . AV counting using 3D model reconstruction for neurons exposed to different treatments . ( C ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV - infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV - infected microglia supernatant . The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres . Scale bar , 20 µm .

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 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

Figure 2(B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments.(C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.


Figure 3 ( A ) The number of AV in neurites decreased significantly in neurons exposed to the SIV - infected microglia supernatant for 3 or 24 hr , however ; pretreatment with rapamycin blocked this effect . * * * P < 0 . 001 , for n = 6 experiments . Scale bar , 20 µm . ( B ) The AV number is unchanged in neuronal soma after exposure to the SIV - infected microglia supernatant for 3 or 24 hr . ( C and D ) Both the total length of neuronal processes ( C ) and number of brunch points ( D ) are decreased after exposure to after SIV - infected microglia supernatant treatment . * * * P < 0 . 001 , for n = 6 experiments . All values are mean ± SEM .

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 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 3(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.(B) The AV number is unchanged in neuronal soma after exposure to the SIV-infected microglia supernatant for 3 or 24 hr.(C and D) Both the total length of neuronal processes (C) and number of brunch points (D) are decreased after exposure to after SIV-infected microglia supernatant treatment. *** P<0.001, for n = 6 experiments. All values are mean±SEM.


Figure 4 ( A ) The LC3 - II level is reduced after 3 or 24 hr exposure to SIV - infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin ( 2 µM ) . The protein level of elongation complex Atg12 - Atg5 is also reduced , and again the effect is blocked in the presence of rapamycin . The protein level of p62 is increased for similar conditions with SIV - infected microglia supernatant , and the increase is blocked in the presence of rapamycin . GAPDH protein was used in these experiments as the loading control . One representative experiment of n = 4 is shown . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 .

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 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 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

Figure 4(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.


Figure 5 ( A ) Total AV counting results show that a significant drop occurs in neurons exposed to TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , * * P < 0 . 01 for n = 6 experiments . All values are mean ± SEM . ( B and C ) Distribution of AV number in soma and neurites respectively . ( B ) The number of AV in neurites decreased significantly in neurons exposed to both TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , for n = 6 experiments . ( C ) The AV number in soma is increased in neurons after exposure to TNF - α for 3 or 24 hr , however it remained unaffected in neurons after exposure to NMDA . * * P < 0 . 01 for n = 6 experiments . ( D ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF - α or NMDA at 25 ng / ml and 35 µM respectively . Scale bar , 20 µm .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively.(B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments.(C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments.(D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.


Figure 6 ( A ) . Neuronal survival tested by MTT . There are significant decreases of neuronal survival after exposure to SIV - infected microglia supernatant for 3 or 24 hr , which is significantly abrogated in the presence of rapamycin . Rapamycin alone has no effect . MK - 801 ( 100 µM ) also prevented the neurotoxicity induced by SIV - infected microglia supernatant . * * * P < 0 . 001 , for n = 6 experiments . ( B ) Neuronal survival was examined with MTT in cultures exposed to SIV - infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3 - MA ( 1 mM ) or Baf ( 100 nM ) . There is no significant difference between cells exposed to SIV - infected microglia supernatant and a pretreatment with 3 - MA followed by exposure to SIV - infected microglia supernatant . However , there is a significant decrease of neuronal survival exposed to SIV - infected microglia supernatant when cells are pretreated with Baf . The SIV - infected microglia supernatant from no infected monkey ( no Sup ) doesn ' t affect neuronal survival and there is no significant difference with control samples . All values are mean ± SEM .

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 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

Figure 6(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.(B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.


Figure 7 ( A ) Relative values ( RV ) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected ( UN ) monkeys , n = 7 for SIVE , n = 12 for SIV and n = 9 for UN , P < 0 . 05 . B ) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects ( NNDS ) , n = 8 for HAD and n = 10 for NNDS , P < 0 . 0001 . ( C ) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys , as well as those with HAD in brain sections from hippocampus . p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues . Higher magnification views are shown in green insets . Black arrows indicate p62 - or ubiquitin - immunoreactive structures within the neuronal cell body or proximal processes , and red arrowheads indicate a dot - like immunoreactivity in the neuropil . Scale bar , 20 µm .

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Figure 7(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05.B) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001.(C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.


Figure 1IFNγ enhances macrophage host cell death after type I ( RH ) and type II ( Pru ) Toxoplasma gondii ( Tg ) infection . Graphs show cell death as measured by LDH release assay ( left ) and viability measured by XTT assay ( right ) in untreated or IFNγ - primed THP - 1 cells infected with type I or type II Tg for 24 h . LDH release assays from THP - 1 cells left untreated or primed with IFNγ , transfected with siRNA against indicated GBPs or non - targeting control ( CTRL ) and infected with indicated strains of Tg for 24 h . LDH release assay from primary monocyte - derived macrophages ( MDM ) left untreated or treated with IFNγ , transfected with siRNA against GBP1 or non - targeting control ( CTRL ) and infected with indicated strain of Tg for 24 hours . Mean ± SEM of n = 4 donors shown . LDH release assay from indicated wildtype , GBP1 knockout ( ∆ GBP1 ) or GBP1 reconstituted ( ∆ GBP1 + Tet - GBP1 ) cells infected with type I or type II Tg for 24 hours . Cells were untreated or treated with IFNγ or additionally treated with doxycycline ( Dox ) as indicated . Realtime propidium iodide ( PI ) uptake assay from indicated THP - 1 cells infected with type I or type II Tg , and untreated or treated with IFNγ and / or Dox as labelled . AU , arbitrary units . LDH release assay from wildtype THP - 1 or ∆ GBP1 cells stably reconstituted with Dox - inducible expression plasmids of the indicated mutants of GBP1 . Cells were pre - treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h .

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 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 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

Figure 1IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h.LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h.LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown.LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.


Figure 2Propidium iodide ( PI ) uptake in indicated wildtype THP - 1 or ∆ CASP - 1 , ∆ CASP - 4 , ∆ ASC cells , or THP - 1 stably expressing GSDMD - targeting miRNA ( GSDMDmiR ) infected with the indicated strains of Toxoplasma gondii ( Tg ) for 24 h . Cells were untreated or treated with IFNγ as indicated . Mean area under the curve ( AUC ) ± SEM from n = 3 experiments are shown . ns , not significant , from two - way ANOVA following adjustment for multiple comparisons . Real - time apoptosis assay ( Annexin V ( AnnV ) - Glo luminescence ) from IFNγ - primed primary human MDMs transfected with non - targeting control ( siCTRL ; left ) or GBP1 siRNA ( siGBP1 ; right ) and left uninfected or infected with type I or type II Tg . Depicted is the mean ± SEM of n = 4 donors . AU , arbitrary units . Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h . Cells were untreated or pre - treated with IFNγ before infection . Treatment with TNFα and cycloheximide ( CHX ) served as positive control for apoptosis . Images are representative of n = 4 biologically independent experiments . Live time - lapse imaging to visualize apoptotic cell death and measure caspase - 3 / 7 activities using a fluorescent caspase substrate . THP - 1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg . Plot in ( D ) shows cumulative caspase - 3 / 7 activity from an 18 - hour time - course ( mean ± SEM from n = 3 independent experiments ) . Representative phase - contrast images overlaid with fluorescence signal for caspase - 3 / 7 activity ( green ) images are shown in ( E ) from IFNγ - primed THP - 1 cells infected with type I Tg captured at indicated times post - infection . Scale bar 20 μm . AnnV - Glo assay from IFNγ - primed THP - 1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg . Area under the curve ( AUC ) from a real - time AnnV - Glo luminescence assays similar to those in ( B ) are plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from IFNγ - primed primary human MDMs transfected with siRNA against CASP8 , CASP10 and non - targeting control ( CTRL ) and infected with type I or type II Tg . Area under the curve ( AUC ) from real - time assays similar to those in ( B ) are plotted from n = 4 independent experiments . Matched shapes and color of symbols indicate donors .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm.AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.


Figure 3AnnV - Glo assay from IFNγ - primed THP - 1 transfected with siRNA against indicated human NLR / ALR sensors and infected with type I or type II Toxoplasma gondii ( Tg ) . Area under the curve ( AUC ) from real - time assays are plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from IFNγ - primed primary human MDMs transfected with siRNA against AIM2 or non - targeting control ( CTRL ) and infected with type I or type II Tg . Area under the curve ( AUC ) from real - time assays are plotted from n = 4 independent experiments . Matched shapes and color of symbols indicate donors . Representative images ( top ) from IFNγ - primed THP - 1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase - 8 . Blue , Nuclei ; Red , ASC ; Green , active caspase - 8 ( IETD - FITC ) and Grey , Tg . Scale bar , 5 μm or 2 μm in the magnified images . Quantification of ASC specks ( bottom ) in Tg - infected THP - 1 WT or ∆ CASP1 treated with IFNγ or left untreated at 8 hours post infection , plotted as mean ± SEM from n = 3 independent experiment . Immunoblots from primary MDMs infected with Tg strains for indicated times . Cells were untreated or pre - treated with IFNγ before infection . Images are representative of n = 4 biologically independent experiments .

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 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

Figure 3AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.


Figure 4Representative images ( A ) and quantification ( B ) from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or monocyte derived macrophages ( MDM ) stained for GBP1 . Cells were infected with type I or type II Toxoplasma gondii ( Tg ) for 2 h . Blue , Nuclei ; Grey , Tg ; Red , mCH - GBP1 or Magenta , GBP1 . Scale bar , 5 μm . Graph in ( B ) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments .

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Figure 4Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments.


Figure 5LDH release assay from indicated THP - 1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium ( STm ) WT or a SPI - 1 mutant ΔinvA for 4 h at indicated multiplicities of infection ( MOI ) . Mean ± SEM of n = 3 experiments are shown . Propidium iodide ( PI ) uptake assay from IFNγ - primed primary MDMs transfected with non - targeting control siRNA ( CTRL ) or siRNA against GBP1 ( left ) or indicated lines of THP - 1 ( right ) infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . PI uptake assay from IFNγ - primed primary MDMs transfected with indicated siRNA or non - targeting control ( CTRL ) and infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 4 independent experiments . PI uptake assay from IFNγ - primed or untreated THP - 1 ΔCASP - 1 cells transfected with indicated siRNA and infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Schematic on right shows overview of the pathway leading to caspase - 1 and caspase - 4 activation . LDH release assay from indicated THP - 1 cells treated with IFNγ and Dox and infected with STm - GFP for 4 h . Shown is the mean ± SEM of n = 3 experiments . Representative images from IFNγ - primed THP - 1 ∆ GBP1 cells stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or primary MDMs stained for GBP1 . Cells were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm . quantification ( G ) from IFNγ - primed THP - 1 ∆ GBP1 cells stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or primary MDMs stained for GBP1 . Cells were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm . Graph in ( G ) shows percentage of STm vacuoles targeted by GBP1 . Quantification of GBP1 recruitment to STm - GFP containing vacuoles ( SCV ) in MDMs pre - treated with IFNγ or left untreated ( UT ) . Mean ± 95 % CI of GBP1 fluorescence intensity measured on the indicated number ( n ) of SCVs for each condition from three independent experiments are shown . Representative images from THP - 1 ∆ GBP1 + Tet - mCH - GBP1 cells stably expressing YFP - CASP4C258S . Cells were infected with STm for 2 h . Blue , Nuclei & STm ( Hoechst dye ) ; Red , mCH - GBP1 ; Green , YFP - CASP4C258S . Scale bar , 5 μm . Quantification of THP - 1 ∆ GBP1 + Tet - mCH - GBP1 cells stably expressing YFP - CASP4C258S and infected with STm for 2 h . Graph shows percentage of STm vacuoles targeted by GBP1 , CASP4 or both from n = 3 experiments . n . d . not detected .

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 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 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 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 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 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 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

Figure 5LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown.Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm.quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown.Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.


Figure 6Cell death measured using Annexin V ( AnnV ) - Glo assay of THP - 1 ∆ CASP1 cells transfected with non - targeting control ( CTRL ) or GBP1 siRNA and treated as indicated . Cells were primed with IFNγ , transfected with soluble Tg antigen ( STAg ) or both , or left untreated ( UT ) . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 cells transfected with soluble Tg antigen ( STAg ) that was untreated ( UT ) or treated as indicated . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Immunoblots from IFNγ - primed THP - 1 ∆ CASP - 1 or THP - 1 ∆ ASC cells transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA and then untreated ( - ) or transfected with STAg for 12 h . Images are representative of n = 2 independent experiments . AnnV - Glo assay from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 cells transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA and then untreated ( UT ) or transfected with STAg as indicated for 18 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 8 activation . Propidium iodide ( PI ) uptake assay from MDMs transfected with non - targeting control ( CTRL ) or GBP1 siRNA and then left untreated ( UT ) , treated with LPS or transfection reagent ( Lipofectamine ) only or transfected with LPS for 4 h . Area under the curve ( AUC ) from real - time assays plotted as mean ± SEM from n = 4 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 4 activation .

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

Figure 6Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation.Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.


Figure 1a , Diagram of the cell cycle composed of G1 , S , G2 , and M phases ( not to scale ) . Phase durations were quantified by time - lapse fluorescence microscopy using a PCNA - mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min . b , Mean phase durations in RPE , U2OS , and H9 cell lines . Error bars represent standard deviations . c , Coefficient of variation ( CV ) of phase durations . d , Percentage of the total variation in cell cycle duration contributed by individual phases . e , Correlations between individual cell cycle phase durations . Sample sizes were adequate to detect correlations n = 125 ( RPE ) , 130 ( U2OS ) , 113 ( H9 ) . R2 , square of Pearson correlation coefficient .

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

Figure 1a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min.b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.c, Coefficient of variation (CV) of phase durations.d, Percentage of the total variation in cell cycle duration contributed by individual phases.e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.