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 ± from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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

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 ± from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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

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).


cholesterol content of ECs freshly isolated from Ldlr + / + Ldlr - / - mice stimulated for 15 min with VEGF - B186 . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Tukey ´ s multiple comparisons , p - value : * * * < 0 . 001 ( compared to Ldlr + / + control ) .

B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

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).


cholesterol content ( left panel ) glucose uptake ( right panel ) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Tukey ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . p - value : # < 0 . 05 , # # # < 0 . 001 ( compared to simvastatin control ) .

B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

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).


surface biotinylated LDLR in HUVECs after 20 min of VEGF - B167 or VEGF - B186 stimulation . Data presented as mean ± from three independent experiments . Western blot shown below . Statistical evaluation using t - , p - value : * < 0 . 05 ( compared to control ) .

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

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 of surface distribution of LDLR by immunofluorescence of non - permeabilized ( , left panels ) , Dil - LDL surface binding ( evaluated at 4 ° C , middle panels ) 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 O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

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.


of non - esterified cholesterol content by means of filipin in blood vessel compartment ( podocalyxin + ) in hearts of 15 - week old C57BL / 6 Vegfb + / - ( n = 5 ) Vegfb - / - mice ( n = 7 ) , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 ( compared to Vegfb + / + mice ) .

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

Quantification 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).


of non - esterified cholesterol content by means of filipin in hearts of 15 - week old C57BL / 6 Vegfb + / - ( n = 5 ) Vegfb - / - ( n = 7 ) mice , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * * < 0 . 01 ( compared to Vegfb + / + mice ) .

O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O

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).


of non - esterified cholesterol content by means of filipin in hearts of 25 - week old CD1 Flt1 + / lacz ( n = 20 ) mice , normalized to wildtype CD1 ( Flt1 + / + ) mice ( n = 16 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using t - , p - value : * * < 0 . 01 .

O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


of non - esterified cholesterol content by means of filipin in hearts of 12 to 16 - week old C57BL / 6 wildtype ( Vegfb + / + ) ( n = 4 ) , BKS . C57BL / 6 db / + Vegfb + / + ( n = 8 ) , db / db Vegfb + / - ( n = 10 ) db / db Vegfb - / - ( n = 4 ) mice , normalized to db / db Vegfb + / + ( n = 6 ) mice . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to db / db Vegfb + / + mice ) .

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

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 : of non - esterified cholesterol content by means of filipin in hearts of C57BL / 6 mice after 25 on HFD , long - term treated with isotype control ( n = 17 ) or VEGF - B blocking antibody ( 2H10 , n = 17 ) initiated after 5 on HFD , normalized to - matched mice on chow diet ( n = 10 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Right panel : weights ( mean ± ) of HFD ( n = 17 + 17 ) chow fed littermate mice ( n = 10 ) . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to HFD control mice ) .

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

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).


of non - esterified cholesterol content by means of filipin in hearts of mice on HFD for 17 followed by treatment for 1 week with 2H10 antibody ( n = 5 ) compared to control antibody treated mice ( n = 5 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using t - , p - value : * * < 0 . 01 .

O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-TISSUE O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( A ) hair coats of Sirt7 − / − Sirt7 + / + mice were clipped on P60 in mid second telogen phase . were captured at P60 , P120 P150 .

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

(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 ) postnatal day when hair coat recovered by 25 % , 50 % , 75 % 100 % in shaved Sirt7 − / − Sirt7 + / + mice . n = 6 mice / genotype . Box - - whisker plots : mid - , median ; box , 25th 75th percentiles ; whiskers , minimum maximum .

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

(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 of skin in unshaved Sirt7 − / − Sirt7 + / + mice on P90 P130 .

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

(C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130.


( D ) Representative immunofluorescence showing Ki67 p - cadherin ( Pcad , labeling HG ) expression in Sirt7 − / − Sirt7 + / + HFs on P74 P90 . HF boundary dermal papilla ( DP ) is denoted by dotted dashed lines , respectively .

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

(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 ) Ki67 positive in each hair germ in D ; n = 3 mice 10 HGs were counted in each mouse .

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

(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 − / − Sirt7 + / + mice . n = 6 mice per genotype . Box - - whisker plots : mid - , median ; box , 25th 75th percentiles ; whiskers , minimum maximum .

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

(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 ) hair coats of Sirt7 - TG WT mice were clipped on P45 were captured on P56 P90 .

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

(G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90.


( H ) postnatal day when hair coat recovered by 25 % , 50 % , 75 % 100 % in shaved Sirt7 - TG WT mice . n = 6 mice per genotype . Box - - whisker plots : mid - , median ; box , 25th 75th percentiles ; whiskers , minimum maximum .

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

(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 showing Ki67 Pcad expression in Sirt7 - TG WT HFs at P60 P65 . HF boundary DP is noted by dotted dashed lines , respectively .

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

(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 ) of Ki67 positive in each HG ; n = 3 mice 10 HGs were counted in each mouse .

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

(J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse.


( K ) H & E of skin tissues in unshaved Sirt7 - TG WT mice on P65 .

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

(K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65.


( L ) Telogen duration in Sirt7 - TG WT mice . n = 6 mice per genotype . Box - - whisker plots : mid - , median ; box , 25th 75th percentiles ; whiskers , minimum maximum .

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

(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.


( A - B ) labeling of Sirt7 Ki67 expression in skin at different HF phases in second hair cycle in depilation - induced hair cycle in WT mice . P56 , P69 , P74 P84 indicate postnatal day in second hair cycle . Pd , post depilation . Dashed indicates bulge ( Bu ) of a hair follicle solid indicates Ki - 67 positive region .

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

(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 ) hair coats of Sirt7f / f Sirt7f / f ; K15 - Cre mice were clipped at P60 were captured on P60 , P125 P150 .

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

(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 ) postnatal day of hair coat recovery by 25 % , 50 % 75 % 100 % in shaved Sirt7f / f Sirt7f / f ; K15 - Cre mice . n = 6 mice per genotype . Box - - whisker plots : mid - , median ; box , 25th 75th percentiles ; whiskers , minimum maximum .

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

(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 of skin in Sirt7f / f Sirt7f / f ; K15 - Cre mice at P100 .

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

(E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100.


( F ) Representative immunofluorescence showing Ki67 / Pcad expression ( upper ) Cd34 / Ki67 expression ( lower ) in Sirt7fl / fl Sirt7f / f ; K15 - Cre HFs on P56 , P74 P85 . dashed indicates bulge of hair follicle .

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

(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 ) of Ki67 - positive per HG ; n = 3 mice 10 HGs were counted in each mouse .

(G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse.


( H ) Ki67 - positive per bulge ; n = 3 mice 10 bulges were counted in each mouse .

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

(H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse.


( A ) Representative colonies formed by Sirt7 + / + , Sirt7 - / - Sirt7 - TG bulge SCs that were puriﬁed from telogen - phase HFs allowed to grow for 2 . ( B - C ) Quantiﬁcations of colony number in ( A ) ; n = 3 mice per genotype .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-ORGANISM O O O

(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 of indicated in HFSC colonies from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice ( D )

B-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O

Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)


in HFSC colonies from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice Nfatc1 were quantified using Image J Plus 6 . 0 ( E , n = 3 mice for each group ) .

O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O

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 of NFATc1 expression level in human keratinocyte HaCaT treated with SIRT7 siRNAs .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE B-CELL_LINE O O B-GENEPROD O O

(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.


( G ) Immunoblot of NFATc1 expression in presence of ectopic SIRT7 expression in HaCaT .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE O

(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 after indicated treatments ( n = 3 ) . relative luciferase activity was determined by dual luciferase reporter .

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

(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 ) analyses of Nfatc1 expression in hair follicles of Sirt7f / f , Sirt7f / f ; K15 - Cre Sirt7 - TG mice at indicted time points . black dashed indicates bulge ( Bu ) of hair follicle .

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

(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 of Nfatc1 - targeted genes in HFSCs colonies cultured from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice , n = 3 for each genotypes .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O

(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.


( A - B ) Immunoblot of interactions between ectopic HA - SIRT7 FLAG - NFATc1in HEK293T .

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

(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.


( C ) Co - immunoprecipitation ( CoIP ) between endogenous NFATc1 SIRT7 in HEK293T .

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

(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.


( D ) Immunoblots showing endogenous NFATc1 pull - down by GST - SIRT7 in HEK293T .

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

(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.


( E ) Immunoblot of HA - SIRT7 WT various mutants in anti His - NFATc1 immunoprecipitates . ∆ N indicates N - terminal deletion ; ∆ C indicates C - terminal deletion ; AC indicates N - terminal C - terminal deletion .

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

(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 of HA - SIRT7 in truncated FLAG - NFATc1 immunoprecipitates . N indicates N - terminal domain ; C indicates C - terminal domain .

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

(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 of interactions between endogenous NFATc1 SIRT7 in keratinocyte HaCaT .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-CELL_TYPE B-CELL_LINE O

(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.


( I - J ) Immunoblot of NFATc1 acetylation in response to indicated treatments .

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

(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.


Immunoblot of NFATc1 - WT / K612R acetylation on SIRT7 deacetylation assays in vitro ( K )

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

Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K)


Immunoblot of NFATc1 - WT / K612R acetylation on SIRT7 deacetylation assays in vivo ( L ) .

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

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 ) acetylation under indicated treatments .

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

Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.


( O ) Immunoblot of NFATc1 phosphorylation among K612 mutants , noting that K612R represents deacetylation while K612Q hyperacetylation .

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

(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.


( A ) Representative immunoblots showing NFATc1 degradation in HEK293T stably overexpressing NFATc1 in presence of cycloheximide ( CHX ) , with or without SIRT7 overexpression . ( B ) of NFATc1 in ( A ) from 3 independent experiments .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

(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 in Sirt7 + / + Sirt7 - / - keratinocytes . ( D ) of Nfatc1 in ( C ) from 3 independent experiments .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O

(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 ) related quantitative data ( F , n = 3 ) showing NFATc1 in HEK293T overexpressing wildtype HA - SIRT7 or a SIRT7 enzyme - dead mutant ( H187Y ) .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O

(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 ) quantitative data ( H , n = 3 ) showing NFATc1 in HEK293T transfected with NFATc1 - CA , with or without ectopic SIRT7 , treated with MG132 .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O

(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 ) related quantitative data ( J , n = 3 ) showing NFATc1 - WT NFATc1 - K612R turnover in HEK293T .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O

(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.


Immunoblots showing interaction ( by co - immunoprecipitation ) between ectopic NFATc1 / PA28γ in HEK293T ( K ) ( L ) after manipulating SIRT7 .

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

Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) (L) after manipulating SIRT7 levels.


Immunoblots showing interaction ( by co - immunoprecipitation ) between endogenous NFATc1 / PA28γ in HaCaT ( L ) after manipulating SIRT7 .

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

Immunoblots showing the interaction (by co-immunoprecipitation) between endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.


( M - N ) Immunoblot ( M ) quantitative data ( N , n = 3 ) of NFATc1 upon overexpression of HA - SIRT7 with or without PA28γ oligo siRNAs treatment .

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

(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 PA28γ in HEK293T cotransfected with WT HA - SIRT7 or an enzyme - dead mutant ( H187Y ) .

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

(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).


( A ) of Nfatc1 expression in HFs on P84 in Sirt7 + / + Sirt7 - / - mice with or without Cyclosporin A ( CsA ) treatment . black dashed indicates bulge ( Bu ) of hair follicle . ( B ) Quantitative of Nfatc1 in ( A , n = 10 hair follicles ) using ImageJ plus 6 . 0 .

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

(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 of Ki67 Pcad at P84 in Sirt7 + / + Sirt7 - / - HFs with or without CsA treatment .

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

(C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.


( D ) of Ki67 - positive per HG in ( C , n = 10 hair follicles ) .

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

(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).


( E ) H & E of skin in indicated treatment groups at P100 .

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

(E) H&E staining of the skin in the indicated treatment groups at P100.


( F ) Representative showing haircoat recovery after indicated treatments . dashed circle indicates shaved ; dotted indicates hair growth boundary .

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

(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.


( A ) Immunoblot of Sirt7 expression in young ( 3M ) aged ( 18M ) HFSCs . # 1 , # 2 , # 3 represented mice . Below , of Sirt7 ; n = 3 mice for each group ; Y - axis represents relative band ( measured with Image J ® ) normalized to signal in young mice .

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O

(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 of Nfatc1 Sirt7 in HFs of young ( 3 months ) old ( 18 months ) mice ( B ) , relative of Sirt7 Nfatc1 - positive in bulge ( C - D ) . / black dashed indicates bulge ( Bu ) of hair follicle .

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

(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 ) were captured of hair coats of Sirt7 + / + Sirt7 - / - mice at 15 months . yellow dashed lines indicate hair thinning or hair loss .

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

(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 of Nfatc1 Sirt7 in HFs of 15 - month - old Sirt7 + / + Sirt7 - / - mice . / black dashed indicates bulge ( Bu ) of hair follicle .

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

(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 ) of Nfatc1 - positive per bulge in ( F , n = 10 hair follicles ) .

(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).


( H ) captured of skin in plucked regions ( blue dotted lines ) of WT ( 3 months ) , aged WT ( 20 months ) aged Sirt7 - TG mice ( 20 months ) , 14 days after depilation . Pigmentation of skin indicates hair regrowth . n = 3 mice per genotype .

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

(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 old Sirt7 - TG mice in ( H ) , n = 3 mice for each group .

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

(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 ) hair coat recovery score ( described in methods ) after back hair of old WT old Sirt7 - TG mice was shaved . n = 3 mice per genotype .

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

(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 ) captured of hair coats of WT Sirt7 - TG mice at 28 months - of - . yellow dashed lines indicate hair thinning or hair loss regions .

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

(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.


( A - J ) Confocal of different z sections ( 0 . 2 µm of thickness ) of a neuron transfected with GFP - LC3 ( A - D ) sections are flattened ( E ) . Higher magnification of outlined in are shown in inserts ( A - D ) . Different sections of 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 , process ( K ) higher magnification of zoomed outlined in ( K - I ) . Reconstructed 3D image of same neuron using IMARIS , which evaluates AV localization in reconstructed 3D image ( M - O ) . Enlarged areas of neuron revealed presence of AV in soma neurites ( N - O ) .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

(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).


( B ) Total AV counting results show that a significant drop in 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 pretreatment with rapamycin followed by SIV - infected microglia supernatant ( Sup ) exposure for 24 hr . * * * P < 0 . 001 , * P < 0 . 05 for n = 6 experiments . values are mean ± . AV counting using 3D model reconstruction for neurons exposed to different treatments .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O

(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 of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . five panels display a typical sample image of a neuron under control conditions as well as 3 hr 24 hr exposure to SIV - infected microglia supernatant or pretreated with rapamycin prior exposure to SIV - infected microglia supernatant . right panels show a snap shot of a 3D outline of neuron with AV marked as green spheres . Scale bar , 20 µm .

O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O O O O O O O O O

(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.


( A ) number of AV in neurites decreased significantly in neurons exposed to 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 .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) AV number is unchanged in neuronal soma after exposure to SIV - infected microglia supernatant for 3 or 24 hr .

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

(B) The AV number is unchanged in neuronal soma after exposure to the SIV-infected microglia supernatant for 3 or 24 hr.


( C D ) Both total of neuronal processes ( C ) number of brunch points ( D ) are decreased after exposure to after SIV - infected microglia supernatant treatment . * * * P < 0 . 001 , for n = 6 experiments . values are mean ± .

O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) LC3 - II level is reduced after 3 or 24 hr exposure to SIV - infected microglia supernatant this decrease effect is blocked when is pretreated with rapamycin ( 2 µM ) . level of elongation Atg12 - Atg5 is also reduced , again effect is blocked in presence of rapamycin . level of p62 is increased for similar conditions with SIV - infected microglia supernatant , increase is blocked in presence of rapamycin . GAPDH was used in these experiments as loading control . One representative experiment of n = 4 is shown .

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

(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 D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± ( n = 4 ) . * * * P < 0 . 001 .

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

(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 O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O


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


( 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 . values are mean ± . ( B C ) Distribution of AV number in soma neurites respectively .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O

(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 ) 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 .

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

(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 ) 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 .

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

(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 of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . four panels display a typical sample image of a neuron under 3 hr 24 hr exposure to TNF - α or NMDA at 25 ng / ml 35 µM respectively . Scale bar , 20 µm .

O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(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.


( 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 presence of rapamycin . Rapamycin alone has no effect . MK - 801 ( 100 µM ) also prevented neurotoxicity induced by SIV - infected microglia supernatant . * * * P < 0 . 001 , for n = 6 experiments .

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

(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 in presence or absence of different drugs such as 3 - MA ( 1 mM ) or Baf ( 100 nM ) . There is no significant difference between exposed to SIV - infected microglia supernatant 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 are pretreated with Baf . SIV - infected microglia supernatant from no infected monkey ( no Sup ) doesn ' t affect neuronal survival there is no significant difference with control samples . values are mean ± .

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

(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.


( A ) Relative values ( RV ) of p62 mRNA level in frontal lobe of SIVE versus SIV uninfected ( UN ) monkeys , n = 7 for SIVE , n = 12 for SIV n = 9 for UN , P < 0 . 05 .

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

(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 n = 10 for NNDS , P < 0 . 0001 .

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

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 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 in human NNDS a dot profile structures in SIVE HAD brain tissues . Higher magnification views are shown in green insets . Black arrows indicate p62 - or ubiquitin - immunoreactive structures within neuronal or proximal processes , red arrowheads indicate a dot - like immunoreactivity in neuropil . Scale bar , 20 µm .

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

(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.


IFNγ enhances macrophage host after type I ( RH ) type II ( Pru ) Toxoplasma gondii ( Tg ) infection . Graphs show as measured by LDH release ( left ) viability measured by XTT ( right ) in untreated or IFNγ - primed THP - 1 infected with type I or type II Tg for 24 h .

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

IFNγ 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 left untreated or primed with IFNγ , transfected with siRNA against indicated GBPs or non - targeting control ( CTRL ) infected with indicated strains of Tg for 24 h .

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

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 from monocyte - derived macrophages ( MDM ) left untreated or treated with IFNγ , transfected with siRNA against GBP1 or non - targeting control ( CTRL ) infected with indicated of Tg for 24 hours . Mean ± of n = 4 donors shown .

B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

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 from indicated wildtype , GBP1 knockout ( ∆ GBP1 ) or GBP1 reconstituted ( ∆ GBP1 + Tet - GBP1 ) infected with type I or type II Tg for 24 hours . were untreated or treated with IFNγ or additionally treated with doxycycline ( Dox ) as indicated .

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

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 from indicated THP - 1 infected with type I or type II Tg , untreated or treated with IFNγ / or Dox as labelled . AU , arbitrary units .

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

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 from wildtype THP - 1 or ∆ GBP1 stably reconstituted with Dox - inducible expression plasmids of indicated mutants of GBP1 . were pre - treated with IFNγ Dox infected with either type I or type II Tg for 24 h .

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

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.


Propidium iodide ( PI ) uptake in indicated wildtype THP - 1 or ∆ CASP - 1 , ∆ CASP - 4 , ∆ ASC , or THP - 1 stably expressing GSDMD - targeting miRNA ( GSDMDmiR ) infected with indicated strains of Toxoplasma gondii ( Tg ) for 24 h . were untreated or treated with IFNγ as indicated . Mean under curve ( AUC ) ± from n = 3 experiments are shown . ns , not significant , from two - way ANOVA following adjustment for multiple comparisons .

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

Propidium 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 ( Annexin V ( AnnV ) - Glo luminescence ) from IFNγ - primed human MDMs transfected with non - targeting control ( siCTRL ; left ) or GBP1 siRNA ( siGBP1 ; right ) left uninfected or infected with type I or type II Tg . Depicted is mean ± of n = 4 donors . AU , arbitrary units .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O

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 MDMs infected with indicated Tg strains for 12 h . were untreated or pre - treated with IFNγ before infection . Treatment with TNFα cycloheximide ( CHX ) served as positive control for apoptosis . are representative of n = 4 biologically independent experiments .

B-EXP_ASSAY O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O

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 measure caspase - 3 / 7 activities using a caspase substrate . THP - 1 WT were primed with IFNγ or left untreated 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 ± from n = 3 independent experiments ) . Representative phase - contrast overlaid with signal for caspase - 3 / 7 activity ( green ) are shown in ( E ) from IFNγ - primed THP - 1 infected with type I Tg captured at indicated times post - infection . Scale bar 20 μm .

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

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 from IFNγ - primed THP - 1 transfected with siRNA against indicated human caspases infected with type I or type II Tg . under curve ( AUC ) from a real - time AnnV - Glo luminescence assays similar to those in ( B ) are plotted as mean ± from n = 3 independent experiments .

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

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 from IFNγ - primed human MDMs transfected with siRNA against CASP8 , CASP10 non - targeting control ( CTRL ) infected with type I or type II Tg . under curve ( AUC ) from real - time assays similar to those in ( B ) are plotted from n = 4 independent experiments . Matched shapes color of symbols indicate donors .

B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

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.


AnnV - Glo from IFNγ - primed THP - 1 transfected with siRNA against indicated human NLR / ALR sensors infected with type I or type II Toxoplasma gondii ( Tg ) . under curve ( AUC ) from real - time assays are plotted as mean ± from n = 3 independent experiments .

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

AnnV-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 from IFNγ - primed human MDMs transfected with siRNA against AIM2 or non - targeting control ( CTRL ) infected with type I or type II Tg . under curve ( AUC ) from real - time assays are plotted from n = 4 independent experiments . Matched shapes color of symbols indicate donors .

B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 ( top ) from IFNγ - primed THP - 1 WT infected with type I Tg for 8 hours stained for ASC active caspase - 8 . Blue , Nuclei ; Red , ASC ; Green , active caspase - 8 ( IETD - FITC ) Grey , Tg . Scale bar , 5 μm or 2 μm in magnified . 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 ± from n = 3 independent experiment .

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

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 MDMs infected with Tg strains for indicated times . were untreated or pre - treated with IFNγ before infection . are representative of n = 4 biologically independent experiments .

B-EXP_ASSAY O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

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.


Representative ( A ) ( 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 . 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 of Tg vacuoles targeted by GBP1 from n = 4 experiments .

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

Representative 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.


LDH release from indicated THP - 1 untreated or treated with IFNγ infected with Salmonella Typhimurium ( STm ) WT or a SPI - 1 mutant ΔinvA for 4 h at indicated multiplicities of infection ( MOI ) . Mean ± of n = 3 experiments are shown .

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

LDH 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 from IFNγ - primed 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 . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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 from IFNγ - primed MDMs transfected with indicated siRNA or non - targeting control ( CTRL ) infected with STm - GFP for 4 h . under curve ( AUC ) from real - time plotted as mean ± from n = 4 independent experiments .

B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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 from IFNγ - primed or untreated THP - 1 ΔCASP - 1 transfected with indicated siRNA infected with STm - GFP for 4 h . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments . Schematic on right shows overview of pathway leading to caspase - 1 caspase - 4 activation .

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

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 from indicated THP - 1 treated with IFNγ Dox infected with STm - GFP for 4 h . Shown is mean ± of n = 3 experiments .

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

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 from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or MDMs stained for GBP1 . were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm .

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

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.


( G ) from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or MDMs stained for GBP1 . 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 of STm vacuoles targeted by GBP1 .

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

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.


of GBP1 recruitment to STm - GFP containing vacuoles ( SCV ) in MDMs pre - treated with IFNγ or left untreated ( UT ) . Mean ± 95 % CI of GBP1 measured on indicated number ( n ) of SCVs for each condition from three independent experiments are shown .

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

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 from THP - 1 ∆ GBP1 + Tet - mCH - GBP1 stably expressing YFP - CASP4C258S . were infected with STm for 2 h . Blue , Nuclei & STm ( Hoechst dye ) ; Red , mCH - GBP1 ; Green , YFP - CASP4C258S . Scale bar , 5 μm .

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

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.


of THP - 1 ∆ GBP1 + Tet - mCH - GBP1 stably expressing YFP - CASP4C258S infected with STm for 2 h . Graph shows of STm vacuoles targeted by GBP1 , CASP4 or both from n = 3 experiments . n . d . not detected .

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

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.


measured using Annexin V ( AnnV ) - Glo of THP - 1 ∆ CASP1 transfected with non - targeting control ( CTRL ) or GBP1 siRNA treated as indicated . were primed with IFNγ , transfected with soluble Tg antigen ( STAg ) or both , or left untreated ( UT ) . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

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

Cell 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 from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 transfected with soluble Tg antigen ( STAg ) that was untreated ( UT ) or treated as indicated . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

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

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 transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA then untreated ( - ) or transfected with STAg for 12 h . are representative of n = 2 independent experiments .

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

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 from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA then untreated ( UT ) or transfected with STAg as indicated for 18 h . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 8 activation .

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

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 from MDMs transfected with non - targeting control ( CTRL ) or GBP1 siRNA then left untreated ( UT ) , treated with LPS or transfection reagent ( Lipofectamine ) only or transfected with LPS for 4 h . under curve ( AUC ) from real - time assays plotted as mean ± from n = 4 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 4 activation .

B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

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.


a , Diagram of cycle composed of G1 , S , G2 , M phases ( not to scale ) . Phase durations were quantified by time - lapse microscopy using a PCNA - mCherry reporter to identify four discrete events during lifetime of an were acquired every 10 min .

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

a, 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 , H9 lines . Error bars represent standard deviations .

O O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O

b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.


c , Coefficient of variation ( CV ) of phase durations .

c, Coefficient of variation (CV) of phase durations.


d , of total variation in cycle duration contributed by phases .

d, Percentage of the total variation in cell cycle duration contributed by individual phases.


e , Correlations between cycle phase durations . Sample sizes were adequate to detect correlations n = 125 ( RPE ) , 130 ( U2OS ) , 113 ( H9 ) . R2 , square of Pearson correlation coefficient .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O

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.


b , Shift in phase durations of RPE overexpressing Myc . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : Myc , n =

O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O

b, Shift in phase durations of RPE cells overexpressing Myc. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: Myc, n = 116


c , Pairwise correlation between cycle phase durations of RPE overexpressing Myc . Number of : Myc , n =

O O O O O B-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O

c, Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc. Number of cells: Myc, n = 116


e , Shift in phase durations of RPE treated with 50 ng / mL aphidicolin . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : aphidicolin , n =

O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

e, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115


f , Pairwise correlation between cycle phase durations under aphidicolin treatment . Number of : aphidicolin , n =

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

f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115


h , Shift in phase durations of RPE incubated at 34 ° C . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : 34 ° C , n =

O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

h, Shift in phase durations of RPE cells incubated at 34°C. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: 34°C, n = 122


i , Pairwise correlation between phase durations for incubated at 34 ° C . Number of : 34 ° C , n =

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

i, Pairwise correlation between phase durations for cells incubated at 34°C. Number of cells: 34°C, n = 122


k , Shift in phase durations of RPE treated with NCS . , boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : NCS , n = 119 .

O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.


l , Pairwise correlation between phase durations for treated with NCS . Number of : NCS , n = 119 .

O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O

l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.


g , Shifts in phase durations for RPE treated with 2 μM CVT - 313 . A boxplot representing distributions of durations in untreated is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov . ( n = 117 ) .

O O O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).


h , Pairwise correlation between cycle phase durations upon treatment with CVT - 313 .

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

h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.


j , Shifts in phase durations for RPE overexpressing cyclin D . A boxplot representing distributions of durations in untreated is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov . ( n = 113 ) .

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

j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells).


k , Pairwise correlation between cycle phase durations upon overexpression of cyclin D . P indicates P - value .

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

k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.


b , Shift in phase durations of U2OS treated with NCS . , boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : NCS , n =

O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

b, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114


c , Pairwise correlation between phase durations for U2OS treated with NCS . Number of : NCS , n =

O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O

c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114


e , Shift in phase durations of U2OS treated with 50 ng / mL aphidicolin . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : aphidicolin , n = 153 .

O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

e, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.


f , Pairwise correlation between cycle phase durations under aphidicolin treatment . Number of : aphidicolin , n = 153 .

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

f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.


B . Comparison of ΔPrP expression . Representative immunoblot is shown for detection of ( 3F4 ) MoPrP ΔPrPs in transiently transfected N2a using mAb 3F4 . ΔPrPs are similar in expression level glycosylation appearance , except for Δ159 - 167 which has an extra fragment larger than diglycoform ( lane 7 , arrow ) .

O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159-167 which has an extra fragment larger than the diglycoform (lane 7, arrow).


C . ΔPrPs are not converted into PK - resistant PrP in prion - infected . 22L - ScN2a were transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , lysates digested with PK or not . Immunoblot was done using mAb 3F4 .

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

C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4.


. D . Pilot study for dominant - negative inhibition ( DNI ) of Δ159 Δ159 - 175 . DNI was assessed by co - transfecting 22L - ScN2a with ( 3F4 ) MoPrP Δ159 or Δ159 - 175 , respectively , testing for PK - resistant ( 3F4 ) MoPrP . Left panel shows representative immunoblot ( mAb 3F4 ) right panel statistical of quantified PK - res of a triplicate experiment . Empty pcDNA3 . 1 plasmid was used as control for co - transfection in lane 1 . error bars indicate standard deviation . * , p < 0 . 05 .

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

. D. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159-175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159-175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.