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 .

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

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 ] .

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

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 .

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

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

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

O O B-EXP_ASSAY O B-GENEPROD 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

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 .

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 O B-SUBCELLULAR O O 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 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 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 .

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

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

O 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 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 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 O 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

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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 ) .

O 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 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 ± 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 .

O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-DISEASE B-ORGANISM O O 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 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 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 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 .

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 O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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 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 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 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 + 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 .

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

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

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 .

O O 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 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 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 .

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

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 .

O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O B-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 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 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 *

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

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*


CD14 + monocytes were cultured with M - CSF , IL - 4 and TNFα and analyzed by scRNA - seq at different time points ( n = 2 biological replicates ) . ( D ) Expression patterns of monocytes ( top row ) , macrophages ( middle row ) and DC ( bottom row ) markers .

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

CD14+ monocytes were cultured with M-CSF, IL-4 and TNFα and analyzed by scRNA-seq at different time points (n=2 biological replicates). (D) Expression patterns of monocytes (top row), macrophages (middle row) and DC (bottom row) markers.


( D ) Expression of phenotypic markers used for UMAP projection .

(D) Expression of phenotypic markers used for UMAP projection.


( E ) Supervised gating strategy for moDC and moMac at each time point ( gated on live CD11b + CD115 + cells ) .

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

(E) Supervised gating strategy for moDC and moMac at each time point (gated on live CD11b+ CD115+ cells).


( F ) Supervised gating strategy to identify monocyte - derived cells .

(F) Supervised gating strategy to identify monocyte-derived cells.


( E ) Transcription factor activity was calculated with Dorothea and Viper . Differentially activated regulons between clusters are shown .

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

(E) Transcription factor activity was calculated with Dorothea and Viper. Differentially activated regulons between clusters are shown.


( A ) Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor .

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

(A) Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.


Monocytes were cultured with M - CSF for 5 days . IRF1 expression was silenced using a lentivirus containing shRNA . ( B ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 8 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor .

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

Monocytes were cultured with M-CSF for 5 days. IRF1 expression was silenced using a lentivirus containing shRNA. (B) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=8 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.


Monocytes were cultured with RPMI medium ( R10 ) , or combinations of M - CSF , IL - 4 and TNFα in the presence or absence of B18R ( type I interferon inhibitor ) . ( G ) ISG expression after 9 hours was analyzed by RTqPCR . Each symbol represents an individual donor ( n = 5 biological replicates ) .

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

Monocytes were cultured with RPMI medium (R10), or combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (G) ISG expression after 9 hours was analyzed by RTqPCR. Each symbol represents an individual donor (n=5 biological replicates).


Monocytes were cultured with combinations of M - CSF , IL - 4 and TNFα in the presence or absence of B18R ( type I interferon inhibitor ) . ( H ) Monocyte differentiation after 5 days . One representative donor is shown ( n = 5 biological replicates ) . Proportion of mo - Mac and mo - DC after 5 days . Each symbol represents an individual donor ( n = 5 biological replicates ) . Paired t - test .

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

Monocytes were cultured with combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (H) Monocyte differentiation after 5 days. One representative donor is shown (n=5 biological replicates). Proportion of mo-Mac and mo-DC after 5 days. Each symbol represents an individual donor (n=5 biological replicates). Paired t-test.


( B ) ZNF366 expression across clusters .

(B) ZNF366 expression across clusters.


Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ( C ) Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova .

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

Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (C) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.


Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ZNF366 expression was silenced using a lentivirus containing shRNA . ( D ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 4 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova .

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

Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. ZNF366 expression was silenced using a lentivirus containing shRNA. (D) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.


Monocytes were cultured with M - CSF , IL - 4 and TNFα for 5 days . ( G ) Protein quantification by Western Blot . Actin was used as loading control . Representative results are shown ( n = 6 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova .

Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (G) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.


MAFF expression was silenced using a lentivirus containing shRNA . ( H ) Protein quantification by Western Blot after 5 days . Actin was used as loading control . Representative results are shown ( n = 4 biological replicates ) . Quantification was performed by densitometry . Each symbol represents an individual donor . Paired one - way Anova .

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

MAFF expression was silenced using a lentivirus containing shRNA. (H) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.


MAFF expression was silenced using a lentivirus containing shRNA . ( I ) Mo - mac and mo - DC differentiation after 5 days was assessed by flow cytometry . One representative donor is shown ( n = 7 biological replicates ) . DN = double negative . Proportion of mo - DC , mo - Mac and DN cells after 5 days . Median is shown ( n = 7 biological replicates ) . Paired one - way Anova .

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

MAFF expression was silenced using a lentivirus containing shRNA. (I) Mo-mac and mo-DC differentiation after 5 days was assessed by flow cytometry. One representative donor is shown (n=7 biological replicates). DN = double negative. Proportion of mo-DC, mo-Mac and DN cells after 5 days. Median is shown (n=7 biological replicates). Paired one-way Anova.


( A ) BECN1 expression of microglia from Becn1 + / - and wild type mouse pups in full medium without Bafilomycin A1 ( Baf ) was quantified by Western blotting . Microglia from Becn1 + / - mice show a signifcant reduction of BECN1 ( 48 % ) compared to microglia from wild type mice ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 7 ; two - tailed t - test * * : p < 0 . 01 . Blot shows representative amounts of BECN1 after various treatments .

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

(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments.


( B ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 ( Baf ) . LC3 and ACTIN levels were determined by Western blot . LC3 - II was significantly reduced in microglia from Becn1 + / - mice after Baf treatment in full medium compared to wild type microglia ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 4 ; two - tailed t - test * : p < 0 . 05 .

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

(B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05.


( C ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 ( Baf ) and stained for endogenous LC3 . Exemplary images and the quantification of LC3 - positive vesicles / µm2 cell area show reduced presence of LC3 - positive vesicles in microglia from Becn1 + / - and induction of autophagy by HBSS ; mean ± SEM , wild type n = 4 , Becn1 + / - n = 4 , 3 - 5 fields per view / n and condition and 73 - 271 cells / n and condition ; two - tailed t - test * : p < 0 . 05 ; scale bar : 20

O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE 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 O O O O O O O O O O O O O O O O O O O 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) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm


( A , B ) Microglia isolated from newborn Becn1 + / - and wild type mouse pups were either non treated or treated with a pro - inflammatory stimulus ( LPS followed by ATP ) . As additional controls cells treated only with LPS or ATP were used in ( A ) . The concentration of the pro - inflammatory cytokines IL - 1beta ( A ) and IL - 18 ( B ) in the cell supernatant was determined by ELISA . Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS / ATP ; mean ± SEM , wild type n ( A / B ) = 9 / 2 , wild type LPS n = 3 / 0 , wild type ATP n = 3 / 0 , wild type LPS / ATP n = 9 / 9 , Becn1 + / - n = 23 / 5 , Becn1 + / - LPS / ATP n = 23 / 23 , two - tailed t - test * : p < 0 . 05 .

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

(A, B) Microglia isolated from newborn Becn1+/- and wild type mouse pups were either non treated or treated with a pro-inflammatory stimulus (LPS followed by ATP). As additional controls cells treated only with LPS or ATP were used in (A). The concentration of the pro-inflammatory cytokines IL-1beta (A) and IL-18 (B) in the cell supernatant was determined by ELISA. Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS/ATP; mean ± SEM, wild type n (A/B) = 9/2, wild type LPS n = 3/0, wild type ATP n = 3/0, wild type LPS/ATP n = 9/9, Becn1+/- n = 23/5, Becn1+/- LPS/ATP n = 23/23, two-tailed t-test *: p < 0.05.


( C ) Microglia isolated from wild type mouse pups were pre - incubated in full medium or starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment . The concentration of the pro - inflammatory cytokine IL - 1beta in the cell supernatant was determined by ELISA . Incubation with HBSS reduced IL - 1beta release significantly while 3 - MA treatment increased it ; mean ± SEM , full medium n = 6 , full medium LPS / ATP n = 6 , full medium LPS / ATP + 3MA n = 3 , HBSS n = 6 , HBSS LPS / ATP n = 6 , HBSS LPS / ATP + 3MA n = 6 ; two - tailed t - test * : p < 0 . 05 ; * * : p < 0 . 01 .

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

(C) Microglia isolated from wild type mouse pups were pre-incubated in full medium or starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment. The concentration of the pro-inflammatory cytokine IL-1beta in the cell supernatant was determined by ELISA. Incubation with HBSS reduced IL-1beta release significantly while 3-MA treatment increased it; mean ± SEM, full medium n = 6, full medium LPS/ATP n = 6, full medium LPS/ATP + 3MA n = 3, HBSS n = 6, HBSS LPS/ATP n = 6, HBSS LPS/ATP + 3MA n = 6; two-tailed t-test *: p < 0.05; **: p < 0.01.


( D ) Microglia isolated from wild type mouse pups were pre - incubated in starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment , microglia were stained for LC3 and images taken by confocal microscopy . Representative images show a reduction of LC3 - positive vesicles in 3 - MA treated cells . Scale bar : 20 µm .

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

(D) Microglia isolated from wild type mouse pups were pre-incubated in starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment, microglia were stained for LC3 and images taken by confocal microscopy. Representative images show a reduction of LC3-positive vesicles in 3-MA treated cells. Scale bar: 20 µm.


( E ) Proteins were extracted from brains of wild type , Becn1 + / - , APPPS1 and APPPS1 Becn1 + / - mice at the indicated ages . IL - 1beta was measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . IL - 1beta was significantly increased in the TBS and TX fraction ; mean ± SEM , wild type : n ( per age group ) = 2 / 6 / 3 , Becn1 + / - : n = 2 / 5 / 2 , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with WT as control group ; * : p < 0 . 05 ; * * : p < 0 . 01 .

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

(E) Proteins were extracted from brains of wild type, Becn1+/-, APPPS1 and APPPS1 Becn1+/- mice at the indicated ages. IL-1beta was measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. IL-1beta was significantly increased in the TBS and TX fraction; mean ± SEM, wild type: n (per age group) = 2/6/3, Becn1+/-: n = 2/5/2, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with WT as control group; *: p < 0.05; **: p < 0.01.


( F ) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1 + / - mice at the age as indicated . Amyloid beta 1 - 40 and 1 - 42 were measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . No difference in the amyloid beta load between the genotypes is apparent ; mean ± SEM , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with APPPS1 as control group , ns .

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

(F) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1+/- mice at the age as indicated. Amyloid beta 1-40 and 1-42 were measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. No difference in the amyloid beta load between the genotypes is apparent; mean ± SEM, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with APPPS1 as control group, ns.


( A ) The expression of NLRP3 protein in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced expression of NLRP3 ; mean ± SEM , wild type n = 4 , wild type LPS / ATP n = 9 , Becn1 + / - n = 4 , Becn1 + / - LPS / ATP n = 23 ; ANOVA with Tukey ´ s post hoc test * : p < 0 . 05 .

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

(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.


( B ) The expression of Nlrp3 mRNA in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by qPCR . No significant differences can be detected between wild type and Becn1 + / - microglia ; mean ± SEM , wild type : n = 7 , Becn1 + / - : n = 3 ; ANOVA with Tukey ´ s post hoc test , ns for LPS / ATP treated wild type versus Becn1 + / - .

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

(B) The expression of Nlrp3 mRNA in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by qPCR. No significant differences can be detected between wild type and Becn1+/- microglia; mean ± SEM, wild type: n = 7, Becn1+/- : n = 3; ANOVA with Tukey ´s post hoc test, ns for LPS/ATP treated wild type versus Becn1+/-.


( C ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for ASC ( red ) and imaged by confocal microscopy to investigate the assembly of inflammasomes . In non stimulated cells ASC is distributed diffusely in the cytoplasm ( upper panels ) . However , after stimulation formation of inflammasomes ( arrowheads ) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis / ejection ( arrow ) . Quantification showed a significantly increased percentage of inflammasomes in or around Becn1 + / - microglia ; mean ± SEM , wild type LPS / ATP n = 4 , Becn1 + / - LPS / ATP n = 7 ; two - tailed t - test * : p < 0 . 05 ; scale bar : 50 µm , insert scale bar : 10 µm .

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

(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.


( D ) The presence of cleaved CASP1 ( p10 ) protein in the supernatant of LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced release of CASP1 ; mean ± SEM , wild type LPS / ATP n = 8 , Becn1 + / - LPS / ATP n = 18 ; two - tailed t - test * : p < 0 . 05 .

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

(D) The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced release of CASP1; mean ± SEM, wild type LPS/ATP n = 8, Becn1+/- LPS/ATP n = 18; two-tailed t-test *: p < 0.05.


( A ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and imaged by confocal microscopy . Stimulation resulted in the appearance of many NLRP3 - containing aggregates of different sizes ( arrows ) . In non treated cells , only a few overlapping signals with LC3 - stained autophagosomes ( arrowheads ) are visible . Stimulated microglia however , showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates ; scale bar : 7 . 5 µm .

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

(A) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for NLRP3 (green) and LC3 (red) and imaged by confocal microscopy. Stimulation resulted in the appearance of many NLRP3-containing aggregates of different sizes (arrows). In non treated cells, only a few overlapping signals with LC3-stained autophagosomes (arrowheads) are visible. Stimulated microglia however, showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates; scale bar: 7.5 µm.


( B ) LPS / ATP treated wild type microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates of different sizes coclustering with LC3 - positive autophagosomes are clearly visible . 3D volume rendering of large ( I ) and small ( II ) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes ; scale bar : 10 µm ; magnified ROIs : 1 µm .

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

(B) LPS/ATP treated wild type microglia were immunolabeled for NLRP3 (green) and LC3 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates of different sizes coclustering with LC3-positive autophagosomes are clearly visible. 3D volume rendering of large (I) and small (II) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes; scale bar: 10 µm; magnified ROIs: 1 µm.


( C ) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize to the same organelle ; mean ± SEM , n = 4 .

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

(C) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize to the same organelle; mean ± SEM, n = 4.


( A ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by confocal microscopy . Colocalizations of NLRP3 aggregates ( arrows ) and CALCOCO2 ( arrowheads ) are apparent ; scale bar : 10 µm .

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

(A) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by confocal microscopy. Colocalizations of NLRP3 aggregates (arrows) and CALCOCO2 (arrowheads) are apparent; scale bar: 10 µm.


( B ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates coclustering with CALCOCO2 - positive signals are visible . 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2 ; scale bar : 10 µm ; magnified ROIs : 1 µm .

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

(B) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates coclustering with CALCOCO2-positive signals are visible. 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2; scale bar: 10 µm; magnified ROIs: 1 µm.


( C ) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize ; mean ± SEM , n = 3 .

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

(C) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize; mean ± SEM, n = 3.


( D ) In SIM images from wild type microglia the sub - autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2 . LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster . While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity , the relative LC3 intensity there was significantly lower ; mean ± SEM , LC3 : n = 4 , CALCOCO2 : n = 3 ; two - tailed t - test * * : p < 0 . 01 .

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

(D) In SIM images from wild type microglia the sub-autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2. LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster. While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity, the relative LC3 intensity there was significantly lower; mean ± SEM, LC3: n = 4, CALCOCO2: n = 3; two-tailed t-test **: p < 0.01.


( E ) Microglia from Becn1 + / - mice were transfected with scrambled siRNA or with siRNA for CALCOCO2 . 6 d after transfection cells were stimulated with LPS / ATP . The expression of CALCOCO2 protein was determined by Western blot . Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2 ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 .

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

(E) Microglia from Becn1+/- mice were transfected with scrambled siRNA or with siRNA for CALCOCO2. 6 d after transfection cells were stimulated with LPS/ATP. The expression of CALCOCO2 protein was determined by Western blot. Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


( F ) The amount of IL - 1beta in the supernatant released from the cells described in ( E ) was determined by ELISA . Microglia with a CALCOCO2 knockdown released significantly more IL - 1beta ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 .

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

(F) The amount of IL-1beta in the supernatant released from the cells described in (E) was determined by ELISA. Microglia with a CALCOCO2 knockdown released significantly more IL-1beta; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


B . Confocal microscopy images of HeLa , RPE , and U2OS cell lines that were pre - extracted with Triton X - 100 before fixed with formaldehyde and co - stained with DAPI and antibodies against PCNA and FAM111A . Scale bars : 10

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

B. Confocal microscopy images of HeLa, RPE, and U2OS cell lines that were pre-extracted with Triton X-100 before fixed with formaldehyde and co-stained with DAPI and antibodies against PCNA and FAM111A. Scale bars: 10 μm


C . U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy . Scale bars : 10 μm .

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

C. U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy. Scale bars: 10 μm.


HEK293 , RPE , or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) , 48 h ( RPE ) , or 72h ( U2OS ) . The colonies were fixed then stained with crystal violet .

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

HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet.


Cell - cycle profiles of HEK293 , RPE and U2OS FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry .

Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.


C . HEK293 cell lines were induced with doxycycline to express wild - type or mutant FAM111A for 24 h ( HEK293 ) Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) .

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

C. HEK293 cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293) Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test).


D . Cell - cycle profiles of HEK293 FAM111A - expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry .

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

D. Cell-cycle profiles of HEK293 FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.


A . HEK293 or U2OS / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from 105 cells was resolved by SDS - PAGE , and probed in western blots for both endogenous FAM111A ( 71 kDa ) and MycBioID - FAM111A ( 110 kDa ) as well as full length and cleaved PARP ( 116 and 89 kDa , respectively ) . The Tubulin western blot is a sample loading control .

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

A. HEK293 or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from 105 cells was resolved by SDS-PAGE, and probed in western blots for both endogenous FAM111A (71 kDa) and MycBioID-FAM111A (110 kDa) as well as full length and cleaved PARP (116 and 89 kDa, respectively). The Tubulin western blot is a sample loading control.


B . HEK293 / MycBioID - FAM111A cell lines with no treatment ( - Dox ) , or treated with doxycycline , along with DMSO or 100 μM Z - VAD - FMK for 24 h ( total inhibition of PARP cleavage was also observed when 50 μM Z - VAD - FMK was used ) . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots with Myc , PARP , or Tubulin antibodies .

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

B. HEK293/MycBioID-FAM111A cell lines with no treatment (- Dox), or treated with doxycycline, along with DMSO or 100 μM Z-VAD-FMK for 24 h (total inhibition of PARP cleavage was also observed when 50 μM Z-VAD-FMK was used). Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots with Myc, PARP, or Tubulin antibodies.


C . HEK293 cell lines were treated with doxycycline and Z - VAD - FMK for 24 h . The colonies were fixed then stained with crystal violet . Cell growth was quantitated as total area on ImageJ . Values are mean ± s . e . m . of independent experiments ( n = 3 ) .

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

C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).


C . Cells were treated with doxycycline along with DMSO or Z - VAD - FMK for 24 h . They were fixed with formaldehyde , and stained with NPC antibody Mab414 and DAPI . The images were captured by confocal microscopy . Scale bar : 10 μm .

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

C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.


D . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from an equal number of cells was incubated with magnetic streptavidin beads , and pull downs were analyzed by western blotting using GANP and Myc antibodies .

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

D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.


E . HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h , while growing in the presence or absence of 50 mM supplemental biotin . Total protein from whole cell lysates was incubated with magnetic streptavidin beads . Pulldowns were analyzed by western blotting using GANP and Myc antibodies .

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

E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.


F . DMSO or Z - VAD - FMK treated HEK293 / MycBioID - FAM111A cell lines were induced with doxycycline for 24 h . Total protein from ~ 105 cells was resolved by SDS - PAGE , and probed in western blots using antibodies against NPC ( Mab414 ) , PARP , cleaved Caspase 3 , Myc , or Tubulin .

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

F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.


A . Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA . Images were taken with a Zeiss Axio Imager . LT is shown in green , and DAPI blue . Scale bar : 100 μm . B . Quantification of LT positive cells described in A and fixed at indicated times post transfection . Five hundred or more cells were counted for each condition . C . Quantification of RFP marker distribution upon co - transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA . Cells were fixed at the indicated times . Five hundred or more cells were counted for each condition . In B & C , values are mean ± s . d . of independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( two - tailed unpaired t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-GENEPROD B-EXP_ASSAY O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD O O O O O O O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm. B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition. C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test).


D . Western blot analysis of control or FAM111A - depleted U2OS cells transfected with SV40 LT variants . Cells were collected and lysed at indicated timepoints after SV40 transfection , immunoblotted with FAM111A , LT , or PARP antibodies . Cdc2 - PSTAIR was used as a loading control .

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

D. Western blot analysis of control or FAM111A-depleted U2OS cells transfected with SV40 LT variants. Cells were collected and lysed at indicated timepoints after SV40 transfection, immunoblotted with FAM111A, LT, or PARP antibodies. Cdc2-PSTAIR was used as a loading control.


A Heatmap depiction of the 3 , 731 DNA hypomethylation loci in ago4 ‐ 6 . Each hypomethylated region corresponds to a colored horizontal bar , and the bars are clustered numerically into a column ( y ‐ axis ) . Cytosines were examined as CG , CHG , and CHH . The color ‐ scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions .

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

A Heatmap depiction of the 3,731 DNA hypomethylation loci in ago4‐6. Each hypomethylated region corresponds to a colored horizontal bar, and the bars are clustered numerically into a column (y‐axis). Cytosines were examined as CG, CHG, and CHH. The color‐scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions.


B Heatmap depiction of the 3 , 678 DNA hypomethylation loci in ago6 ‐ 2 .

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

B Heatmap depiction of the 3,678 DNA hypomethylation loci in ago6‐2.


B Box plots showing methylation patterns of different groups of loci as categorized in ( A ) .

B Box plots showing methylation patterns of different groups of loci as categorized in (A).


B DNA methylation levels in different mutants . The 2 , 174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered ( y ‐ axis ) . Cytosines were examined as CG , CHG , and CHH .

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

B DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH.


C DNA methylation levels at TAS1a , TAS1c , and TAS3a loci . Snapshots from whole ‐ genome bisulfite sequencing results are shown .

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C DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown.


D RT - qPCR measurements of transposon RNA levels . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . Actin 2 was used as an internal control . RNA levels in the mutants are relative to those in the wild ‐ type ( Col ‐ 0 ) . Means ± SD are shown , n = 3 .

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D RT-qPCR measurements of transposon RNA levels. Double: ago4‐6 ago6‐2 double mutant. Actin 2 was used as an internal control. RNA levels in the mutants are relative to those in the wild‐type (Col‐0). Means ± SD are shown, n = 3.


A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies . Yellow signals would be expected in the merged images if the two proteins co ‐ localize , as a result of the overlap of red and green channels . DNA ( blue ) was stained with DAPI .

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A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies. Yellow signals would be expected in the merged images if the two proteins co‐localize, as a result of the overlap of red and green channels. DNA (blue) was stained with DAPI.


B The largest subunit of Pol IV , NRPD1 ( red ) , was visualized by its specific antibody in cells expressing FLAG ‐ tagged AGO4 or AGO6 ( green ) .

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B The largest subunit of Pol IV, NRPD1 (red), was visualized by its specific antibody in cells expressing FLAG‐tagged AGO4 or AGO6 (green).


A The largest subunit of Pol V , NRPE1 ( red ) , was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG ‐ tagged AGO4 ( green ) . AGO4 ‐ NRPE1 co ‐ localized to nucleolar dot ( yellow dot , marked with white arrow , upper panel ) but not in nucleoplasmic foci ( lower panel ) .

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A The largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG‐tagged AGO4 (green). AGO4‐NRPE1 co‐localized to nucleolar dot (yellow dot, marked with white arrow, upper panel) but not in nucleoplasmic foci (lower panel).


B NRPE1 ( red ) was visualized by its specific antibody in cells expressing FLAG ‐ tagged AGO6 ( green ) . The yellow signals due to the overlap of red and green channels in merged images indicate protein co ‐ localization . DNA ( blue ) was stained with DAPI . AGO6 ‐ NRPE1 co ‐ localized to nucleoplasmic foci ( yellow dots , marked with white arrow ) .

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B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).


A AGO6 dysfunction reduces levels of Pol V ‐ dependent transcripts . RNA levels were measured by RT - qPCR . Means ± SD are shown , n = 3 .

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A AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT-qPCR. Means ± SD are shown, n = 3.


B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6 . Anti ‐ NRPE1 antibody was used for ChIP assays . Means ± SD are shown , n = 3 .

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B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6. Anti‐NRPE1 antibody was used for ChIP assays. Means ± SD are shown, n = 3.


A Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol IV . Identities of siRNAs were retrieved from Havecker et al ( ) , and siRNAs were grouped into 1 , 210 AGO4 ‐ bound clusters and 1 , 486 AGO6 ‐ bound clusters ( see Materials and Methods for details ) , each of which corresponds to a colored horizontal bar ; the bars are stacked numerically into a column ( y ‐ axis ) . Information on siRNA levels in Pol IV mutant and wild ‐ type plants was retrieved from Zhang et al ( ) .

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A Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al ().


B Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol V .

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B Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V.


C Quantification of individual 24 ‐ nt siRNAs by TaqMan small RNA assays . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . snoR101 was used as an internal control . RNA levels in the mutants were relative to those of the wild ‐ type ( Col ‐ 0 ) . Error bars indicate SD , n ≥ 3 .

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C Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.


. C - J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice . Data are expressed as CSS - normalized bacterial

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. C-J Bar Chart with data points showing the abundance of indicated bacterial families in the indicated group of mice. Data are expressed as CSS-normalized bacterial counts


A - C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle , measured by NMR or GC - MS . Data are expressed as μg / ml or bin intensity ( arbitrary unit - a . u . ) D - F Bar chart with individual points showing the levels of pyruvate , succinate and lactate in wt and mdx mice receiving vehicle or DFZ , measured by NMR .

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A-C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle, measured by NMR or GC-MS. Data are expressed as μg/ml or bin intensity (arbitrary unit - a.u.) D-F Bar chart with individual points showing the levels of pyruvate, succinate and lactate in wt and mdx mice receiving vehicle or DFZ, measured by NMR. G