Figure 5C , ChIP - qPCR analysis of ARF2 - GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4 - 2 pif5 - 10 backgrounds . In C fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results . D , RT - qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , pif4 - 2 pif5 - 10 , and pARF2 : ARF2 - GFP OEs in the pif4 - 2 pif5 - 10 background before and after 4 d C - deprivation . Fold changes were calculated with respect to the expression level in the WT before C - deprivation . In D data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results . E , ChIP - qPCR analysis of PIF5 - GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2 - 20 backgrounds . In fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results . F , RT - qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background before and after 4 d C - deprivation . Fold changes were calculated as in D . In F , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results . G , Hypocotyl elongation in seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background . Seedlings were grown under continuous light for one week . H , RT - qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G . Fold changes were calculated with respect to the expression level in the WT . Color legends in H are the same as in F . In H , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results . J , ChIP - qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2 - 20 backgrounds . In J , fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - tailed t test , n . s . , not significant , * * * * , p < 0 . 0001 . The experiment has been independently repeated with similar results .

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

Figure 5C, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4-2 pif5-10 backgrounds. In C fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.D, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. In D data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.E, ChIP-qPCR analysis of PIF5-GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2-20 backgrounds. In fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.F, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 4 d C-deprivation. Fold changes were calculated as in D. In F, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.G, Hypocotyl elongation in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background. Seedlings were grown under continuous light for one week.H, RT-qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G. Fold changes were calculated with respect to the expression level in the WT. Color legends in H are the same as in F. In H, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.J, ChIP-qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2-20 backgrounds. In J, fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


Figure 6A , RT - qPCR analysis of ARF2 expression levels in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , mateq , and pARF2 : ARF2 - GFP OEs in the mateq background . Fold changes were calculated with respect to the expression level in the WT . B , RT - qPCR analysis of PIF5 expression levels in 7 - d - old seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , mateq , and pPIF5 : PIF5 - GFP OEs in the mateq background . Fold changes were calculated as in A . In A , B , data are means ± s . d . of 3 biological replicates . C , Seedlings of the same genotypes as in A before and after 6 d and 9 d C - deprivation . D , Seedlings of the same genotypes as in B before and after 6 d and 11 d C - deprivation . E and F , Chlorophyll and total cellular protein content reduction in genotypes shown in A ( E ) after 6 d and 9 d C - deprivation and in genotypes shown in B ( F ) after 6 d and 11 d C - deprivation . Color legends in E are the same as in A . Color legends in F are the same as in B . data are means ± s . d . of 3 biological replicates . G and H , RT - qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A ( G ) and B ( H ) before and after 4 d C - deprivation . Fold changes were calculated with respect to the expression level in the WT before C - deprivation . Color legends in G are the same as in A . Color legends in are the same as in B . data are means ± s . d . of 3 biological replicates . I , RT - qPCR analysis of PIL1 and YUC8 expression levels in 7 - d - old seedlings of the same genotypes as shown in B . Fold changes were calculated as in A . Color legends in I are the same as in B . data are means ± s . d . of 3 biological replicates .

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

Figure 6A, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, mateq, and pARF2:ARF2-GFP OEs in the mateq background. Fold changes were calculated with respect to the expression level in the WT. B, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, mateq, and pPIF5:PIF5-GFP OEs in the mateq background. Fold changes were calculated as in A. In A, B, data are means ± s.d. of 3 biological replicates.C, Seedlings of the same genotypes as in A before and after 6 d and 9 d C-deprivation. D, Seedlings of the same genotypes as in B before and after 6 d and 11 d C-deprivation.E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) after 6 d and 9 d C-deprivation and in genotypes shown in B (F) after 6 d and 11 d C-deprivation. Color legends in E are the same as in A. Color legends in F are the same as in B. data are means ± s.d. of 3 biological replicates.G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. Color legends in G are the same as in A. Color legends in are the same as in B. data are means ± s.d. of 3 biological replicates.I, RT-qPCR analysis of PIL1 and YUC8 expression levels in 7-d-old seedlings of the same genotypes as shown in B. Fold changes were calculated as in A. Color legends in I are the same as in B. data are means ± s.d. of 3 biological replicates.


Figure 7B , ChIP - qPCR analysis of ARF2 - GFP binding to the promoters of ABS3 . Fold enrichments were calculated with respect to the input . PP2A served as a non - binding control . Data are means ± s . d . of 4 technical replicates . The experiment has been independently repeated 3 times with similar results . C , pABS3 : GFP or pABS3 ∆ S2 : GFP was expressed in WT mesophyll protoplasts with or without p35S : ARF2 - HA . p35S : mCherry was co - transfected and served as a transfection control . Protoplasts were examined with fluorescence microscopy . Experiments were independently repeated 3 times with similar results . Bars , 100 μm . D , Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3 : GFP with or without p35S : ARF2 - HA . Data are median with interquartile range , n is indicated in parentheses , two - tailed Mann - Whitney test , n . s . , not significant , * * * * , p < 0 . 0001 . E , RT - qPCR analysis of ABS3 expression levels in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , pif4 - 2 pif5 - 10 , and pARF2 : ARF2 - GFP OEs in the pif4 - 2 pif5 - 10 background . Fold changes were calculated with respect to the expression level in the WT . Data are means ± s . d . of 3 biological replicates .

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

Figure 7B, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ABS3. Fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 4 technical replicates. The experiment has been independently repeated 3 times with similar results.C, pABS3:GFP or pABS3∆S2:GFP was expressed in WT mesophyll protoplasts with or without p35S:ARF2-HA. p35S:mCherry was co-transfected and served as a transfection control. Protoplasts were examined with fluorescence microscopy. Experiments were independently repeated 3 times with similar results. Bars, 100 μm. D, Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3:GFP with or without p35S:ARF2-HA. Data are median with interquartile range, n is indicated in parentheses, two-tailed Mann-Whitney test, n.s., not significant, ****, p<0.0001.E, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background. Fold changes were calculated with respect to the expression level in the WT. Data are means ± s.d. of 3 biological replicates.


Figure 1 ( B ) Time course of SUMO - 1 modification of His - tagged HTTex1p ( 25Q ) - purified proteins ( WT , K6R , K9R , K6 , 9R , K6 , 9 , 15R , and S13 , 16D ) was performed in vitro . SUMOylation was visualized using anti - His antibody . WT HTTex1p is SUMOylated within 16 min , K6R or K9R mutations delay SUMOylation , and combined mutations ( K6 , 9R or K6 , 9 , 15R ) greatly reduce SUMOylation . Mutations that mimic phosphorylation ( S13 , 16D ) alter kinetics of SUMO - 1 modification with SUMO modification observed beginning by 4 min .

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

Figure 1(B) Time course of SUMO-1 modification of His-tagged HTTex1p (25Q)-purified proteins (WT, K6R, K9R, K6,9R, K6,9,15R, and S13,16D) was performed in vitro. SUMOylation was visualized using anti-His antibody. WT HTTex1p is SUMOylated within 16 min, K6R or K9R mutations delay SUMOylation, and combined mutations (K6,9R or K6,9,15R) greatly reduce SUMOylation. Mutations that mimic phosphorylation (S13,16D) alter kinetics of SUMO-1 modification with SUMO modification observed beginning by 4 min.


Figure 2 ( A ) qRT - PCR analysis of SUMO - modifying proteins and enzymes in cortex and striata of WT mice at 12 weeks . Relative expression for all the SUMO enzymes is normalized to mouse β - actin . ( B and C ) qRT - PCR of SUMO mRNAs from 12 - week - old WT and R6 / 2 mouse cortex ( B ) and striatum ( C ) . SUMO enzyme mRNAs are differentially expressed in R6 / 2 versus control with statistically significant increases in SENP1 ( p = 0 . 01 ) , SENP3 ( p = 0 . 02 ) , and SUMO - 1 ( p = 0 . 003 ) in cortex and SENP1 ( p = 0 . 02 ) , SENP6 ( p = 0 . 04 ) , PIAS3 ( p = 0 . 007 ) , SUMO - 1 ( p = 0 . 02 ) , and SUMO - 2 ( p = 0 . 02 ) in striatum . Samples were analyzed in quadruplicate and normalized to mouse β - actin . Data are shown as R6 / 2 expression relative to WT levels set at 1 for each enzyme with ± SD ( n = 4 ) . * p < 0 . 05 . n . s . , not significant . ( D and E ) Western blot analysis of SUMO - 2 in 14 - week R6 / 2 cortex ( D ) and striatum ( E ) versus aged - matched controls . SUMO - 2 is upregulated in R6 / 2 striatum versus control ( p = 0 . 026 ; n = 4 ) . Protein is normalized to α - tubulin and quantitated using ImageJ .

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

Figure 2(A) qRT-PCR analysis of SUMO-modifying proteins and enzymes in cortex and striata of WT mice at 12 weeks. Relative expression for all the SUMO enzymes is normalized to mouse β-actin.(B and C) qRT-PCR of SUMO mRNAs from 12-week-old WT and R6/2 mouse cortex (B) and striatum (C). SUMO enzyme mRNAs are differentially expressed in R6/2 versus control with statistically significant increases in SENP1 (p = 0.01), SENP3 (p = 0.02), and SUMO-1 (p = 0.003) in cortex and SENP1 (p = 0.02), SENP6 (p = 0.04), PIAS3 (p = 0.007), SUMO-1 (p = 0.02), and SUMO-2 (p = 0.02) in striatum. Samples were analyzed in quadruplicate and normalized to mouse β-actin. Data are shown as R6/2 expression relative to WT levels set at 1 for each enzyme with ± SD (n = 4). *p < 0.05. n.s., not significant.(D and E) Western blot analysis of SUMO-2 in 14-week R6/2 cortex (D) and striatum (E) versus aged-matched controls. SUMO-2 is upregulated in R6/2 striatum versus control (p = 0.026; n = 4). Protein is normalized to α-tubulin and quantitated using ImageJ.


Figure 3 ( A ) HeLa cells transfected with His - tagged HTTex1p ( 46Q ) or 46QP - K6 , 9 , 15R ( 3R ) along with GFP - SUMO - 1 or GFP - SUMO - 2 , lysed under denaturing conditions , and nickel purified ( Ni - NTA ) . Unmodified HTT - 46Q is indicated by the arrow and SUMO - modified HTT by the boxed region . The lysine mutant ( 3R ) serves as a negative control . Ni - NTA represents nickel - purified His - tagged HTT , and WC TCA represents 10 % of the whole - cell lysate expression of HTT and myc - actin ( transfection control ) . HTT is modified by SUMO - 1 ( left ) and SUMO - 2 ( right ) . ( B ) SUMO isopeptidases ( SENP1 , SENP2 , SENP3 , SENP5 , and SENP6 ) modulate SUMO - modified HTT when overexpressed together with HTT ( 46QP - H4 or 3R ) and SUMO - 1 ( GFP - SUMO - 1 ) . SENP1 , SENP2 , and SENP6 decrease HTT SUMOylation . Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System ( LI - COR ) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100 . ( C ) Titration of SUMO - 1 . Denaturing nickel purification of HTTex1p ( 46QP - H4 ) following transfection with decreasing amounts of SUMO - 1 reduces the amount of SUMO - modified HTT to undetectable levels . The Ni - NTA blot displayed in the gray scale shows purified HTTex1 and SUMO - modified HTTex1 using HTT antibody . Note that 0 . 5 μg of SUMO - 1 ( ¼ the amount of SUMO - 1 cDNA ) was used for identifying the SUMO - 1 E3 ligase for HTTex1p . Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100 . Note that all experiments were performed in triplicate , and a representative figure is shown .

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

Figure 3(A) HeLa cells transfected with His-tagged HTTex1p (46Q) or 46QP-K6,9,15R (3R) along with GFP-SUMO-1 or GFP-SUMO-2, lysed under denaturing conditions, and nickel purified (Ni-NTA). Unmodified HTT-46Q is indicated by the arrow and SUMO-modified HTT by the boxed region. The lysine mutant (3R) serves as a negative control. Ni-NTA represents nickel-purified His-tagged HTT, and WC TCA represents 10% of the whole-cell lysate expression of HTT and myc-actin (transfection control). HTT is modified by SUMO-1 (left) and SUMO-2 (right).(B) SUMO isopeptidases (SENP1, SENP2, SENP3, SENP5, and SENP6) modulate SUMO-modified HTT when overexpressed together with HTT (46QP-H4 or 3R) and SUMO-1 (GFP-SUMO-1). SENP1, SENP2, and SENP6 decrease HTT SUMOylation. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.(C) Titration of SUMO-1. Denaturing nickel purification of HTTex1p (46QP-H4) following transfection with decreasing amounts of SUMO-1 reduces the amount of SUMO-modified HTT to undetectable levels. The Ni-NTA blot displayed in the gray scale shows purified HTTex1 and SUMO-modified HTTex1 using HTT antibody. Note that 0.5 μg of SUMO-1 (¼ the amount of SUMO-1 cDNA ) was used for identifying the SUMO-1 E3 ligase for HTTex1p. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. Note that all experiments were performed in triplicate, and a representative figure is shown.


Figure 4 ( A ) Under limiting SUMO conditions ( 1 / 4 SUMO - 1 , lanes 3 - 9 ) , PIAS1 increases HTT - SUMO modification above 1 / 4 SUMO alone . Purified HTT ( arrow ) and SUMO - HTT ( boxed region ) were detected using anti - HTT . Graph depicts quantitation of the Ni - NTA western blot using the Odyssey Infrared Imaging Software ( LI - COR ) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100 . ( B ) Western analysis of overexpression of HTTex1 ( 46QP - H4 or 3R ) , SUMO - 2 ( GFP - SUMO - 2 ) , and all the PIAS proteins ( PIAS1 , PIASxα , PIASxβ , PIAS3 , and PIASy ) . Under nonlimiting SUMO - 2 conditions , PIAS1 enhances SUMO modification of HTT . WC TCA shows overall myc - actin ( transfection control ) and HTT levels . Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey ( LICOR ) . ( C ) Left panel is the autoradiography results of a GST pull - down assay showing that radiolabeled human PIAS1 interacts with HTTex1p . Right panel is a phosphorimager analysis of GST pull - downs , performed in triplicate , showing the percentage of 35S - labeled PIAS protein that bound the GST proteins : GST alone , unexpanded HTT with and without the proline - rich region ( 20QP and 20Q ) , expanded HTT with and without the proline - rich region ( 51QP and 51Q ) , and the proline - rich region alone ( Pro ) . Error bars were calculated as an estimate of SE = STDEV ( n ) / SQRT ( n - 1 ) .

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

Figure 4(A) Under limiting SUMO conditions (1/4 SUMO-1, lanes 3-9), PIAS1 increases HTT-SUMO modification above 1/4 SUMO alone. Purified HTT (arrow) and SUMO-HTT (boxed region) were detected using anti-HTT. Graph depicts quantitation of the Ni-NTA western blot using the Odyssey Infrared Imaging Software (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.(B) Western analysis of overexpression of HTTex1 (46QP-H4 or 3R), SUMO-2 (GFP-SUMO-2), and all the PIAS proteins (PIAS1, PIASxα, PIASxβ, PIAS3, and PIASy). Under nonlimiting SUMO-2 conditions, PIAS1 enhances SUMO modification of HTT. WC TCA shows overall myc-actin (transfection control) and HTT levels. Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey (LICOR).(C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1).


Figure 5 ( B ) Longer HTT polypeptides are modified by SUMO - 1 . Western blot overexpressing HTTex1p ( 46QP - H4 or 3R ) , unexpanded HTT - 586 fragment ( 25Q - 586 or 25Q - 3R - 586 aa ) , and expanded HTT - 586 fragment ( 137Q - 586 or 137Q - 3R - 586 aa ) with SUMO - 1 ( GFP - SUMO - 1 ) . Cell lysates were subjected to HTT immunoprecipitation ( IP ) using HTT antibody . Western analysis performed with the Odyssey ( LI - COR ) allows detection of HTT ( data not shown ) and SUMO simultaneously and shows that all forms of HTT are SUMO - 1 modified using the anti - GFP antibody . HTTex1p is covalently SUMO - 1 modified ( lane 3 ) , and the modification disappears when Lys are mutated to Arg ( 3R ) ( lane 4 ) . Both unexpanded ( lane 4 and 5 ) and expanded HTT - 586 fragments ( lane 6 and 7 ) are covalently SUMO - 1 modified in both the presence and absence of the three Lys in the N - terminal region of HTT ( 3R ) . Free SUMO - 1 is indicated with the arrow , and SUMO - modified HTT is indicated by the boxes . Inset on the right , from a replicate experiment , shows comigration of expanded HTT ( anti - HTT ) and SUMO - 1 ( anti - GFP ) displayed in the gray scale and in color when the two antibodies are merged ( HTT in red , SUMO - 1 in green , and yellow when colocalizing ) . ( C ) Bait plasmids ( HTT - 586 - 25Q or HTT - 586 - 73Q aa ) were transformed into the L40ccua MATa yeast strain . Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16 , 888 prey proteins ( with Gal4 activation domain fusions ) using pipetting and spotting robots . Diploid yeasts were spotted onto SDIV ( - Leu - Trp - Ura - His ) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β - galactosidase assays . After 5 - 6 days of incubation at 30ºC , digitized images of the agar plates and nylon membranes were assessed for growth and β - galactosidase activity using the software Visual Grid ( GPC Biotech ) . ( D ) Overexpression of PIAS1 alone , with unexpanded HTT constructs ( 25Q - 586 aa ) plus SUMO - 1 ( GFP - SUMO - 1 ) , shows that PIAS1 binds both full - length HTT and HTT - 586 fragment ( 25Q - 586 aa ) . WT HTT was used in these experiments to preclude confounding aggregation effects . Western analysis detection was performed using Odyssey and is displayed in the gray scale but is shown in color on the merge ( HTT is red , and PIAS1 is green ) . WB , western blot . ( E ) HeLa cells overexpressing either expanded 586 aa - HTT or the phosphomimetic ( S13 , 16D = DD ) with SUMO - 2 plus and minus PIAS1 . HTT was purified by IP using hydrazide beads ( Bioclone ) crosslinked to HTT ( Enzo ) antibody and subjected to western analysis using anti - HTT ( MAB5490 ) . Arrows indicate SUMO - 2 - modified HTT . ( F ) IP of HTT with hydrazide - linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics ( S13 , 16D - 586 aa ) are modified by SUMO - 2 . Arrows indicate SUMO - conjugated HTT . Note that all experiments including the Y2H assay were done in triplicate ; representative experiments are shown . Arrows indicate SUMO - 2 - modified HTT .

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

Figure 5(B) Longer HTT polypeptides are modified by SUMO-1. Western blot overexpressing HTTex1p (46QP-H4 or 3R), unexpanded HTT-586 fragment (25Q-586 or 25Q-3R-586 aa), and expanded HTT-586 fragment (137Q-586 or 137Q-3R-586 aa) with SUMO-1 (GFP-SUMO-1). Cell lysates were subjected to HTT immunoprecipitation (IP) using HTT antibody. Western analysis performed with the Odyssey (LI-COR) allows detection of HTT (data not shown) and SUMO simultaneously and shows that all forms of HTT are SUMO-1 modified using the anti-GFP antibody. HTTex1p is covalently SUMO-1 modified (lane 3), and the modification disappears when Lys are mutated to Arg (3R) (lane 4). Both unexpanded (lane 4 and 5) and expanded HTT-586 fragments (lane 6 and 7) are covalently SUMO-1 modified in both the presence and absence of the three Lys in the N-terminal region of HTT (3R). Free SUMO-1 is indicated with the arrow, and SUMO-modified HTT is indicated by the boxes. Inset on the right, from a replicate experiment, shows comigration of expanded HTT (anti-HTT) and SUMO-1 (anti-GFP) displayed in the gray scale and in color when the two antibodies are merged (HTT in red, SUMO-1 in green, and yellow when colocalizing).(C) Bait plasmids (HTT-586-25Q or HTT-586-73Q aa) were transformed into the L40ccua MATa yeast strain. Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16,888 prey proteins (with Gal4 activation domain fusions) using pipetting and spotting robots. Diploid yeasts were spotted onto SDIV (-Leu-Trp-Ura-His) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β-galactosidase assays. After 5-6 days of incubation at 30ºC, digitized images of the agar plates and nylon membranes were assessed for growth and β-galactosidase activity using the software Visual Grid (GPC Biotech).(D) Overexpression of PIAS1 alone, with unexpanded HTT constructs (25Q-586 aa) plus SUMO-1 (GFP-SUMO-1), shows that PIAS1 binds both full-length HTT and HTT-586 fragment (25Q-586 aa). WT HTT was used in these experiments to preclude confounding aggregation effects. Western analysis detection was performed using Odyssey and is displayed in the gray scale but is shown in color on the merge (HTT is red, and PIAS1 is green). WB, western blot.(E) HeLa cells overexpressing either expanded 586 aa-HTT or the phosphomimetic (S13,16D = DD) with SUMO-2 plus and minus PIAS1. HTT was purified by IP using hydrazide beads (Bioclone) crosslinked to HTT (Enzo) antibody and subjected to western analysis using anti-HTT (MAB5490). Arrows indicate SUMO-2-modified HTT.(F) IP of HTT with hydrazide-linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics (S13, 16D-586 aa) are modified by SUMO-2. Arrows indicate SUMO-conjugated HTT. Note that all experiments including the Y2H assay were done in triplicate; representative experiments are shown. Arrows indicate SUMO-2-modified HTT.


Figure 6 ( A ) Western analysis of whole - cell lysates from HeLa cells transfected with His - SUMO - 1 or SUMO - 2 and / or 97Q - HTT exon 1 and treated with 5 μM MG132 for 18 hr . Lysates were separated using differential centrifugation into a detergent - soluble fraction ( SOLUBLE ) with 1 % Triton X - 100 and a detergent - insoluble fraction ( INSOLUBLE ) with 4 % SDS . Western blot probed with anti - HTT shows full - length endogenous HTT in the SOLUBLE fraction ( upper arrow ) and 97Q - HTTex1 ( lower arrow ) ( left panel ) . In the INSOLUBLE fraction , HTT HMW species are indicated by the bracket and asterisks ( right panel ) . ( B ) MG132 and SUMO - 2 cause mutant HTT to accumulate as HMW species ( bracke and asterisk ) . Western blot showing IP with HTT antibody crosslinked beads from the detergent - insoluble fraction probed with the anti - HTT antibody . ( C ) Mutant HTT ( 97Q - Httex1 ) fibrils are detected with anti - HTT in the insoluble fraction with treatment of MG132 or addition of exogenous SUMO - 2 . ( D ) Western blot with increasing concentrations of SUMO - 2 detected with anti - His antibody ( left panel ) . Middle panel is the same western blot probed with anti - HTT showing soluble forms of HTT . Right panel presents western blot from detergent - insoluble fraction with monomeric HTT ( 97Q ) at 55 kDa , and the asterisk ( * ) indicates the HMW species . Note that all experiments were performed in triplicate ; representative figures are shown .

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

Figure 6(A) Western analysis of whole-cell lysates from HeLa cells transfected with His-SUMO-1 or SUMO-2 and/or 97Q-HTT exon 1 and treated with 5 μM MG132 for 18 hr. Lysates were separated using differential centrifugation into a detergent-soluble fraction (SOLUBLE) with 1% Triton X-100 and a detergent-insoluble fraction (INSOLUBLE) with 4% SDS. Western blot probed with anti-HTT shows full-length endogenous HTT in the SOLUBLE fraction (upper arrow) and 97Q-HTTex1 (lower arrow) (left panel). In the INSOLUBLE fraction, HTT HMW species are indicated by the bracket and asterisks (right panel).(B) MG132 and SUMO-2 cause mutant HTT to accumulate as HMW species (bracke and asterisk). Western blot showing IP with HTT antibody crosslinked beads from the detergent-insoluble fraction probed with the anti-HTT antibody.(C) Mutant HTT (97Q-Httex1) fibrils are detected with anti-HTT in the insoluble fraction with treatment of MG132 or addition of exogenous SUMO-2.(D) Western blot with increasing concentrations of SUMO-2 detected with anti-His antibody (left panel). Middle panel is the same western blot probed with anti-HTT showing soluble forms of HTT. Right panel presents western blot from detergent-insoluble fraction with monomeric HTT (97Q) at 55 kDa, and the asterisk (*) indicates the HMW species. Note that all experiments were performed in triplicate; representative figures are shown.


Figure 7 ( A ) Western blot analysis of HeLa cells over - expressing exogenous PIAS1 in the presence of mutant HTT ( 97Q ) when separated into detergent - soluble and detergent - insoluble fractions . No difference is detected in monomeric HTT ( top panel , Soluble ) , but HMW HTT levels increase with PIAS1 overexpression . Anti - PIAS1 antibody ( Invitrogen ) was used to detect PIAS1 . ( B ) Acute knockdown of PIAS1 decreases HMW HTT species in the detergent - insoluble fraction . PIAS1 knockdown is detected in detergent - soluble and - insoluble fractions using anti - PIAS1 antibody . ( C ) Drosophila melanogaster expressing mutant HTTex1p ( 93Q ) in a reduced Su ( var ) 2 - 10 / dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration ( left panel , p = 0 . 033 ) when comparing dPIAS / + to + / + flies and increased overall survival ( right panel , p = 0 . 047 ) when comparing dPIAS1 / + to + / + flies . Significance was measured by Student ' s t test . ( D ) HMW SUMO - 2 accumulates in postmortem HD striata . Western blot analysis of the insoluble fraction from three control and three HD postmortem striata as described ( Experimental Procedures ) .

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

Figure 7(A) Western blot analysis of HeLa cells over-expressing exogenous PIAS1 in the presence of mutant HTT (97Q) when separated into detergent-soluble and detergent-insoluble fractions. No difference is detected in monomeric HTT (top panel, Soluble), but HMW HTT levels increase with PIAS1 overexpression. Anti-PIAS1 antibody (Invitrogen) was used to detect PIAS1.(B) Acute knockdown of PIAS1 decreases HMW HTT species in the detergent-insoluble fraction. PIAS1 knockdown is detected in detergent-soluble and -insoluble fractions using anti-PIAS1 antibody.(C) Drosophila melanogaster expressing mutant HTTex1p (93Q) in a reduced Su(var)2-10/dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration (left panel, p = 0.033) when comparing dPIAS/+ to +/+ flies and increased overall survival (right panel, p = 0.047) when comparing dPIAS1/+ to +/+ flies. Significance was measured by Student's t test.(D) HMW SUMO-2 accumulates in postmortem HD striata. Western blot analysis of the insoluble fraction from three control and three HD postmortem striata as described (Experimental Procedures).


Figure 1A . Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP ( 4 hours plus 30 minutes ) treatment as indicated . B . Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 3 biological repeats in one experiments ) . C . Size distributions for vesicles as analyzed in D . D . Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells . F . Flow cytometry analysis for CD63 + exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants . Quantification was done by mean fluorescence intensity . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 4 ) .

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

Figure 1A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).C. Size distributions for vesicles as analyzed in D.D. Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells.F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).


Figure 2B . Co - immunoprecipitation of GSDMD with full - length and deletion mutants of IQGAP1 tested in HEK293 cells . C . Co - immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells . D . Co - immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant ( D276A ) restored GSDMD KO YAMC cells . Cells were treated as indicated . E . Co - immunoprecipitation of C - terminal GSDMD ( CT ) with IQGAP1 overexpressed in HEK293 cells . F . Co - immunoprecipitation of pyroptosis mutants full - length ( I105N FL ) and N - terminal GSDMD defective of pyroptosis ( I105N - NT ) with IQGAP1 .

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

Figure 2B. Co-immunoprecipitation of GSDMD with full-length and deletion mutants of IQGAP1 tested in HEK293 cells.C. Co-immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells.D. Co-immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant (D276A) restored GSDMD KO YAMC cells. Cells were treated as indicated.E. Co-immunoprecipitation of C-terminal GSDMD (CT) with IQGAP1 overexpressed in HEK293 cells.F. Co-immunoprecipitation of pyroptosis mutants full-length (I105N FL) and N-terminal GSDMD defective of pyroptosis (I105N-NT) with IQGAP1.


Figure 3A . Imaging analysis for co - localization of CD63 with IQGAP1 in IQGAP1 ( FL ) and IQ domain truncation ( ΔIQ ) restored YAMC cell . Cells were treated with LPS plus ATP . The IQGAP1 - CD63 co - localization dots ( shown by yellow ) were counted , and Pearson ' s correlation coefficient was calculated for the quantification of co - localization . B . Imaging analysis for co - localization of CD63 with GSDMD in IQGAP1 full length ( FL ) and IQ domain truncation restored YAMC cell ( ΔIQ ) after LPS plus ATP treatment . The GSDMD - CD63 co - localized dots were counted and Pearson ' s correlation coefficient was calculated for quantification . C . Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells . D . Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells , IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells . Cells were treated as indicated and the supernatants were subjected to IP with anti - IL - 1β . E . Western blots analysis for whole cell lysate collected from panel D . F . ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated . G . Vesicle size distributions for exosome samples collected from panel E .

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

Figure 3A. Imaging analysis for co-localization of CD63 with IQGAP1 in IQGAP1 (FL) and IQ domain truncation (ΔIQ) restored YAMC cell. Cells were treated with LPS plus ATP. The IQGAP1-CD63 co-localization dots (shown by yellow) were counted, and Pearson's correlation coefficient was calculated for the quantification of co-localization.B. Imaging analysis for co-localization of CD63 with GSDMD in IQGAP1 full length (FL) and IQ domain truncation restored YAMC cell (ΔIQ) after LPS plus ATP treatment. The GSDMD-CD63 co-localized dots were counted and Pearson's correlation coefficient was calculated for quantification.C. Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells.D. Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells, IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells. Cells were treated as indicated and the supernatants were subjected to IP with anti-IL-1β. E. Western blots analysis for whole cell lysate collected from panel D.F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated.G. Vesicle size distributions for exosome samples collected from panel E.


Figure 4A . Imaging analysis for early endosome ( shown by marker EEA1 ) change in WT and IQGAP1 KO YAMC cells upon indicated treatments . Quantification was done by counting the EEA1 puncta from each cell . Five field views of cells ' puncta were counted . Three independent experiments were repeatedB . Co - localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells . Yellow dots indicated the co - localization signal . C . Co - localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells . D . Co - localization of CD63 with IQGAP1 - GSDMD complex , as measured by PLA , shown by the yellow dots after LPS and ATP treatment . E . Co - localization of CD63 and pro - IL - 1β in YAMC and IQGAP1 KO YAMC cells . F . Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments . Secreted proteins were pulled down by IL - 1β in the supernatant and the indicated proteins were probed for . G . Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment . H . Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments . I . Co - immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments .

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Figure 4A. Imaging analysis for early endosome (shown by marker EEA1) change in WT and IQGAP1 KO YAMC cells upon indicated treatments. Quantification was done by counting the EEA1 puncta from each cell. Five field views of cells' puncta were counted. Three independent experiments were repeatedB. Co-localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells. Yellow dots indicated the co-localization signal.C. Co-localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells.D. Co-localization of CD63 with IQGAP1-GSDMD complex, as measured by PLA, shown by the yellow dots after LPS and ATP treatment.E. Co-localization of CD63 and pro-IL-1β in YAMC and IQGAP1 KO YAMC cells.F. Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments. Secreted proteins were pulled down by IL-1β in the supernatant and the indicated proteins were probed for.G. Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment.H. Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments.I. Co-immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments.


Figure 5A . Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO ( IQ1 KO ) , IQGAP1 full length restored ( FL ) and GRD domain truncation restored ( ΔGRD ) YAMC cells upon LPS or LPS plus ATP treatment . B . PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell . The green dots showed the interaction of IQGAP1 and CDC42 . C . Colocalization of CD63 with IQGAP1 - CDC42 complex ( shown by PLA signal ) in WT and GSDMD KO cells under LPS treatment . Quantification was performed by counting PLA green dots . D . Co - immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild - type and GSDMD KO cells upon LPS or LPS plus ATP treatment . E . Co - immunoprecipitation analysis of GTP - bound CDC42 in indicated cells following LPS treatment . F . Co - immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild - type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment . G . Western blots for exosome secretion upon CDC42 GTPase inhibition . Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity , followed by LPS and ATP treatment as indicated . H . Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G . I . Co - immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 ( 10µM for 24 hours ) treated cells following LPS or LPS plus ATP treatment .

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Figure 5A. Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO (IQ1 KO), IQGAP1 full length restored (FL) and GRD domain truncation restored (ΔGRD) YAMC cells upon LPS or LPS plus ATP treatment.B. PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell. The green dots showed the interaction of IQGAP1 and CDC42.C. Colocalization of CD63 with IQGAP1-CDC42 complex (shown by PLA signal) in WT and GSDMD KO cells under LPS treatment. Quantification was performed by counting PLA green dots.D. Co-immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild-type and GSDMD KO cells upon LPS or LPS plus ATP treatment.E. Co-immunoprecipitation analysis of GTP-bound CDC42 in indicated cells following LPS treatment.F. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild-type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment.G. Western blots for exosome secretion upon CDC42 GTPase inhibition. Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity, followed by LPS and ATP treatment as indicated. H. Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G.I. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 (10µM for 24 hours) treated cells following LPS or LPS plus ATP treatment.


Figure 6A . Histology staining for IQGAP1 and GSDMD in naive and inflamed ( 3 days of DSS - challenge ) colon tissues . B . Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls . C . Western blot analysis for supernatant collected from colon explants of DSS - treated wild - type ( WT ) , Gsdmd - / - ( GSDMD KO ) and Iqgap1 - / - ( IQGAP1 KO ) mice . Supernatant was first pulled down by IL - 1β antibody and samples were subjected to Western blot analysis . The indicated proteins were probed . D . Immunofluorescent staining with anti - GTP - CDC42 in DSS treated colons from Gsdmd + / + ( WT ) and Gsdmd - / - ( GSDMD KO ) , IQGAP1 + / + and IQGAP1 - / - mice .

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Figure 6A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues.B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls.C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice.


Figure 1 ( A ) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC - labeled zymosan ( 3 mg / ml , 10 µl ) in one hind paw . White dotted lines show the position of zymosan . Quantification of the number of macrophages and their subtypes 8 , 24 , 48 and 72 hours after zymosan injection based on the MELC images . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * * p < 0 . 001 . # p < 0 . 05 , # # # p < 0 . 001 as compared to naïve mice . Quantification of the number of macrophages and their subtypes 8 , 24 , 48 and 72 hours after zymosan injection based on the MELC images . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * * p < 0 . 001 . # p < 0 . 05 , # # # p < 0 . 001 as compared to naïve mice . Quantification of the number of macrophages and their subtypes 8 , 24 , 48 and 72 hours after zymosan injection based on the MELC images . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * * p < 0 . 001 . # p < 0 . 05 , # # # p < 0 . 001 as compared to naïve mice . FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 . FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 . FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 . FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 1(A) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC-labeled zymosan (3 mg/ml, 10 µl) in one hind paw. White dotted lines show the position of zymosan.Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.


Figure 2 ( A ) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM ( n . d . , not determinable ) .

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Figure 2(A) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM (n.d., not determinable).


Figure 3Representative MELC images showing the IL - 4 and Siglec F expression 4 hours ( panel C ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs . Representative MELC images showing the IL - 4 and Siglec F expression 24 hours ( panel D ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs . ( E ) Number of IL - 4 - expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , two - tailed Students t - test * * * p < 0 . 001 . ( F ) Number of IL - 4 - expressing eosinophils in the three regions 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 3Representative MELC images showing the IL-4 and Siglec F expression 4 hours (panel C) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.Representative MELC images showing the IL-4 and Siglec F expression 24 hours (panel D) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.(E) Number of IL-4-expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, two-tailed Students t-test ***p<0.001.(F) Number of IL-4-expressing eosinophils in the three regions 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.(G) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, **p<0.01, ***p<0.001.


Figure 4FACS analysis of eosinophils in the blood B ; n = 6 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice . FACS analysis of eosinophils in the paws ( panel C n = 8 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice . Zymosan - induced mechanical ( panel E ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice . Zymosan - induced thermal ( panel F ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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Figure 4FACS analysis of eosinophils in the blood B; n= 6 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.FACS analysis of eosinophils in the paws (panel C n= 8 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.Zymosan-induced mechanical (panel E) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.Zymosan-induced thermal (panel F) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.


Figure 5Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , as well as efferocytosis ( panel H ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . Quantification of eosinophils , the sum of all macrophages , neutrophils ( panel I ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 5Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, as well as efferocytosis (panel H) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.Quantification of eosinophils, the sum of all macrophages, neutrophils (panel I) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


Figure 6 ( A ) IL - 4 levels in paws from naive and zymosan - injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil - depleted mice . Data are shown as mean ± SEM ( n = 6 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * * p < 0 . 01 , * * * p < 0 . 001 . ( B ) FACS analysis of the eosinophil number in paws from control and eosinophil - depleted mice with or without IL - 4c treatment 24h prior zymosan injection . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni * * * p < 0 . 001 . ( C ) Same as panel B except that resident ( Ly6C - ) and monocyte - derived macrophages ( Ly6C + ) were determined . Same as panel B except that M2 - like ( panel D ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 . Same as panel B except that M1 - like ( panel E ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 . Same as panel B except that M0 macrophages ( panel F ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

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Figure 6(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.(C) Same as panel B except that resident (Ly6C-) and monocyte-derived macrophages (Ly6C+) were determined.Same as panel B except that M2-like (panel D) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.Same as panel B except that M1-like (panel E) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.Same as panel B except that M0 macrophages (panel F) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


Figure 7 ( A ) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil - depleted mice . Data are mean ± SEM ( n = 6 mice ) . Student ' s One - way ANOVA , significance between control and anti - Siglec F antibody treatment * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . FACS analysis of the neutrophil count in paws ( panel B ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 . FACS analysis of the neutrophil count in peripheral blood ( panel C ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) Same as panel B except that efferocytosis was determined . * p < 0 . 05 ; * * * p < 0 . 001 ( E ) FACS analysis of alive and dying neutrophils in paws from control or eosinophil - depleted mice . Data are shown as the mean ± SEM ( n = 5 mice ) .

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Figure 7(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.FACS analysis of the neutrophil count in paws (panel B) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.FACS analysis of the neutrophil count in peripheral blood (panel C) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.(D) Same as panel B except that efferocytosis was determined. *p<0.05; ***p<0.001(E) FACS analysis of alive and dying neutrophils in paws from control or eosinophil-depleted mice. Data are shown as the mean ± SEM (n=5 mice).


Figure 8 ( C ) Zymosan - induced thermal hypersensitivity in control or eosinophil - depleted mice with or without IL - 4c treatment 24h hours prior zymosan injection . The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points . Data are shown as the mean ± SEM ( n = 6 mice ) . Two - way ANOVA / Bonferroni , * p < 0 . 05 , * * p < 0 . 01 ; ns , not significant .

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Figure 8(C) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.


Figure 1E Distribution of endogenous WIPI2 and STING in MEFs . F Statistical analysis of the number of STING - and WIPI2 - positive puncta in cells treated as in ( E ) . G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING - HA . H Statistical analysis of the number of STING - and ATG16L1 - positive puncta in cells treated as in ( G ) . All the cells were treated with or without cGAMP for 2 h . All statistical data are presented as mean ± SEM of three independent experiments .

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Figure 1E Distribution of endogenous WIPI2 and STING in MEFs. F Statistical analysis of the number of STING- and WIPI2-positive puncta in cells treated as in (E). G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING-HA. H Statistical analysis of the number of STING- and ATG16L1-positive puncta in cells treated as in (G). All the cells were treated with or without cGAMP for 2 h. All statistical data are presented as mean ± SEM of three independent experiments.


Figure 2A Co - immunoprecipitation of endogenous STING with WIPI1 / 2 - Myc in MEFs transfected with Myc - tagged WIPI1 or WIPI2 . The cells were treated with or without cGAMP for 2 h and the WIPI1 / 2 - Myc precipitates were analyzed by western blot using anti - STING . B Co - immunoprecipitation of endogenous WIPI2 with HA - tagged STING , STING - R238A or STING - S366A from HEK293T cells stably expressing STING - HA STING - R238A - HA or STING - S366A - HA . The cells were treated with or without cGAMP for 2 h . Co - immunoprecipitation of endogenous STING with Myc - WIPI2 or the indicated WIPI2 mutants from MEFs transfected with the indicated plasmids . J Purified GST - tagged STING - 153 - 379 or STING - 153 - 379 - 3A - 2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants , and then precipitated with glutathione sepharose beads . The bound WT or mutant WIPI2 was detected by western blot using anti - WIPI2 . K Statistical analysis of ( J ) .

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Figure 2A Co-immunoprecipitation of endogenous STING with WIPI1/2-Myc in MEFs transfected with Myc-tagged WIPI1 or WIPI2. The cells were treated with or without cGAMP for 2 h and the WIPI1/2-Myc precipitates were analyzed by western blot using anti-STING. B Co-immunoprecipitation of endogenous WIPI2 with HA-tagged STING, STING-R238A or STING-S366A from HEK293T cells stably expressing STING-HA STING-R238A-HA or STING-S366A-HA. The cells were treated with or without cGAMP for 2 h.Co-immunoprecipitation of endogenous STING with Myc- WIPI2 or the indicated WIPI2 mutants from MEFs transfected with the indicated plasmids.J Purified GST-tagged STING-153-379 or STING-153-379-3A-2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants, and then precipitated with glutathione sepharose beads. The bound WT or mutant WIPI2 was detected by western blot using anti-WIPI2. K Statistical analysis of (J).


Figure 3A WIPI2 - GFP puncta in MEFs stably expressing GFP - tagged WT or mutant WIPI2 . The cells were treated with cGAMP for 2 h or Torin1 for 1 h . B Statistical analysis of the number of WIPI2 - GFP puncta in cells treated as in ( A ) . F LC3 and p62 level in HEK293T cells stably expressing HA - tagged WT or mutant STING . The cells were treated with or without cGAMP for 4 h .

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Figure 3A WIPI2-GFP puncta in MEFs stably expressing GFP-tagged WT or mutant WIPI2. The cells were treated with cGAMP for 2 h or Torin1 for 1 h. B Statistical analysis of the number of WIPI2-GFP puncta in cells treated as in (A).F LC3 and p62 level in HEK293T cells stably expressing HA-tagged WT or mutant STING. The cells were treated with or without cGAMP for 4 h.


Figure 4C Co - immunoprecipitation of endogenous WIPI2 with STING - HA from HEK293 cells stably expressing STING - HA with or without BECN1 deletion . The cells were treated with or without Torin1 for 3 h . E Co - immunoprecipitation of endogenous STING with Myc - tagged WT or mutant WIPI2 from MEFs treated with or without VPS34 - IN1 for 2 h . G GFP - LC3 punctum formation in MEFs stably expressing GFP - LC3 and STING - HA . The cells were treated with or without cGAMP or VPS34 - IN1 for 2 h . Scale bars , 10 µm . I Western blot assay of p62 and phospho - TBK1 ( Ser172 ) in MEFs . The cells were pretreated with or without VPS34 - IN1 for 1 h and then co - treated with cGAMP or Torin1 for another 4 h along with or without CQ . CQ , chloroquine . J Statistical analysis of p62 protein level in cells treated as in ( I ) .

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Figure 4C Co-immunoprecipitation of endogenous WIPI2 with STING-HA from HEK293 cells stably expressing STING-HA with or without BECN1 deletion. The cells were treated with or without Torin1 for 3 h.E Co-immunoprecipitation of endogenous STING with Myc-tagged WT or mutant WIPI2 from MEFs treated with or without VPS34-IN1 for 2 h.G GFP-LC3 punctum formation in MEFs stably expressing GFP-LC3 and STING-HA. The cells were treated with or without cGAMP or VPS34-IN1 for 2 h. Scale bars, 10 µm.I Western blot assay of p62 and phospho-TBK1 (Ser172) in MEFs. The cells were pretreated with or without VPS34-IN1 for 1 h and then co-treated with cGAMP or Torin1 for another 4 h along with or without CQ. CQ, chloroquine. J Statistical analysis of p62 protein level in cells treated as in (I).


Figure 5A , B Localization of endogenous WIPI2 , WIPI1 , ATG16L1 ( A ) or Myc - tagged WT WIPI2 or WIPI2 mutants ( B ) in MEFs transfected with Cy3 labeled - ISD ( 1 μg / ml ) . ISD , interferon stimulatory DNA . D Statistical analysis of cytoplasmic DNA puncta in cells treated as in ( C ) . F Statistical analysis of cytoplasmic DNA puncta in cells treated as in ( E ) .

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Figure 5A, B Localization of endogenous WIPI2, WIPI1, ATG16L1 (A) or Myc-tagged WT WIPI2 or WIPI2 mutants (B) in MEFs transfected with Cy3 labeled-ISD (1 μg/ml). ISD, interferon stimulatory DNA.D Statistical analysis of cytoplasmic DNA puncta in cells treated as in (C).F Statistical analysis of cytoplasmic DNA puncta in cells treated as in (E).


Figure 6A Wipi2 - or Sting - deleted MEFs were treated with Ara - C for 12 h , and then cultured in Ara - C - free medium . After 24 h , the cells were lysed and analyzed by western blot using the indicated antibodies . B Statistical analysis of the protein level of phospho - TBK1 ( Ser172 ) and STING in cells treated as in ( A ) . The relative phospho - TBK1 level was normalized to TBK1 , the relative STING level was normalized to Actin . C - E MEFs with Wipi2 or Sting deletion ( C ) , Wipi2 - KO MEFs with transfection of Myc - tagged WT or mutant WIPI2 ( D ) , or Sting - KO MEFs with transfection of HA - tagged WT or mutant STING ( E ) , were treated with Ara - C for 12 h and then cultured in Ara - C - free medium for another 24 h . Total mRNA was extracted and the Ifnb1 mRNA level was measured by real - time PCR and normalized to Actb mRNA . 2RE , WIPI2 - R108E / R125E . F Sting - KO MEFs with transfection of HA - tagged WT or mutant STING were treated with cGAMP for the indicated time . The cells were lysed and analyzed by western blot using the indicated antibodies .

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

Figure 6A Wipi2- or Sting-deleted MEFs were treated with Ara-C for 12 h, and then cultured in Ara-C-free medium. After 24 h, the cells were lysed and analyzed by western blot using the indicated antibodies. B Statistical analysis of the protein level of phospho-TBK1 (Ser172) and STING in cells treated as in (A). The relative phospho-TBK1 level was normalized to TBK1, the relative STING level was normalized to Actin.C-E MEFs with Wipi2 or Sting deletion (C), Wipi2-KO MEFs with transfection of Myc-tagged WT or mutant WIPI2 (D), or Sting-KO MEFs with transfection of HA-tagged WT or mutant STING (E), were treated with Ara-C for 12 h and then cultured in Ara-C-free medium for another 24 h. Total mRNA was extracted and the Ifnb1 mRNA level was measured by real-time PCR and normalized to Actb mRNA. 2RE, WIPI2-R108E/R125E.F Sting-KO MEFs with transfection of HA-tagged WT or mutant STING were treated with cGAMP for the indicated time. The cells were lysed and analyzed by western blot using the indicated antibodies.


figf1 ( a ) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression ( 18 h cultures ) . Values are means ± s . e . m . of three independent experiments . ( b ) The cell death regulated by Atg5 is apoptosis . All data presented are from 5 h cultures . Caspase - 3 - like activity analysed in HeLa cells ( graph ) . Morphology of Jurkat cells is also shown . The numbers in the lower right corner indicate the percentage of cell death , which was determined in parallel experiments using flow cytometryc ) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z - VAD - fmk . Numbers indicate the results of the quantitative analysis ( percentage ) of each dot blot . d ) Reduction of drug - induced cell death by silencing of Atg5 . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) . The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting . The asterisks indicate P 0 . 05 . n . s . , no significant difference .

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

figf1(a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments.(b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometryc) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot.d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.


figf2a ) Spontaneous neutrophil apoptosis . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein . 33K Atg5 disappeared during apoptosis in a time - dependent manner . HeLa cells overexpressing Atg5 served as positive control . b ) Anti - CD95 - induced apoptosis of Jurkat cells . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein and a reduction of 33K Atg5 in a time - dependent manner . The 24K anti - Atg5 - reactive protein was also visible in Jurkat cells after treatment with actinomycin D ( Act . D ) or cycloheximide ( CHX ; 5 h treatment in both cases ) . GAPDH expression indicates equal loading in each panel . All immmunoblots are representative of at least three independent experiments .

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

figf2a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control.b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.


figf3e ) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation . Calpain cleaved Atg5 at Thr 193 . Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice . The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank . The position of the six - amino - acid deletion causing calpain resistance is indicated in red . ( d ) Following calpain - mediated cleavage of recombinant Atg5 in vitro , the sample was fractionated by preparative reversed - phase HPLC and analysed by mass spectrometry . The exact relative molecular masses for full - length and truncated Atg5 are indicated . ( f ) A six - amino - acid deletion surrounding the cleavage site ( 35S - Atg5Δ191 - 196 ) led to a loss of the calpain cleavage fragment . ( g ) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor - mediated apoptosis of neutrophils . In contrast , Atg6 did not show evidence for cleavage and its levels remained unchanged in this system . Results of 7 h cultures are shown . ( h ) Atg5 is not cleaved in apoptotic HeLa cells ( 5 h staurosporine treatment ) lacking the small subunit of calpain , which was silenced by using specific siRNA . ( i ) Calpain resistant Atg5Δ191 - 196 was unable to sensitize HeLa cells lacking endogeneous Atg5 ( due to gene silencing , see Fig . 1c ) to staurosporine - induced cell death . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) .

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

figf3e) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation. Calpain cleaved Atg5 at Thr 193. Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice. The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank. The position of the six-amino-acid deletion causing calpain resistance is indicated in red.(d) Following calpain-mediated cleavage of recombinant Atg5 in vitro, the sample was fractionated by preparative reversed-phase HPLC and analysed by mass spectrometry. The exact relative molecular masses for full-length and truncated Atg5 are indicated.(f) A six-amino-acid deletion surrounding the cleavage site (35S-Atg5Δ191-196) led to a loss of the calpain cleavage fragment.(g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown.(h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA.(i) Calpain resistant Atg5Δ191-196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).