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 <

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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 - 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 <

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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 the prototypical Wnt target genes , AXIN2 and LEF1 ( A ) , and at the promoter regions of HOXC4 and 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 cells . The data represent the 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 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

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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 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 <

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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 ) cells . Samples were compared using Student " s t - test . 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 the amount of β - catenin detected in the 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 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

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 . 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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 ) 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 .

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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 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 * * * .

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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 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 * * * .

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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 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

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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 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

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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 ) .

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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 the 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 the 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 ± 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 .

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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 ) and overexpression ( N ) .

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


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 .

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

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 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 .

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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 analysis ( GESA ) for inflammatory and immune responses .

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


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

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

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


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 + + +

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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 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 .

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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 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 .

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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 the RNA - seq data . The 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 protein expression . Data are represented as mean ± SEM . n = 3 . Student ' s t - test . Significantly different at p = 0 . 0077 * in graph C .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

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

(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 ± SEM . 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 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 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 .

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 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 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 .

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

O O O B-CELL_TYPE I-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 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O 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 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 ± SEM . 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 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 - 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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 ) , and the 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 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 ) , and the 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 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 ) , 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 .

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 O O B-CELL_TYPE B-GENEPROD O 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 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 ) , 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 .

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

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

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 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 .

O O O O O B-GENEPROD O 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 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 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 .

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

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

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

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 O B-GENEPROD O B-DISEASE B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 O B-TISSUE O O B-GENEPROD O B-DISEASE B-ORGANISM O 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 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 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 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 .

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 O B-TISSUE O O O O O O 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 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 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 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 .

O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-TISSUE O B-ORGANISM O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O 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 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 levels were estimated in the midbrain SN of the 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 I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O 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


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

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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 ± 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 .

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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 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 .

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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 + 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 *

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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 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 ) .

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(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 ( 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 ) .

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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).


B . Quantitative analyses of fragmented mitochondria size ( mean area ( μm2 ) per mitochondrion ) after Myc - hFis1 overexpression in WT and Drp1 - / - 293T cells using Image J software ( Particle analysis ) in three independent experiments . In each cell , only dispersed individual mitochondria were analyzed . The total number of mitochondria ( mito ) analyzed is indicated for each condition .

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

B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.


C . Quantitative analysis of mean mitochondria number per cell in WT and Drp1 - / - 293T cells overexpressing Myc - hFis1 using Image J software ( Particle analysis ) in three independent experiments .

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

C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.


E . Percentages of cells with indicated mitochondrial morphologies in WT and Drp1 - / - 293T cells transfected with empty vector ( control ) , Myc - hFis1 and either Myc - or GFP - tagged mutants as indicated in three independent experiments .

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

E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA ( control ) and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) as indicated . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Western blot of Dyn2 , Myc - hFis1 , Drp1 and GAPDH in WT 293T cells collected from the experiments in ( A ) .

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B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A).


C . Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector or Myc - hFis1 in three independent experiments .

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

C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments.


D . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) . Representative examples of tubular , fragmented and tubular cluster phenotypes are indicated .

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D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.


E . Western blot of Dyn2 , Myc - hFis1 and GAPDH in Drp1 - / - 293T cells collected from the experiments in ( D ) .

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E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D).


F . Percentages of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells treated with scrambled siRNA or Dyn2 siRNA , followed by transfection with empty vector or Myc - hFis1 as indicated in three independent experiments .

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

F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with scrambled siRNA ( control ) or with hFis1 - siRNA and then stained with MitoTracker ( red ) .

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

A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).