( a ) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes ( APHL ) isolated from livers of 18Q - htt ( 18Q ) and 111Q - htt ( 111Q ) mice . Cyt , cytochrome ; Dyn IC , dynein intermediate chain ; ADRP , adipose differentiation - related protein ; Polyubq , polyubiquitin . Bottom : amount of each protein in 111Q - htt sample , as a multiple of its amount in 18Q - htt sample . Mean + s . d . ; n = 4 .

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(a) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4.


( b ) Immunoblots for htt and LC3 in homogenates ( Homog ) , cytosol and fractions enriched in APHs and enriched in autophagolysosomes ( APHL ) isolated from livers of wild - type ( 18Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) . Representative one of four experiments with duplicated samples . * P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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(b) Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Immunoblot for htt or p62 from total APHs ( T ) and their corresponding membranes ( Mbr ) and matrices ( Mtx ) isolated from livers of wild - type htt ( 18Q ) and 111Q - htt mutant huntingtin knock - in mice ( 111Q ) . Left , representative immunoblots . Right , distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots . AV , autophagic vacuoles . Mean + s . d . * P 0 . 05 compared to wild - type values .

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(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values.


( b ) Filter retardation analysis of the same fractions as in a , blotted for htt . Negative signal indicated absence of aggregates retained in the filter .

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(b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter.


( c ) Immunoblot for ubiquitin in homogenates ( Homog ) , cytosol and the autophagic fractions described in a , using 6 % ( top ) and 16 % ( bottom ) gels . Dotted line separates stacking and running parts of gel .

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(c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel.


( d ) Immunoblot for htt , LC3 and p62 , of brain homogenates ( Homog ) and fractions enriched in APHs from the same mouse groups as in a .

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(d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a.


( e ) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co - immunoprecipitation for htt under low stringency . Input for 111Q - htt autophagic vacuoles was one - third of that for 18Q - htt to avoid antibody saturation . Levels of htt ( top ) and p62 ( bottom ) in the input , immunoprecipitate ( IP ) and flow through ( FT ) are shown . Full - length blots in Supplementary Figure 21 .

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(e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.


( a ) Neutral lipids in MEFs from 18Q - htt and 111Q - htt mice stained with BODIPY 493 / 503 .

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(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.


( b ) Electron micrographs of livers from 18Q - htt and 111Q - htt mice . Right : number of lipid droplets ( LD ) per cell profile ( prof . ) , mean area of LDs and percentage of cellular area occupied by LDs . Mean + s . d . ; n = 3 .

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(b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3.


( c - e ) Neutral lipids in striatal cells from 7Q - htt and 111Q - httmice ( c ) , grown on a monolayer of their own

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(c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes


( c - e ) Neutral lipids in primary striatal neurons from 18Q - htt and 111Q - httmice grown on a monolayer of their own astrocytes ( d )

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(c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d)


( c - e ) Neutral lipids in lymphoblasts from a control ( UC ) and HD - affected human ( e ) stained with BODIPY 493 / 503 . MAP2 staining highlights neurons . Extended study in Supplementary Figure 15b .

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(c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b.


( f ) Electron micrographs of a lymphoblast from an individual with HD ( with 78 and 15 polyglutamine repeats in the two alleles ) . Green arrows , LDs .

(f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs.


( g ) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects . Numbers of polyglutamine repeats are shown below . Mean + s . d . of > 100 cell profiles . Dotted red line , mean value of the respective samples . * P 0 . 05 .

(g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05.


( h ) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals ( top ) and two individuals with HD ( bottom ) . Nuclei are highlighted with hematoxylin . Lipid droplets are visible as red puncta . Subject identifiers are shown in each panel .

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(h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel.


( i ) The percentage of total cellular area occupied by lipid droplets ( left ) and the average number of lipid droplets per cell ( right ) was calculated for each of the samples by quantification of eight or nine different fields . * P 0 . 001 ; analysis of variance , P 0 . 01 for each person with HD versus each control individual by Tukey post hoc test . Subject identifiers are as in h .

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(i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.


( a ) Top : immunofluorescence for the mitochondrial marker COX IV in 18Q - htt and 111Q - htt MEFs maintained in the presence or absence of serum . Bottom : number of mitochondria per cell . Mean + s . d . of 10 - 20 cells in three different experiments .

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(a) Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10-20 cells in three different experiments.


( b ) Electron micrographs of striatal neurons from wild - type ( Ctr ) and HD94 mice grown over a wild - type ratastrocyte monolayer . Arrows , abnormally short ( black ) or abnormally long ( green ) mitochondria . Right : number of mitochondria per cell profile . Mean + s . d . of ten cells per group in triplicates .

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(b) Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type ratastrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates.


( c ) Left : electron micrographs of lymphoblasts from controls ( UC ) or people with HD . Arrows as in b . Right : percentage of cellular area occupied by mitochondria in different individuals . Line indicates population mean .

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(c) Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in b. Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean.


( d , e ) Striatal cells from 7Q - htt and 111Q - htt mice ( d ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in

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(d,e) Striatal cells from 7Q-htt and 111Q-htt mice (d) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d


MEFs from 18Q - htt and 111Q - httmice ( e ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e . CCCP was added to control cells in e as a positive control for depolarization . * P 0 . 05 .

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MEFs from 18Q-htt and 111Q-httmice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05.


( f , g ) Striatal cells from 7Q - htt and 111Q - htt mice untreated or treated with vinblastine were co - stained for LC3 and Mitotracker ( f ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f). Arrows, colocalization events. Extended study in Supplementary Figure 16.


( f , g ) Striatal cells from 7Q - htt and 111Q - httmice untreated or treated with vinblastine were co - stained for LC3 and BODIPY 493 / 503 ( g ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.


Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in ( B - F ) . B - F Immunolabeling results of Kcnq1 ( green ) in cochlear cryosections are shown for uninjected WT ( B ) , untreated Kcnq1 − / − ( C ) , injected WT ( D ) , and Kcnq1 − / − mice given injections into the ST ( E ) or SM ( F ) . Cell nuclei were outlined by counterstaining with either DAPI ( B - D , F ) or Qnuclear deep red ( E ) . Scale bars represent approximately 100 μm . Meaning of white arrows are given in the text .

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Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B-F).B-F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B-D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.


Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were , respectively , labeled green and purple . The SV consists of three layers of cells ; marginal cells are the first layer of cells on the side of the endolymphatic space . Cryosection through the SV of WT mice . The large arrow points to the native Kcnq1 ( labeled green ) in WT mice . Smaller arrows show nuclei of the marginal cells . Cryosection through the SV of a Kcnq1 − / − mouse injected with AAV expressing Kcnq1 . Labeled in green ( bigger arrow ) is the AAV1 - expressed Kcnq1 , found only in the apical membrane of the marginal cells . Smaller arrows show nuclei of the marginal cells . Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV . Cryosection through the lateral wall of Kcnq1 − / − mice showing intracellular distribution of Kcnq1immunolabeling ( green ) in fibrocytes . Cryosection through the spiral ganglia of cochlea of treated Kcnq1 − / − mice showing immunolabeling ( green ) in the cells of spiral ganglia ( arrows ) . Data information : Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei . Scale bars represent appropriately 50 μm .

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Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.


Quantification of Kcnq1 - positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled ( red ) by phalloidin conjugated with rhodamine . A Immunolabeling results ( Kcnq1 labeled in green ) in WT mice . B , C Immunolabeling results ( Kcnq1 labeled in green ) of treated Kcnq1 − / − mice , middle ( B ) and apical ( C ) turns , respectively . D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT ( gray bars , left ) , untreated Kcnq1 − / − ( middle ) , and treated Kcnq1 − / − mice ( black bars , right ) . Data are given as mean ± SD ( n = 6 ) . E , F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine . Results from WT ( E ) and untreated Kcnq1 − / − mice ( F ) are compared . Data information : Scale bars represent approximately 50 μm .

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Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.


Comparison of cochlear morphologyA - C Cochlear sections obtained from WT ( A ) , nontreated Kcnq1 − / − ( B ) , and treated Kcnq1 − / − mice ( C ) were compared . Major landmarks of the cochlear sections are labeled and pointed by arrows . Scale bars represent approximately 50 μm .

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Comparison of cochlear morphologyA-C Cochlear sections obtained from WT (A), nontreated Kcnq1−/− (B), and treated Kcnq1−/− mice (C) were compared. Major landmarks of the cochlear sections are labeled and pointed by arrows. Scale bars represent approximately 50 μm.


Waveforms of ABRs are compared in WT , untreated Kcnq1 − / − , and treated Kcnq1 − / − mice , as labeled above data traces . Series of averaged ABR data traces were evoked from tone - burst sounds with intensities ranging from 20 to 90 dB SPL .

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Waveforms of ABRs are compared in WT, untreated Kcnq1−/−, and treated Kcnq1−/− mice, as labeled above data traces. Series of averaged ABR data traces were evoked from tone-burst sounds with intensities ranging from 20 to 90 dB SPL.


Summary of averaged ABR thresholds at various frequencies for different groups of mice ; untreated Kcnq1 − / − ( open triangles ) , treated Kcnq1 − / − ( filled triangles ) , injected WT ( filled circles ) , and uninjected WT ( filled squares ) . Plot legends are given in the figure . The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1 − / − mice . Error bars represent standard error of the mean .

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Summary of averaged ABR thresholds at various frequencies for different groups of mice; untreated Kcnq1−/− (open triangles), treated Kcnq1−/− (filled triangles), injected WT (filled circles), and uninjected WT (filled squares). Plot legends are given in the figure. The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1−/− mice. Error bars represent standard error of the mean.


Click - evoked ABR thresholds of WT ( filled squares ) , treated Kcnq1 − / − mice ( filled triangles ) , untreated Kcnq1 − / − mice ( open squares ) measured 4 - 30 weeks after mice were born . Error bars represent standard error of the mean . Upward arrows indicate that click - ABR thresholds were at the maximal sound level that could be reliably measured by the system .

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Click-evoked ABR thresholds of WT (filled squares), treated Kcnq1−/− mice (filled triangles), untreated Kcnq1−/− mice (open squares) measured 4-30 weeks after mice were born. Error bars represent standard error of the mean. Upward arrows indicate that click-ABR thresholds were at the maximal sound level that could be reliably measured by the system.


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice and an equal volume of PBS was injected in left hind paw . After 1 hour of formalin injection , Balb / c mice were treated with a single dose of either PBS ( vehicle control ) or Diclofenac ( 10 mg / kg ) or different concentrations of rPPE2 ( 2 mg / kg and 3 mg / kg ) via intraperitoneal route and after three hours of treatment , mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population . ( A ) Photographs of representative sections were visualized at 20X magnification ( scale bar = 50 µm ) . ( B ) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . For ( B , unpaired t - test was applied to calculate p values .

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A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.


( B - E ) In another experiment , NIH - 3T3 cells ( B , C ) or MEFs ( D , E ) were incubated with either PBS or rPPE2 ( 3 µg / ml ) . After 45 minutes of rPPE2 treatment , cells were fixed , permeabilized , and stained with anti - PPE2 Ab . DAPI was used to stain the nucleus . Nuclear localization of rPPE2 in NIH - 3T3 ( C ) and MEFs ( E ) was measured by calculating the colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure C , E unpaired t - test was applied to calculate p values .

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(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.


( F - G ) ( F ) NIH - 3T3 cells were transiently transfected with either pEGFP or pEGFP - PPE2 . After 24 hours of transfection , cells were fixed and observed under a confocal microscope . DAPI was used to stain the nucleus . ( G ) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure G , unpaired t - test was applied to calculate p values .

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(F-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.


( D ) Lysates prepared from the same samples used in C were resolved on SDS - PAGE and checked for GFP expression by Western blotting using anti - GFP Ab . GAPDH was used as a loading control .

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(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.


( E ) Varying concentrations of rPPE2 protein were incubated with [ γ - 32P ] - ATP oligonucleotides of 60 bp spanning SCF promoter . DNA - protein complexes were resolved on 7 % native polyacrylamide gel . In cold competition reactions , equimolar cold / unlabeled 60 bp oligonucleotides were used ( arrow represents PPE2 - oligo complex ) . The data shown are representative of 3 independent experiments .

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(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . B ) ( B ) Next , Toluidine blue staining was done to check the mast cell population . Photographs of representative sections were visualized at 20X magnification ( scale bar = 100 µm ) .

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A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. B) (B) Next, Toluidine blue staining was done to check the mast cell population. Photographs of representative sections were visualized at 20X magnification (scale bar = 100 µm).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . ( C ) Counting of mast cells was performed in Toluidine blue - stained paw sections using ImageJ software and was normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . unpaired t - test was applied to calculate p values .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. (C) Counting of mast cells was performed in Toluidine blue-stained paw sections using ImageJ software and was normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. unpaired t-test was applied to calculate p values.


A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 24 , 48 and 72 hours and imaged after DNA staining with Hoechst . Green arrowheads indicate examples of prometaphase arrested cells , and the red arrowheads indicate examples of dead cells due to Plk1 downregulation . Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments . Scale bar , 20 μm .

A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.


B ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . C ) Quantification of nuclear size measurements from Fig 2B . Data derived from 3 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test .

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B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.


D ) Solid phase transfection of nontargeting ( scrambled ) , GOLGA2 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Golga2 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . E ) Quantification of experiments in Fig 2D . Data derived from 3 independent experiments . P value ( scrambled vs GOLGA2 ) < 2e - 16 , Mann - Whitney U test .

D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test.


F ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . G ) Quantification of experiments in Fig 2F . Data derived from 3 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test .

F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.


H ) Comparison of phenotypic penetrance with liquid and solid phase transfection . RPE - 1 , HEK293T , NCI - H358 and NCI - N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers ( 2250 or 20000 cells / well ) . 5 days post transfection cell viability were measured by CellTiter - Glo . Boxplots represent values from at least 3 independent experiments containing 3 technical replicates .

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H) Comparison of phenotypic penetrance with liquid and solid phase transfection. RPE-1, HEK293T, NCI-H358 and NCI-N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers (2250 or 20000 cells/well). 5 days post transfection cell viability were measured by CellTiter-Glo. Boxplots represent values from at least 3 independent experiments containing 3 technical replicates.


I ) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines . Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days . The raw values are background subtracted and normalized to the mock controls . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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I) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines. Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days. The raw values are background subtracted and normalized to the mock controls. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


A ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . Scale bar , 50μm . B ) Quantification of experiments in Fig 3A . Data derived from 2 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test .

A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.


C ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . D ) Quantification of experiments in Fig 3C . Data derived from 2 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test .

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C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test.


E ) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


A ) Viability of NCI - N87 and NCI - H358 cells upon transfection of 45 different gRNAs by solid phase transfection . Cas9 expressing NCI - N87 and NCI - H358 cell were seeded on pre - coated plates . 5 days post transfection , cell viability in each well were measured by CellTiter - Glo . Values were background subtracted , normalized to scrambled controls and plotted against each other . Blue dots represent scrambled controls , whereas the yellow dots represent POLR2A gRNAs . Green dots represent the genes that affect the viability in a cell line dependent manner . Results are representative of 3 independent experiments .

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A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments.


B , C ) Logarithm of the odds ( LOD ) scores of cancer associated gene KOs in NCI - N87 and NCI - H358 cells derived from screens with gRNAs ( B ) and RNPs ( C ) . Bottom panels show distribution of positive ( POLR2A ) and negative ( scrambled ) controls for each experiment . LOD score of 3 is highlighted with a dashed line , indicating statistical significance .

B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.


A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting RNP complexes into human primary lung fibroblasts ( HLF - 1 ) , primary lung adenocarcinoma cells ( LAD - 1 ) or RPE - 1TP53 - / - cells . 72 hours post transfection cells were stained with Hoechst and imaged . Boxplots represent values from 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs Plk1 ) < 0 . 05 . In the right panels , representative images for each cell line is shown after RNP transfection targeting PLK1 . Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation . Scale bars indicate 20μm .

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A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting RNP complexes into human primary lung fibroblasts (HLF-1), primary lung adenocarcinoma cells (LAD-1) or RPE-1TP53 -/- cells. 72 hours post transfection cells were stained with Hoechst and imaged. Boxplots represent values from 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs Plk1) < 0.05. In the right panels, representative images for each cell line is shown after RNP transfection targeting PLK1. Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation. Scale bars indicate 20μm.


B ) Two human primary lung fibroblasts ( HLF ) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma ( LSC ) or lung adenocarcinoma ( LAD ) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


A , Seedlings of WT , abs3 - 1D , FD11 - 55 , and arf2 - 20 before and after 6 d C - deprivation . B , Seedlings of WT , abs3 - 1D , FD18 - 70 , and pif5 - 10 before and after 6 d C - deprivation .

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A, Seedlings of WT, abs3-1D, FD11-55, and arf2-20 before and after 6 d C-deprivation. B, Seedlings of WT, abs3-1D, FD18-70, and pif5-10 before and after 6 d C-deprivation.


C and D , RT - qPCR analysis of ABS3 expression levels in 7 - d - old seedlings of the same genotypes as shown in A ( C ) and B ( D ) . Fold changes were calculated with respect to the expression level in the WT . two - tailed t test , * * * * , p < 0 . 0001 , n . s . , not significant . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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C and D, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of the same genotypes as shown in A (C) and B (D). Fold changes were calculated with respect to the expression level in the WT. two-tailed t test, ****, p<0.0001, n.s., not significant. data are means ± standard deviation (s.d.) of 3 biological replicates.


E and F , Chlorophyll and total cellular protein content reduction in genotypes shown in A ( E ) and B ( F ) after 6 d C - deprivation . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) and B (F) after 6 d C-deprivation. data are means ± standard deviation (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 . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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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. data are means ± standard deviation (s.d.) of 3 biological replicates.


C , Heatmap of log2FC values of 2 , 587 ABS3 - regulated genes ( abs3 - 1D vs . WT ) under C - deprivation in indicated comparisons .

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C, Heatmap of log2FC values of 2,587 ABS3-regulated genes (abs3-1D vs. WT) under C-deprivation in indicated comparisons.


F , Heatmap of log2FC values of 1 , 326 ARF2 , PIF5 , and C - deprivation co - regulated genes in indicated comparisons . C - deprivation : abs3 - 1D 4 d C - deprivation vs . abs3 - 1D 0 d C - deprivation ; ARF2 : abs3 - 1D vs . arf2 - 20 abs3 - 1D after 4 d C - deprivation ; PIF5 : abs3 - 1D vs . abs3 - 1D pif5 - 10 after 4 d C - deprivation . Enlarged were expression changes of senescence genes identified in DAVID GO

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F, Heatmap of log2FC values of 1,326 ARF2, PIF5, and C-deprivation co-regulated genes in indicated comparisons. C-deprivation: abs3-1D 4 d C-deprivation vs. abs3-1D 0 d C-deprivation; ARF2: abs3-1D vs. arf2-20 abs3-1D after 4 d C-deprivation; PIF5: abs3-1D vs. abs3-1D pif5-10 after 4 d C-deprivation. Enlarged were expression changes of senescence genes identified in DAVID GO analysis


A , Interaction between ARF2 and PIF5 in the yeast two - hybrid assay . Growth of yeast cells co - transformed with indicated BD and AD vectors was monitored on quadruple dropout medium ( QDO , SD - Ade / - His / - Leu / - Trp ) and QDO medium containing X - α - gal .

A, Interaction between ARF2 and PIF5 in the yeast two-hybrid assay. Growth of yeast cells co-transformed with indicated BD and AD vectors was monitored on quadruple dropout medium (QDO, SD-Ade/-His/-Leu/-Trp) and QDO medium containing X-α-gal.


B , Interaction between ARF2 and PIF5 in protoplast BiFC assays . YN - ARF2 and PIF5 - YC were coexpressed with the nuclear marker NLS - mCherry in Arabidopsis mesophyll protoplasts . Coexpression of YN - ARF2 and YC alone or YN alone with PIF5 - YC served as negative controls . Bars , 10 μm .

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B, Interaction between ARF2 and PIF5 in protoplast BiFC assays. YN-ARF2 and PIF5-YC were coexpressed with the nuclear marker NLS-mCherry in Arabidopsis mesophyll protoplasts. Coexpression of YN-ARF2 and YC alone or YN alone with PIF5-YC served as negative controls. Bars, 10 μm.


C , Direct interaction between GST - ARF2 and MBP - PIF5 - His in in vitro GST pull - down assays .

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C, Direct interaction between GST-ARF2 and MBP-PIF5-His in in vitro GST pull-down assays.


D , Co - immunoprecipitation of ARF2 - HA with PIF5 - GFP in vivo . ARF2 - HA was coexpressed with PIF5 - GFP or GFP alone in protoplasts . Proteins co - immunoprecipitated by GFP - Trap beads were immunoblotted with anti - GFP and anti - HA antibodies .

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D, Co-immunoprecipitation of ARF2-HA with PIF5-GFP in vivo. ARF2-HA was coexpressed with PIF5-GFP or GFP alone in protoplasts. Proteins co-immunoprecipitated by GFP-Trap beads were immunoblotted with anti-GFP and anti-HA antibodies.


A , 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 6 d C - deprivation .

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A, 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 6 d C-deprivation.


B , RT - qPCR analysis of ARF2 expression levels in 7 - d - old seedlings of the same genotypes as shown in A . Fold changes were calculated with respect to the expression level in the WT . data are means ± s . d . of 3 biological replicates .

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B, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of the same genotypes as shown in A. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.


C , Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C - deprivation . Color legends in C are the same as in B . data are means ± s . d . of 3 biological replicates .

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C, Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C-deprivation. Color legends in C are the same as in B. data are means ± s.d. of 3 biological replicates.


D , 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 6 d C - deprivation .

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D, 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 6 d C-deprivation.


E , RT - qPCR analysis of PIF5 expression levels in 7 - d - old seedlings of the same genotypes as shown in D . Fold changes were calculated with respect to the expression level in the WT . data are means ± s . d . of 3 biological replicates .

E, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of the same genotypes as shown in D. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.


F , Chlorophyll and total cellular protein content reduction in genotypes shown in D after 6 d C - deprivation . Color legends in F are the same as in E . data are means ± s . d . of 3 biological replicates .

F, Chlorophyll and total cellular protein content reduction in genotypes shown in D after 6 d C-deprivation. Color legends in F are the same as in E. data are means ± s.d. of 3 biological replicates.


G, Seedlings of WT, arf2-20, pif5-10, and arf2-20 pif5-10 before and after 12 d C-deprivation.


H , Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C - deprivation . data are means ± s . d . of 3 biological replicates .

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H, Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C-deprivation. data are means ± s.d. of 3 biological replicates.


I, Seedlings of WT, abs3-1D, arf2-20 abs3-1D, pif5-10 abs3-1D, and arf2-20 pif5-10 abs3-1D before and after 8 d C-deprivation.


J , Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C - deprivation . data are means ± s . d . of 3 biological replicates .

J, Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C-deprivation. data are means ± s.d. of 3 biological replicates.


C , 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 .

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C, 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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.


A , 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 .

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A, 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 .

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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 .

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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 .

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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 .

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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.


B , 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 .

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B, 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 .

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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 .

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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.


( 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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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.


( 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 ) .

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(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 .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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 ) .

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(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).


( 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 ) .

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(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 ) .

(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 .

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(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 .

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(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 .

(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.


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

(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 .

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(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 .

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(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 ) .

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(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).


A . 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 .

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A. 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 ) .

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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 .

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 .

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 ) .

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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).


B . Co - immunoprecipitation of GSDMD with full - length and deletion mutants of IQGAP1 tested in HEK293 cells .

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B. 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 .

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 .

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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 .

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 .

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F. Co-immunoprecipitation of pyroptosis mutants full-length (I105N FL) and N-terminal GSDMD defective of pyroptosis (I105N-NT) with IQGAP1.


A . 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 .

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A. 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.