top panel depicts experimental approach used to tether MS2 ‐ loop ‐ containing RNAs to lacO arrays via a GFP ‐ tagged LacI ‐ MS2 coat fusion . lacO arrays were stably integrated in genome of U2OS . In bottom panel , CLSM of U2OS are shown that were transfected with MS2 ‐ aluRNAR . They reveal localization of LacI ‐ GFP ‐ MS2 NCL ( immunofluorescence , red ) . arrow indicates lacO array , which is associated with a nucleolar domain .

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The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.


MS2 ‐ loop ‐ containing forward aluRNA or aluRNAL or aluRNAR were recruited to single stably integrated lacO array in U2OS F4IIB8 . propensity for nucleolar localization was evaluated as average number of lacO arrays with tethered detected in nucleoli ( ± 95 % CI ) is plotted in bar chart . Calculations are on of more than 100 . * * P ‐ value 0 . 01 , t ‐ , from of two independent biological replicates . As controls , transcripts of MS2 loops only , MS2 ‐ CDV3 , MS2 ‐ RepA , MS2 ‐ STARD7 MS2 ‐ CORO1C RNAs were used . inset shows a CLSM image with MS2 ‐ GFP ‐ LacI ( green ) NPM ( red , immunofluorescence ) after recruitment of MS2 ‐ aluRNA .

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MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.


Top : Scheme illustrating experimental approach used to tether GFP ‐ tagged LacI fusion via a GFP ‐ binding ( GBP ) to lacO arrays . lacO arrays are stably integrated in genome of U2OS . Bottom : CLSM showing localization of GBP ‐ LacI ‐ RFP , GFP ‐ NCL , GFP ‐ NPM or GFP ‐ TIP5 ‐ RBD fusion relative to aluRNA that was visualized by FISH . insets contain an enlarged image of one of lacO loci .

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Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.


CLSM showing GBP ‐ LacI ‐ RFP GFP ‐ NCL ( green ) recruited to lacO arrays as indicated by arrows . Immunofluorescence of endogenous NPM ( red ) marks nucleoli . inset shows decondensation of lacO arrays in nucleolus . graph at bottom depicts average number of lacO loci tethered to nucleoli ( ± 95 % CI ) by indicated ( n = 72 ) . NCL ‐ ∆ is an NCL deletion mutant lacking RBD GAR domains required for binding . * * P ‐ value 0 . 01 , t ‐ .

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CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.


CLSM of UBF ( immunofluorescence , green ) or rDNA ( FISH , green ) ( DAPI , blue ) after knockdown of NCL NPM by siRNA in HeLa .

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CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.


Western blots for detection of TCF7 , LEF1 , TCF7L1 , TCF7L2 β - catenin ( B " ) in wild - type ( WT ) knockout ( K . O . )

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Western blots for detection of TCF7, LEF1, TCF7L1, TCF7L2 and β-catenin (B") proteins in wild-type (WT) and knockout (K.O.)


TCF - reporter TOP - flash performed on indicated lines treated with 10 μM CHIR , for 24 hrs . control FOP - flash reporter is not activated upon CHIR administration . data represent mean ± s . e . m . of averages of nine independent experiments ( N = 9 ) . dBcat , β - catenin knockout ; d4TCF , TCF / LEF quadruple knockout Transfection of TCF - expressing plasmids could restore ability of d4TCF to respond to Wnt3 as measured in a TOP - flash

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TCF-reporter TOP-flash assay performed on the indicated cell lines treated with 10 μM CHIR, for 24 hrs. The control FOP-flash reporter is not activated upon CHIR administration. The data represent the mean ± s.e.m. of averages of nine independent experiments (N = 9). dBcat, β-catenin knockout cells; d4TCF, TCF/LEF quadruple knockout cells Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP-flash assa


Quantitative RT - PCR analyses of Axin2 transcripts , performed on different lines treated with 10 μM CHIR or DMSO ( control ) for 24 hrs . Error bars show that standard deviation obtained from 3 independent experiments Transfection of TCF - expressing plasmids could restore ability of d4TCF to respond t CHI as measured by Axin2 mRNA

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Quantitative RT-PCR analyses of Axin2 transcripts, performed on the different cell lines treated with 10 μM CHIR or DMSO (control) for 24 hrs. Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA abundance


a Heat map showing upregulated genes in wild - type ( WT ) parental HEK 293T upon CHIR treatment , ranked on log2 fold change . Increased decreased transcription is depicted in red blue , respectively . Gene names are indicated along y - axis . Canonical Wnt targets are among most upregulated genes in WT ( red boxes ) . genotypes of clones used is indicated on bottom of heat map . Triplicates of each are included . b Hierarchical clustering of differentially expressed genes with at least a 2 - fold change in any comparison . Dendrogram clustering indicates relationships between different experimental conditions . c Volcano plot displays differentially regulated genes in d4TCF ( C ) dBcat ( C " ) compared to WT parental . Red dots indicate significantly regulated genes on adjusted p - value log - fold - change ( logFC ) . d Smear plots for every indicating behaviour of every gene after CHIR treatment . Differential gene expression is indicated by log2 fold change ( logFC ) , average logCPM ( logarithm of per million reads ) was used to estimate accuracy of

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a Heat map showing the upregulated genes in wild-type (WT) parental HEK 293T cells upon CHIR treatment, ranked based on the log2 fold change. Increased and decreased transcription is depicted in red and blue, respectively. Gene names are indicated along the y-axis. Canonical Wnt targets are among the most upregulated genes in WT cells (red boxes). The genotypes of the cell clones used is indicated on the bottom of the heat map. Triplicates of each cell line are included. b Hierarchical clustering of differentially expressed genes with at least a 2-fold change in any comparison. Dendrogram clustering indicates the relationships between the different experimental conditions. c Volcano plot displays differentially regulated genes in d4TCF (C) and dBcat (C") compared to WT parental cells. Red dots indicate significantly regulated genes based on adjusted p-value and log-fold-change (logFC). d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment. Differential gene expression is indicated by the log2 fold change (logFC), and the average logCPM (logarithm of counts per million reads) was used to estimate the accuracy of the expression


e RT - qPCR - validation of three differentially expressed genes , upon CHIR stimulation in WT , dBcat or d4TCF lines . data represent mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - . Asterisks ( * ) indicate a p - value < 0 . 05 , ( * * ) a p - value <

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e RT-qPCR-based validation of three differentially expressed genes, upon CHIR stimulation in WT, dBcat or d4TCF cell lines. The data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<0.05, and (**) a p-value<


b , c RT - qPCR - validation of selected TCF - independent β - catenin - dependent target genes ( B ) upregulated or ( C ) downregulated in d4TCF ( TCF - independent ) treated with 10μM CHIR or DMSO for 24hr . Data represent mean ± s . e . m . of averages of three independent experiments ( N = 3 ) . Samples were compared using Student " s t - . Asterisks ( * ) indicate a p - value <

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b, c RT-qPCR-based validation of selected TCF-independent β-catenin-dependent target genes (B) upregulated or (C) downregulated in d4TCF cells (TCF-independent) treated with 10μM CHIR or DMSO for 24hr. Data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


( A , B ) β - catenin occupancy on regulatory regions of prototypical Wnt target genes , AXIN2 LEF1 ( A ) , at promoter regions of HOXC4 ZNF503 ( B )

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(A, B) β-catenin occupancy on regulatory regions of the prototypical Wnt target genes, AXIN2 and LEF1 (A), and at the promoter regions of HOXC4 and ZNF503 (B)


( C ) β - catenin ChIP - seq enrichment over ENCODE TCF7L2 ( also known as TCF4 ) ChIP peaks in HEK293T . data represent mean ± 95 % C . I . obtained from three independent experiments ( N = 3 )

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(C) β-catenin ChIP-seq enrichment over ENCODE TCF7L2 (also known as TCF4) ChIP peaks in HEK293T cells. The data represent the mean ± 95% C.I. obtained from three independent experiments (N = 3)


b RT - qPCR validation of β - catenin - GHOST response . A selection of 7 genes ( colour coded ) that are activated upon CHIR treatment in d4TCF , or in wild - type ( WT ) in combination with ICAT overexpression , but not in pentaKO - indicating that their regulation is dependent on β - catenin but can only occur in absence of TCF / LEF - β - catenin interaction . genotype treatment for each condition is indicated in x - axis . Error bars show that standard deviation obtained from 3 independent

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b RT-qPCR validation of the β-catenin-GHOST response. A selection of 7 genes (colour coded) that are activated upon CHIR treatment in d4TCF, or in wild-type (WT) in combination with ICAT overexpression, but not in pentaKO cells - indicating that their regulation is dependent on β-catenin but can only occur in the absence of TCF/LEF-β-catenin interaction. The genotype and the treatment for each condition is indicated in the x-axis. Error bars show that standard deviation obtained from 3 independent experiments


c FOXO4 but not FOXO3 overexpression upregulates β - catenin - GHOST target GADD45 both in CHIR - treated WT ( black bars ) or d4TCF ( grey bars ) . Samples were compared using Student " s t - . Asterisks ( * ) indicate a p - value <

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c FOXO4 but not FOXO3 overexpression upregulates the β-catenin-GHOST target GADD45 both in CHIR-treated WT (black bars) or d4TCF (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


d FOXO4 overexpression induced GADD45 transcription in WT ( black bars ) but not in dBcat ( grey bars ) . Samples were compared using Student " s t - . Asterisks ( * ) indicate a p - value <

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d FOXO4 overexpression induced GADD45 transcription in WT (black bars) but not in dBcat (grey bars) cells. Samples were compared using Student"s t-test. Asterisks (*) indicate a p-value<


e Endogenous FOXO4 , when immunoprecipitated ( IP ) pulls down β - catenin . Overexpressed FOXO4 increases amount of β - catenin detected in immunoprecipitated , indicating physical

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e Endogenous FOXO4, when immunoprecipitated (IP) pulls down β-catenin. Overexpressed FOXO4 increases the amount of β-catenin detected in the immunoprecipitated, indicating physical association


f GADD45 is upregulated by CHIR in d4TCF ; this positive transcriptional regulation is blocked by FOXO4 - specific siRNA but not control scrambled siRNA . experiments were done at least three times ( N = 3 ) . Samples were compared using Student " s t - . Asterisks ( * * ) indicate a p - value < 0 . 02 ; ns , non - statistically significant change was

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f GADD45 is upregulated by CHIR in d4TCF cell; this positive transcriptional regulation is blocked by FOXO4-specific siRNA but not control scrambled siRNA. All experiments were done at least three times (N=3). Samples were compared using Student"s t-test. Asterisks (**) indicate a p-value<0.02; ns, non-statistically significant change was obs


A ) U2OS were harvested at indicated time points after treatment with ionizing radiation ( 15 Gy ) followed by by SDS - PAGE immunoblotting with antibodies recognizing PALB2 Vinculin . ( NT , no treatment ) . lysate from 24 hours time point was treated with phosphatase ( Ppase ) .

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A) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase).


B ) U2OS were left untreated or treated with ATM ( KU 55933 , 10 μM ) , ATR ( ETP - 464 , 1 μM ) , or - PK ( NU7441 , 1 μM ) inhibitors 30 minutes prior to exposure to IR ( 15 Gy , 2h recovery ) . Immunoblots were performed with indicated antibodies .

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B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.


C ) U2OS were left untreated ( NT ) or exposed to HU ( 2 mM ) for indicated times . lysate from 24 hour time point was treated with phosphatase ( Ppase ) . Immunoblots were performed with indicated antibodies .

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C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.


D ) Untreated U2OS or exposed to either HU ( 2 mM , 24h ) or IR ( 15 Gy , 2h recovery ) alone or together with ATM inhibitor ( KU55933 , 10 μM ) or inhibitors to ATR ( ATR # 1 : ETP - 464 , 1 μM ; ATR # 2 : AZ - 20 , 3 μM ) were harvested lysates were prepared . inhibitors were added 30 minutes prior to HU or IR treatment . Immunoblots were performed with indicated antibodies .

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D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.


E ) ATR kinase was performed with N - terminal PALB2 ( aa 1 - 560 ; GST - PALB2 - N ) as a substrate HA - tagged ATR as kinase visualized by Fujifilm Phosphoimager . 293T transfected with empty vector ( EV ) , GST , ATR inhibition ( AZ - 20 , 30 μM ) are controls . HA - ATR GST - PALB2 - N was controlled by immunoblotting with HA GST antibodies .

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E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.


B ) Characterization of Doxycycline - inducible lines expressing siRNA resistant wild type ( WT ) or mutant ( TMA or TMD ) FLAG / HA - tagged PALB2 . lines were transfected with UNC ( Negative Control ) or PALB2 siRNA for 24 hours prior to induction with Doxycycline . Immunoblots were performed with indicated antibodies .

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B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.


C ) characterization of FLAG / HA - tagged PALB2 localization 2 hours following exposure to 15 Gy of IR . Z - stack max image projection background subtraction was done using ImageJ / Fiji . Scale bar = 5μm .

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C) Cell line characterization of FLAG/HA-tagged PALB2 localization 2 hours following exposure to 15 Gy of IR. Z-stack max intensity image projection and background subtraction was done using ImageJ/Fiji. Scale bar = 5μm.


D ) Kinase was performed as described in Figure 1E except that SDS - PAGE gel was Coomassie stained following electrophoresis . WT was used at two different concentrations ( 1x 0 . 75x ) .

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D) Kinase assay was performed as described in Figure 1E except that the SDS-PAGE gel was Coomassie stained following electrophoresis. WT was used at two different concentrations (1x and 0.75x).


E ) Doxycycline - inducible stable U2OS lines were left untreated or treated with Doxycycline / or IR ( 15 Gy , 2h recovery ) . Following extraction lysates were immunoprecipitated ( IP ) with IgG or antibody recognizing phosphorylated S / Q sites ( pS / Q ) analyzed by SDS - PAGE . Immunoblots were performed with indicated antibodies .

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E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.


A ) PALB2 lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR fixed for IF 2 hours later . Z - stack were acquired with a 60X oil objective ( Deltavision ) . Z stack max projection , background subtraction , foci was done using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - ) . Scale bar = 5μm .

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A) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar = 5μm.


B ) PALB2 lines were transfected exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU . 24 hours later were fixed processed for IF as in Figure 3A . in S - phase ( EdU + ) at time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide RAD51 foci in EdU positive were enumerated using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - ) . Scale bar = 5μm .

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B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm.


C ) HeLa DR - GFP was used to analyze homologous recombination activity . were transfected with PALB2 siRNA followed by transfection of I - SceI together with empty vector ( EV ) , WT - , TMA - , or TMD - PALB2 next day . 72 hours later were fixed analyzed by FACS . of with GFP signal from empty vector sample was set to one ( dotted ) . graph shows a representative image from three biological repeats ( error bars = ) .

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C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM).


D ) Phosphorylation of PALB2 supports RAD51 foci . were transfected with siPALB2 overnight followed by forward transfection of siFBH1 / siBLM for 6 hours then induced for expression of PALB2 ( WT TMA ) . 24 hours following induction were pulsed with EdU exposed to HU as in Figure 3B . in S - phase ( EdU + ) at time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide RAD51 foci in EdU positive were enumerated using ImageJ / Fiji ( * p < 0 . 0001 , unpaired Student ' s t - ) . Representative of displaying RAD51 ( green ) HA - PALB2 ( red ) localization in HU treated EdU positive ( not shown ) . Scale bar = 5μm .

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D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.


A ) U2OS PALB2 lines were transfected with UNC ( Negative Control ) or PALB2 siRNA 24 hours later Doxycycline was added to PALB2 lines . Left panel , were treated following day with IR ( 5 Gy ) 2 hours later Nocodazole was added for 6 hours , or treated with only Nocodazole for 6 hours . Right panel , were treated with IR ( 5 Gy ) left to recover for 16 hours . were fixed stained with pMPM2 antibody to detect mitotic by flow cytometry . percentages of mitotic in IR + Nocodazole samples were normalized to Nocodazole samples ( left panel , n = 3 , error bars = ) . For samples with 16 hour recovery after IR of mitotic in WT - PALB2 expressing was set to one ( right panel ) . ( n = 3 , * * p < 0 . 005 , * p < 0 . 05 , unpaired Student ' s t - , error bars = ) .

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A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM).


B ) relative of phosphorylated H2AX ( pS139 ) was examined in total population of WT - TMA - PALB2 expressing lines 18 hours post exposure to IR ( 5 Gy ) . lines were prepared as in above , fixed at indicated time point , stained for pH2AX . were imaged with a 20X air objective on a Scan ^ R workstation ( Olympus ) , mean relative pH2AX was calculated from background subtracted using Scan ^ R software . ( * p < 0 . 0001 , unpaired Student ' s t - ) .

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B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student's t-test).


C ) Number of 53BP1 in TMA - PALB2 expressing G1 daughter . lines were prepared as above left untreated ( NT ) or exposed to IR ( 3 Gy ) , 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis . irradiated bi - nucleated were scored for 53BP1 bodies . Scale bar = 10μm .

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C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.


Nurr1 ( N ) Foxa2 ( F ) synergistically downregulate SGK1 expression in cultured glia . ( A ) Up - downregulated genes in microarray data for cultured glia transduced with N + F ( vs . mock - transduced control ) . Sgk1 is marked with an arrow in top downregulated genes .

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Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N+F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes.


Effect of N F on SGK1 expression in glial shown in microarray ( B ) , - seq ( C ) , Data in ( B C ) represent values of fold change ( FC ) or read ( RC ) with color intensities . n = 3 . One - way ANOVA . Data are represented as mean ± . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

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Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n=3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Effect of N F on SGK1 expression in glial shown in RT - PCR analyses . One - way ANOVA . Data are represented as mean ± . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * .

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Effect of N and F on SGK1 expression in glial cells shown in the RT-PCR analyses. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation

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Downregulation of SGK1 is responsible for N+F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation


Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB

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WB exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation ( E ) thus blocking release of NFκB from NFκB - IκB inhibitory ( F ) .

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WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F).


N + F - mediated downregulation of NFκB signaling ( phosphorylation of p65 , p - p65 , G ) were alleviated by forced SGK1 expression in VM - glial cultures .

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N+F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) were alleviated by forced SGK1 expression in the VM-glial cultures.


H ) N + F - mediated downregulation of pro - inflammatory cytokine expression ( IL - 1β , TNFα , H ) were alleviated by forced SGK1 expression in VM - glial cultures .

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H) N+F-mediated downregulation of pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures.


( I ) SGK1 inhibition blocks glial NFκB signaling . Data are represented as mean ± . n = 4 - 6 . One - way ANOVA . Significantly different at p = 0 . 002 * , 0 . 00004 * * , 0 . 00001 # , 3 . 01E - 10 # # , 3 . 78E - 06 + , 1 . 59E - 07 + + in graph I .

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(I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n=4-6. One-way ANOVA. Significantly different at p=0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I.


Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition

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Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition


Glial pro - inflammatory cytokine expression regulated by SGK1

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Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition ( J - M ) overexpression ( N ) .

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Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J-M) and overexpression (N).


In - seq data ( GSE145490 , normalized read ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in VM - glia treated with SGK1 inhibitor GSK - 650394 vs vehicle ( DMSO ) - treated control were analyzed . A B , Top gene ontologies ( GOs ) KEGG pathways of genes downregulated in VM - glia by SGK1 inhibitor treatment . purple bar indicates number of genes under designated GO term / KEGG pathway . red bar indicates p - value , negative log of p - value ( bottom ) is plotted on X - axis .

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In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed. A and B, Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis.


In - seq data ( GSE145490 , normalized read ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in VM - glia treated with SGK1 inhibitor GSK - 650394 vs vehicle ( DMSO ) - treated control were analyzed .

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In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed.


C , Gene set enrichment ( GESA ) for inflammatory immune responses .

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C, Gene set enrichment analysis (GESA) for inflammatory and immune responses.


D , Scatterplots for differentially expressed genes ( DEGs ) highlighting inflammatory / immune genes that are most significantly downregulated in inhibiter - treated cultures .

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D, Scatterplots for the differentially expressed genes (DEGs) highlighting the inflammatory/immune genes that are most significantly downregulated in the inhibiter-treated cultures.


E , for selected inflammatory genes in SGK1 inhibitor - treated - untreated glial cultures . Data represent RC values ( inside box ) log2 [ SGK1 inhibitor - treated / control ] ( color ) .

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E, Heatmap for selected inflammatory genes in the SGK1 inhibitor-treated and -untreated glial cultures. Data represent the RC values (inside box) and log2[SGK1 inhibitor-treated/control] (color intensity).


F , Microglia astrocyte reactivation - specific marker expressions in - seq data .

F, Microglia and astrocyte reactivation-specific marker expressions in the RNA-seq data.


mRNA expression of NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , Caspase1 . Decreased expression of components shown in - seq Data are expressed as mean ± . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + +

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mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the RNA-seq Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++


B , mRNA expression of NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , Caspase1 . Decreased expression of components shown in q - PCR analyses in other independent VM - glial cultures treated with inhibitor ( GSK - 650394 ) sh - Sgk1 si - Sgk1 ( vs . DMSO , sh - control , si - control , respectively ) ( B ) . n = 3 ; Student ' s t - . Data are expressed as mean ± . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + + in graph B .

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B, mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the q-PCR analyses in other independent VM-glial cultures treated with the inhibitor (GSK-650394) and sh-Sgk1 and si-Sgk1 (vs. DMSO, sh-control, and si-control, respectively) (B). n=3; Student's t-test. Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++ in graph B.


C , Immunoblot to assess NLRP3 inflammasome activation . To activate inflammasome pathway , VM - derived glial were treated with LPS ( 0 . 25 µg / ml , 3h ) then ATP ( 2 . 5 mM , 30 min ) in presence or absence of SGK1 inhibitor GSK - 650394 . Two days later , intracellular released of pro - activated caspase - 1 IL - 1β were determined in culture media lysates , respectively .

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C, Immunoblot analysis to assess NLRP3 inflammasome activation. To activate the inflammasome pathway, VM-derived glial cells were treated with LPS (0.25 µg/ml, 3h) and then ATP (2.5 mM, 30 min) in the presence or absence of the SGK1 inhibitor GSK-650394. Two days later, intracellular and released levels of pro- and activated caspase-1 and IL-1β proteins were determined in the culture media and cell lysates, respectively.


D , Released level of IL - 1β ( activated ) was further quantified using ELISA . n = 3 ; ANOVA . Significantly different at p = 0 . 0002 # , 0 . 0012 * , 0 . 0004 * * in graph D .

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D, Released level of IL-1β (activated) was further quantified using ELISA. n=3; ANOVA. Significantly different at p=0.0002#, 0.0012*, 0.0004** in graph D.


Expression of CGAS - STING pathway genes in - seq data . graph represents log2 RC ratios of [ GSK - 650394 - treated / DMSO - treated control ] .

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Expression of CGAS-STING pathway genes in the RNA-seq data. The graph represents log2 RC ratios of [GSK-650394-treated/DMSO-treated control].


G , WB demonstrating suppression of CGAS - STING signaling by SGK1 inhibitor in VM - glial cultures . Representative blots are shown on left . of active forms of CGAS - STING signaling molecules are quantified from 5 - 10 independent blots ( right ) . Data are represented as mean ± . One - way ANOVA . Significantly different at p = 0 . 001 # , 0 . 014 * ( CGAS ) , 0 . 00004 # , 0 . 0002 * ( STING ) , 0 . 017 # , 0 . 037 * ( pTBK1 ) , 0 . 009 # , 0 . 045 * ( pIRF3 ) , 0 . 012 # , 0 . 036 * ( pP65 ) in graph G .

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

G, WB analysis demonstrating suppression of CGAS-STING signaling by the SGK1 inhibitor in VM-glial cultures. Representative blots are shown on the left. The levels of the active forms of CGAS-STING signaling molecules are quantified from 5-10 independent blots (right). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.001#, 0.014* (CGAS), 0.00004#, 0.0002* (STING), 0.017#, 0.037* (pTBK1), 0.009#, 0.045* (pIRF3), 0.012#, 0.036* (pP65) in graph G.


H , Expression of Ifnb mRNA , a final product of CGAS - STING pathway , determined by real - time PCR analyses . Data are represented as mean ± . n = 3 ; student ' s t - . Significantly different at p = 0 . 0459 # , 0 . 0305 * in graph H .

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

H, Expression of Ifnb mRNA, a final product of the CGAS-STING pathway, determined by real-time PCR analyses. Data are represented as mean ± SEM. n=3; student's t-test. Significantly different at p=0.0459#, 0.0305* in graph H.


Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with SGK1 inhibitors in - seq data Data are represented as mean ± . n = 3 ; Student ' s t - . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B .

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

Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the RNA-seq data Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.


B , Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with SGK1 inhibitors in q - PCR Data are represented as mean ± . n = 3 ; Student ' s t - . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B .

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

B, Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the q-PCR analysis Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.


C , Immunoblot for PGC1α expression . Data are represented as mean ± . n = 3 . Student ' s t - . Significantly different at p = 0 . 0077 * in graph C .

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

C, Immunoblot for PGC1α protein expression. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0077* in graph C.


D , Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM - glia . Green indicates newly synthesized mitochondria , red indicates old or damaged mitochondria . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± . One - way ANOVA . Significantly different at p = 3 . 78E - 08 * , 0 . 00003 * * in graph D .

O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM-glia. Green fluorescence indicates newly synthesized mitochondria, and red fluorescence indicates old or damaged mitochondria. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=3.78E-08*, 0.00003** in graph D.


E , Mitochondrial ROS estimated using MitoSox red probe . was measured using ImageJ , ROS are presented as mean ( MFI ) . Mitochondrial damage was induced by treatment with mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± . One - way ANOVA . Significantly different at p = 0 . 0013 * , 0 . 0273 # in graph E .

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

E, Mitochondrial ROS levels estimated using the MitoSox red probe. Fluorescence intensity was measured using ImageJ, and ROS levels are presented as mean fluorescent intensity (MFI). Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0013*, 0.0273# in graph E.


F , Mitochondrial potential ( JC - 1 ) . red ( JC - 1 Aggregate ) indicates disrupted mitochondrial potential , green ( JC - 1 Monomer , insets ) indicates intact potential . inset were taken from same microscopic fields of respective JC - 1 aggregate . Mitochondrial damage was induced by treatment with mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± . One - way ANOVA . Significantly different at p = 0 . 0006 * , 0 . 0093 # in graph F .

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

F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F.


G , Intracellular of ATP , an indicator of mitochondrial oxidative phosphorylation . Data are expressed as mean ± . n = 3 cultures ; one - way ANOVA . Significantly different at p = 0 . 0009 * , 0 . 00004 * * , 0 . 04913 # , 0 . 0024 + in graph G .

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

G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G.


Glial senescence rescued by SGK1 inhibition . ( H ) Expression of genes associated with senescence in - seq data ( VM - glia with vs . without GSK - 650394 treatment ) .

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

Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment).


( I ) Intracellular ROS estimated by DCF - DA . Data are represented as mean ± . n = 3 ; one - way ANOVA . Significantly different at p = 0 . 0074 * , 0 . 012 # in graph I .

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

(I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I.


( J ) Senescence - associated β - galactosidase ( SA - β - gal ) activity . Data are represented as mean ± . n = 3 ; one - way ANOVA . Significantly different at p = 2 . 05E - 06 * , 0 . 0042 # , 0 . 0006 + in graph J .

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

(J) Senescence-associated β-galactosidase (SA-β-gal) activity. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=2.05E-06*, 0.0042#, 0.0006+ in graph J.


( K ) Immunoblots for associated with senescence . Significantly different at p = 0 . 008 * , 0 . 001 # ( TNFA ) , 0 . 01 * , 0 . 009 # ( HMGB1 ) , 7 . 32E - 10 * , 1 . 23E - 8 # ( IL6 ) , 9 . 62E - 11 * , 1 . 01E - 5 # ( MMP3 ) , 0 . 001 * , 5 . 72E - 4 # ( LAMIN B ) in graph K .

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

(K) Immunoblots for proteins associated with cell senescence. Significantly different at p=0.008*, 0.001# (TNFA), 0.01*, 0.009# (HMGB1), 7.32E-10*, 1.23E-8# (IL6), 9.62E-11*, 1.01E-5# (MMP3), 0.001*, 5.72E-4# (LAMIN B) in graph K.


( L ) IL - 6 secreted from cultured VM - glia . cytokine were estimated using ELISA of media from VM - glia with without SGK1 inhibitor . Data are represented as mean ± . n = 3 cultures . One - way ANOVA . Significantly different at p = 0 . 0028 * , 0 . 0062 # in graph L .

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

(L) IL-6 levels secreted from cultured VM-glia. The cytokine levels were estimated using ELISA of the media from the VM-glia with and without the SGK1 inhibitor. Data are represented as mean ± SEM. n=3 cultures. One-way ANOVA. Significantly different at p=0.0028*, 0.0062# in graph L.


( M ) Glial viability following treatment with senolytic compounds ( azithromycin , fisetin ) . Data are represented as mean ± . n = 3 ; Student ' s t - . Significantly different at p = 0 . 0073 * , 0 . 0018 * * , 0 . 0286 # , 0 . 0269 # # , 0 . 0045 # # # in graph M .

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

(M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M.


B , Western blot for detecting SNCA oligomers . Data are represented as mean ± . n = 4 ; one - way ANOVA . Significantly different at p = 2 . 5E - 07 * , 1 . 65E - 07 * * , 0 . 022 # , 0 . 029 # # , 0 . 046 + , 0 . 015 + + , 0 . 0136 & , 0 . 0016 & & in graph B .

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

B, Western blot analysis for detecting SNCA oligomers. Data are represented as mean ± SEM. n=4; one-way ANOVA. Significantly different at p=2.5E-07*, 1.65E-07**, 0.022#, 0.029##, 0.046+, 0.015++, 0.0136&, 0.0016&& in graph B.


C , Immunocytochemical detection of p - 129 - SNCA , associated with SNCA aggregation . Data are represented as mean ± . n = 6 . Significantly different at p = 0 . 0363 * in graph C .

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

C, Immunocytochemical detection of p-129-SNCA, associated with SNCA aggregation. Data are represented as mean ± SEM. n=6. Significantly different at p=0.0363* in graph C.


E , Bi - FC - assessment of inter - neuronal propagation aggregation of SNCA . Bi - FC + inclusions ( ) were counted in E . Data are represented as mean ± . n = 5 . Significantly different at p = 0 . 0446 * in graph E .

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

E, Bi-FC-based assessment of the inter-neuronal propagation and aggregation of SNCA. Bi-FC+ inclusions (puncta) were counted in E. Data are represented as mean ± SEM. n=5. Significantly different at p=0.0446* in graph E.


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by WB

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

VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation were assessed by WB


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by

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


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation p - 129 - SNCA immunocytochemical analyses . Data are represented as mean ± . n = 3 . Significantly different at p = 0 . 00003 * , 0 . 0003 * * , 0 . 0012 * * * , 0 . 00004 * * * * , 0 . 0321 # in graph G p = 0 . 0052 * in graph H .

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

VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation and p-129-SNCA immunocytochemical analyses. Data are represented as mean ± SEM. n=3. Significantly different at p=0.00003*, 0.0003**, 0.0012***, 0.00004****, 0.0321# in graph G and p=0.0052* in graph H.


Glia derived from mouse VM were transduced with sh - Sgk1 ( B ) or SGK1 ( CMV - Sgk1 , C ) , co - cultured with VM - derived mDA neurons . Seven days after co - culture , were exposed to H2O2 ( 500µM , 3hr ) or not ( - ) , then mDA neuron viability ( TH + ) neurite degeneration ( TH + fiber lengths ) were estimated . Data are represented as mean ± ; n = 4 culture coverslips . Student ' s t - . Significantly different at p = 0 . 033 * , 0 . 0412 * * , 7 . 44E - 23 # , 1 . 39E - 23 # # in graph B p = 0 . 0489 * , 0 . 0366 * * , 0 . 0078 # # in graph C .

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

Glia derived from mouse VM were transduced with sh-Sgk1 (B) or SGK1 (CMV-Sgk1, C), and co-cultured with VM-derived mDA neurons. Seven days after co-culture, cells were exposed to H2O2 (500µM, 3hr) or not (-), and then mDA neuron viability (TH+ cell counts) and neurite degeneration (TH+ fiber lengths) were estimated. Data are represented as mean ± SEM; n=4 culture coverslips. Student's t-test. Significantly different at p=0.033*, 0.0412**, 7.44E-23#,1.39E-23## in graph B and p=0.0489*, 0.0366**, 0.0078## in graph C.


Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner . VM - glia transduced with sh - Sgk1 ( or sh - cont as a control ) were challenged with H2O2 + LPS for 3hr or not . Glial conditioned medium ( GCM ) was prepared in each glial culture condition administered to mDA neurons primarily cultured from mouse VM ( E ) viability of neuronal was estimated using CCK8 assays TuJ1 + . Data are represented as mean ± . n = 3 cultures ; One - way ANOVA . Significantly different at p = 0 . 0002 * , 3 . 29E - 08 * * , 0 . 0003 * * * , 3 . 66E - 06 # , 0 . 0029 # # ( TuJ1 + counting ) , 0 . 0127 + , 5 . 98E - 06 + + , 0 . 0002 + + + , 0 . 0005 & , 0 . 0078 & & ( CCK8 assays ) in graph E .

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

Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (E) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1+ cell counts. Data are represented as mean ± SEM. n=3 cultures; One-way ANOVA. Significantly different at p=0.0002*, 3.29E-08**, 0.0003***, 3.66E-06#, 0.0029## (TuJ1+ cell counting), 0.0127+, 5.98E-06++, 0.0002+++, 0.0005&, 0.0078&& (CCK8 assays) in graph E.


F , Effect of SGK1 inhibitor treatment in mixed mDA neuron + VM - glia co - cultures . Data are represented as mean ± . n = 3 . Student ' s t - . Significantly different at p = 0 . 0068 * , 0 . 0368 * * , 0 . 0249 # , 1 . 48E - 27 # # in graph F .

O O O O B-GENEPROD O O O O O O O B-TISSUE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Effect of the SGK1 inhibitor treatment in the mixed mDA neuron + VM-glia co-cultures. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0068*, 0.0368**, 0.0249#, 1.48E-27## in graph F.


( B - D ) Behaviors of MPTP - PD mice assessed by pole ( B ) , beam ( C ) at 3 - 6 post - MPTP injection , locomotor activity ( total & average speed , D ) at 6 post - MPTP injection . n = 20 GSK - 650394 - treated MPTP - PD mice , 14 vehicle - treated MPTP - PD mice , 8 MPTP - untreated non - PD mice . Data are represented as mean ± . Significantly different at p = 0 . 0012 # , 0 . 0001 * , 3 . 09E - 08 # # , 8 . 21E - 08 * * in graph B p = 0 . 0009 # , 0 . 0035 * , 1 . 03E - 06 # # , 0 . 00003 * * in graph C p = 0 . 0001 * , 0 . 0284 * * , 0 . 0101 # ( total ) , 1 . 49E - 08 * , 0 . 0005 * * , 0 . 0001 # ( average speed ) in graph D .

O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-DISEASE B-ORGANISM O O O O O B-SMALL_MOLECULE O B-DISEASE B-ORGANISM O O B-SMALL_MOLECULE O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Behaviors of MPTP-PD mice assessed by pole (B), beam (C) at 3-6 weeks post-MPTP injection, and locomotor activity (total distance & average speed, D) tests at 6 weeks post-MPTP injection. n= 20 GSK-650394-treated MPTP-PD mice, 14 vehicle-treated MPTP-PD mice, and 8 MPTP-untreated non-PD mice. Data are represented as mean ± SEM. Significantly different at p=0.0012#, 0.0001*, 3.09E-08##, 8.21E-08** in graph B and p=0.0009#, 0.0035*, 1.03E-06##, 0.00003** in graph C and p=0.0001*, 0.0284**, 0.0101# (total distance), 1.49E-08*, 0.0005**, 0.0001# (average speed) in graph D.


Histologic assessment of number of TH + mDA neurons ( E , F ) , neurite lengths ( E , G ) , sizes ( E , H ) Shown are representative for TH + DA neurons in SN TH + DA neurons in boxed areas of ( E ) are enlarged in right .

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

Histologic assessment of the number of TH+ mDA neurons (E, F), neurite lengths (E, G), and cell body sizes (E, H) Shown are the representative images for TH+ DA neurons in the SN TH+ DA neurons in boxed areas of (E) are enlarged in right.


Histologic assessment of number of TH + mDA neurons in SN TH + fiber intensities in striatum Shown are representative for TH + DA neurons in TH - immunoreactivity in striatum ,

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

Histologic assessment of the number of TH+ mDA neurons in the SN and TH+ fiber intensities in the striatum Shown are the representative images for TH+ DA neurons in the TH-immunoreactivity in the striatum,


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . Behaviors ( L - N ) of SNCA - PD mice were assessed at 6 - 12 post - SNCA injection Significantly different at p = 0 . 008 # , 0 . 021 * , 0 . 00001 # # , 0 . 0009 * * in graph L p = 0 . 00002 # , 0 . 0344 * , 0 . 0005 # # , 0 . 0067 * * in graph M p = 0 . 0057 # , 0 . 0345 * ( total ) , 0 . 0001 # , 0 . 0375 # # , 0 . 0338 * ( average speed ) in graph N .

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

Therapeutic effects of GSK-650394 in SNCA-PD model mice. Behaviors (L-N) of the SNCA-PD mice were assessed at 6-12 weeks post- SNCA injection Significantly different at p=0.008#, 0.021*, 0.00001##, 0.0009** in graph L and p=0.00002#, 0.0344*, 0.0005##, 0.0067** in graph M and p=0.0057#, 0.0345* (total distance), 0.0001#, 0.0375##, 0.0338* (average speed) in graph N.


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in SN of SNCA - PD mice were assessed at 12 post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , 8 SNCA - untreated non - PD mice .

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

Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN of the SNCA-PD mice were assessed at 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in SN ( O - U ) of SNCA - PD mice were assessed 12 post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , 8 SNCA - untreated non - PD mice .

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

Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.


A B , Efficiency of AAV9 - mediated gene delivery . AA9 expressing reporter GFP gene were stereotaxically injected into SN of mice , GFP expression in astrocytes , microglia , neuronal was quantified . Data are represented as mean ± . n = 3 mice ( > 5 randomly selected microscopic fields per were counted ) . Significantly different at p = 0 . 006 * , 5 . 1E - 09 # , 5 . 36E - 09 + in graph B .

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

A and B, Efficiency of AAV9-mediated gene delivery. AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice, and GFP expression in astrocytes, microglia, and neuronal cells was quantified. Data are represented as mean ± SEM. n=3 mice (>5 randomly selected microscopic fields per animal were counted). Significantly different at p=0.006*, 5.1E-09#, 5.36E-09+ in graph B.


SGK1 knockdown effect in vivo by treatment with shSgk1 - AAV9 . Total SGK1 mRNA were estimated in midbrain SN of PD mice injected with shSgk1 ( shControl as control ) AAV9 using real time - PCR ( D ) .

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

SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1 mRNA levels were estimated in the midbrain SN of the PD mice injected with shSgk1(shControl as control) AAV9 using real time-PCR (D).


SGK1 knockdown effect in vivo by treatment with shSgk1 - AAV9 . Total

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

SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1


type - specific SGK1 knockdown was estimated by counting SGK1 - immuonoreactivity in neuron ( MAP2 + ) , astrocyte ( GFAP + ) , microglia ( Iba1 + ) populations ( E ) .

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

Cell type-specific SGK1 knockdown was estimated by counting SGK1-immuonoreactivity in neuron (MAP2+), astrocyte (GFAP+), and microglia (Iba1+) populations (E).


Data are represented as mean ± . n = 4 mice in each group . Significantly different at p = 0 . 0004 # in graph D p = 0 . 049 * , 0 . 007 * * , 0 . 002 * * * in graph E . ( F - H ) Behavior of PD mice assessed by pole ( F ) , beam ( G ) at 6 - 12 , locomotor activity ( H ) at 12 post - SNCA injection . Significantly different at p = 0 . 0003 # , 0 . 0428 * , 7 . 46E - 06 # # , 0 . 0012 * * in graph F p = 8 . 04E - 08 # , 0 . 004 * , 1 . 62E - 09 # # , 3 . 3E - 06 * * in graph G p = 0 . 0002 # , 0 . 0194 * ( total ) , 9 . 54E - 06 # , 0 . 0047 * ( average speed ) in graph H .

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

Data are represented as mean ± SEM. n=4 mice in each group. Significantly different at p=0.0004# in graph D and p=0.049*, 0.007**, 0.002*** in graph E. (F-H) Behavior of the PD mice assessed by pole (F), beam (G) at 6-12 weeks, and locomotor activity (H) tests at 12 weeks post- SNCA injection. Significantly different at p=0.0003#, 0.0428*, 7.46E-06##, 0.0012** in graph F and p=8.04E-08#, 0.004*, 1.62E-09##, 3.3E-06** in graph G and p=0.0002#, 0.0194* (total distance), 9.54E-06#, 0.0047* (average speed) in graph H.


Histologic assessment of p129 - SNCA immunoreactivity ( I , J ) , TH + mDA neuron ( I , K ) , neurite lengths ( L ) , sizes ( M ) in SN Significantly different at p = 0 . 0001 * in graph J , p = 2 . 35E - 25 # , 1 . 09E - 16 # # , 4 . 36E - 08 * in graph K p = 1 . 5E - 40 # , 1 . 08E - 24 # # , 2 . 32E - 07 * in graph L p = 9 . 07E - 20 # , 2 . 59E - 16 # # , 0 . 05 * in graph M p = 4 . 62E - 22 # , 5 . 11E - 12 # # , 3 . 49E - 06 * ( TH ) , n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , 8 SNCA - untreated mice . Data are represented as mean ± . One - way ANOVA . Scale bar , 100µm .

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

Histologic assessment of p129-SNCA immunoreactivity ( I,J), TH+ mDA neuron counts (I,K), neurite lengths (L), and cell body sizes (M) in the SN Significantly different at p=0.0001* in graph J, p=2.35E-25#, 1.09E-16##, 4.36E-08* in graph K and p=1.5E-40#, 1.08E-24##, 2.32E-07* in graph L and p=9.07E-20#, 2.59E-16##, 0.05* in graph M and p=4.62E-22#, 5.11E-12##, 3.49E-06* (TH), n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.


Histologic assessment of TH + DA transporter ( DAT ) fiber intensities in striatum n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , 8 SNCA - untreated mice . Data are represented as mean ± . One - way ANOVA . Scale bar , 100µm .

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

Histologic assessment of TH+ and DA transporter (DAT) fiber intensities in the striatum n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.


A - C , Quantitative morphometric analyses of microglia in SN of SNCA - PD mice . Shown in A are representative views of Iba1 + microglia neighboring TH + mDA neurons in SNs of PD mice treated with GSK - 650394 vehicle . Insets , enlarged views for boxed areas . Data are represented as mean ± . n = 3 mice per group . Significantly different at p = 1 . 49E - 11 * in graph B p = 0 . 000003 * in graph C .

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

A-C, Quantitative and morphometric analyses of microglia in the SN area of SNCA-PD mice. Shown in A are representative views of Iba1+ microglia neighboring TH+ mDA neurons in the SNs of PD mice treated with GSK-650394 and vehicle. Insets, enlarged views for the boxed areas. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=1.49E-11* in graph B and p=0.000003* in graph C.


D E , M1 marker CD16 / 32 expression in Iba1 + microglia . Data are represented as mean ± . n = 3 mice per group . Significantly different at p = 0 . 0016 * in graph E .

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

D and E, The M1 marker CD16/32 expression in Iba1+ microglia. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=0.0016* in graph E.


F G , mRNA expression of NLRP3 - inflammasome components pro - inflammatory cytokines in MPTP ( F ) SNCA ( G ) PD mice injected with SGK1 inhibitor or vehicle ( control ) . Each bar represents mean ± of 3 PCR values from each , n = 3 mice per group . Significantly different at p = 0 . 0372 * , 0 . 0173 * * , 0 . 0003 * * * in graph F p = 0 . 002 # , 0 . 0113 # # , 0 . 0053 # # # , 0 . 0001 # # # # in graph G .

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

F and G, mRNA expression of NLRP3-inflammasome components and pro-inflammatory cytokines in MPTP (F) and SNCA (G) PD mice injected with the SGK1 inhibitor or vehicle (control). Each bar represents the mean ± SEM of 3 PCR values from each animal, n=3 mice per group. Significantly different at p=0.0372*, 0.0173**, 0.0003*** in graph F and p=0.002#, 0.0113##, 0.0053###, 0.0001#### in graph G.


H - J , ASC expression in SNs SN microglia of SNCA - PD mice . n = 5 mice per group . Data are represented as mean ± . Significantly different at p = 1 . 91E - 09 * in graph I p = 0 . 0079 * in graph J .

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

H-J, ASC expression in the SNs and SN microglia of SNCA-PD mice. n=5 mice per group. Data are represented as mean ± SEM. Significantly different at p=1.91E-09* in graph I and p=0.0079* in graph J.


K L , β - gal - stained senescence in SNCA - PD mouse SN . Data are represented as mean ± . n = 5 mice per group . Significantly different at p = 0 . 0001 *

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

K and L, β-gal-stained cell senescence in SNCA-PD mouse SN. Data are represented as mean ± SEM. n=5 mice per group. Significantly different at p=0.0001*


( a ) Mitochondrial tubules in HeLa ( top , TMRE stained ) exceeded dimensions of that for autophagosomes ( labelled by EGFP - LC3B ) under either normal ( middle , same field as TMRE image ) or starvation conditions ( right ) .

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

(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right).


( c ) Illuminating a selective region ( dotted circle , top ) within a KR - dMito / EBFP2 - parkin - expressing HeLa led to immediate loss of KR ( top two panels ) . Third to fifth row : magnified view of photo - impaired region , EBFP2 - parkin translocated onto impaired mitochondrial tubules synchronously ( 38 - 60 min after 559 illumination , arrows ) . Scale bars ( panels ) , 10 μm .

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

(c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38-60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.


( a ) A HeLa expressing KR - dMito , EGFP - LC3B EBFP2 - parkin ( cyan ) were 559 nm illuminated ( dotted circle region ; immediate loss of KR ) . ( b - d ) Magnified view of photo - inactivated region in a 34 - 93 min after 559 nm illumination . ( b ) Induction of parkin - mediated mitophagy on a single , long mitochondria tubule ( arrows ) . ( c ) LC3B - coated structures were recruited onto distinct spots on photo - impaired mitochondrial tubule ( arrows : location of original impaired single mitochondrial tubule ) . ( d ) dotted region outlined in c , LC3 structures segregated from main parkin - labelled cluster , carrying away bits of parkin - labelled mitochondria with them . Two events are labelled in image with yellow arrows , respectively . Scale bars ( panels ) , 5 μm .

O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O 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) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b-d) Magnified view of the photo-inactivated region in a 34-93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.


( a ) A HeLa expressing KR - dMito ( red ) , EGFP - DFCP1 ( green ) EBFP2 - parkin ( cyan ) were illuminated with 559 nm at dotted circle region , leading to generation of a long parkin - labelled mitochondria tubule ( right , magnified view of - activated region ) for autophagic turnover ( arrow ) . Scale bars , 10 μm .

O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR 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) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 μm.


( b ) After generation of parkin - labelled mitochondrial tubules following treatments in a , discrete DFCP1 foci ( green , middle panel , ER - associated omegasomes ) formed along parkin - labelled mitochondria ( arrows indicate DFCP1 spots that colocalized with parkin ) . Bottom panel : merged image of parkin DFCP1 . Scale bar , 10 μm .

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

(b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 μm.


( c ) Time - lapse imaging showing DCFP1 formation on parkin - labelled mitochondria . A HeLa expressing KR - dMito , EGFP - DFCP1 ( green ) EBFP2 - parkin ( pseudo - coloured red ) were 559 nm illuminated , leading to generation of parkin - labelled mitochondria tubules . Time - lapse imaging ( 1 frame per min ) revealed that DFCP1 ( green ) formed transiently at discrete sites on parkin - labelled mitochondrial tubules ( red ) . arrows mark two DFCP1 ( ER - associated omegasomes ) formation / disassembly events directly on parkin - labelled tubules . Scale bar , 5 μm .

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

(c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm.


( d ) A COS - 7 expressing EBFP2 - parkin ( cyan ) , AcGFP - sec61β ( green ) TagRFP - DFCP1 ( red ) were treated with 10 μM CCCP for 4 h imaged . Omegasomes ( DFCP1 ) appeared where ER ( sec61β ) impaired mitochondria ( parkin ) overlap . Scale bar , 10 μm .

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

(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.


( e ) Histogram of lifetime of DFCP1 foci on parkin - labelled mitochondria in CCCP - treated HeLa expressing TagRFP - DFCP1 , EGFP - LC3B EBFP2 - parkin .

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

(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.


( f ) Wortmannin treatments delayed initial LC3 recruitment times onto parkin - labelled mitochondria in HeLa expressing KR - dMito , EGFP - LC3B EBFP2 - parkin . Each ' + ' marks times measured in HeLa ( red ' □ ' ( square ) denotes average from five ) . P = 0 . 0172 ( Student ' s t - ) .

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

(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).


( a ) A COS - 7 expressing EBFP2 - Ub ( red ) , AcGFP - sec61β ( green ) mcherry - parkin ( cyan ) was treated with 10 μM CCCP for 1 h imaged . Impaired mitochondria ( parkin ) displayed preferentially ubiquitinated foci ( arrows ) at selected regions that overlap with ER ( sec61β ) .

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

(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).


( b ) HeLa expressing KR - dMito ( red ) , EGFP - LC3 ( green ) EBFP2 - parkin ( cyan ) were illuminated with 559 nm at dotted square regions , generating parkin - labelled mitochondria ( bottom right , magnified view of - activated regions ) . ( c ) Magnified view of photo - inactivated region in b 80 min after illumination ( post - stained with anti - ubiquitin antibody ) . LC3 ubiquitin colocalized at discrete sites along parkin - labelled mitochondria ( arrows ) .

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

(b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows).


( d ) A MG - 132 treated HeLa expressing KR - dMito , EBFP2 - parkin ( green ) EGFP - LC3B ( red ) was 559 nm illuminated , leading to appearance of parkin - labelled mitochondria ( arrows one to three indicated structures , top panels ) . parkin - labelled structures did not fragment nor became engulfed by LC3 structures ( bottom panels ) in presence MG - 132 . Scale bars ( panels ) , 5 μm .

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

(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.


A . Confocal of mitochondrial in wild - type ( WT ) Drp1 - / - 293T transfected with empty vector ( left panel ) Myc - hFis1 ( right panel ) , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).