C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . The translation efficiency was measured by a SUnSET assay .

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C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. The translation efficiency was measured by a SUnSET assay.


D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . mtDNA - encoded proteins were analysed by western blot . β - Actin was used as the loading control .

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D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.


E PLC cell lysates were immunoprecipitated with an anti - MRPL44 antibody or IgG prior to western blot analysis of MRPL44 , MRPL46 , MRPL48 , MRPS15 , MRPS16 , and MRPS35 .

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E PLC cell lysates were immunoprecipitated with an anti-MRPL44 antibody or IgG prior to western blot analysis of MRPL44, MRPL46, MRPL48, MRPS15, MRPS16, and MRPS35.


F Binding of mtDNA - encoded mRNA by endogenous MRPL44 ( representing mitochondrial ribosomes ) was evaluated by RNA immunoprecipitation ( RIP ) in PLC cells with PHGDH knockdown .

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F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.


G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient . After centrifugation , all fractions were subjected to western blot analysis of MRPS35 , MRPL44 , and MRPL48 proteins . RNA concentrations of ND6 were measured .

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G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.


A PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 . The OCR was measured after successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve .

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A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.


B PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were constructed by cell counting with trypan blue exclusion ( right ) .

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B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).


C PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were determined by cell counting with trypan blue exclusion ( right ) .

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C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).


D - F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2 . Then , these cells were subcutaneously injected into male nude mice ( n = 5 in each group ) . Tumour sizes were measured beginning 14 days after inoculation ( D ) . At the end of the experiment , the tumours were excised ( E ) , and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting ( F ) .

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D-F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2. Then, these cells were subcutaneously injected into male nude mice (n = 5 in each group). Tumour sizes were measured beginning 14 days after inoculation (D). At the end of the experiment, the tumours were excised (E), and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting (F).


G - I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice ( n = 5 in each group ) . Mice were intraperitoneally injected with 100 mg / kg tigecycline daily beginning 14 days after inoculation . Tumour sizes were measured beginning 14 days after inoculation ( G ) . At the end of the experiment , the tumours were excised ( H ) , and PHGDH expression in tumour lysates was analysed by western blotting ( I ) .

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G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).


( A - F ) TNF - α , Cxcl1 and Cxcl2 ( A , C , and E respectively ) were measured at 24 h after stimulation of bone - marrow - derived neutrophils ( 5 x 105 cells ) with increasing concentrations of bacterial nucleic acids ( 1 , 5 , 10 and 20 µg of DNA + RNA in equal proportions ) , HK - bacteria ( 1 , 5 , 10 or 20 μg / ml ) or live bacteria ( MOIs of 2 , 5 , 10 and 20 ) . LPS ( 1 , 10 , 100 and 1000 ng / ml ) was used as a positive control stimulus . ( B , D and F ) , Effect of FPR antagonist Boc - 2 on GBS - induced TNF - α , Cxcl1 and Cxcl2 production , respectively . Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 50 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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(A-F) TNF-α, Cxcl1 and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on GBS-induced TNF-α, Cxcl1 and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to Boc-2 (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 10 µM ) or WRW4 ( 1 , 5 and 10 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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Bone-marrow-derived neutrophils were exposed to Boc-2 (10 µM) or WRW4 (1, 5 and 10 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


( A - F ) Cxcl1 and Cxcl2 concentrations in 24 - h culture supernatants of neutrophils ( A and B ) , CSF - polarized ( C and D ) or GM - CSF - polarized ( E and F ) BMDMs ( ΜΦ ) from WT or Fpr - deficient mice stimulated with live GBS ( MOI of 2 , 5 , and 10 ) or HK - GBS ( 10 µg / ml ) . LPS ( 100 ng / ml ) was used as a positive control stimulus . Means + SD of data from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 vs WT mice , determined by unpaired t - test .

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(A-F) Cxcl1 and Cxcl2 concentrations in 24-h culture supernatants of neutrophils (A and B), CSF-polarized (C and D) or GM-CSF-polarized (E and F) BMDMs (ΜΦ) from WT or Fpr-deficient mice stimulated with live GBS (MOI of 2, 5, and 10) or HK-GBS (10 µg/ml). LPS (100 ng/ml) was used as a positive control stimulus. Means + SD of data from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT mice, determined by unpaired t-test.


( E ) , Kaplan - Meier analysis of survival rates in WT , Fpr1 and Fpr2 KO mice after i . p . challenge with 2x105 bacteria . Data are the cumulative results from two experiments , each involving 8 animals per group . * * P < 0 . 01 , * * * * P < 0 . 0001 vs WT mice , as determined by Kaplan - Meier analysis . Data information : means + SDs of three duplicate determinations , each conducted in a different animal .

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(E), Kaplan-Meier analysis of survival rates in WT, Fpr1 and Fpr2 KO mice after i.p. challenge with 2x105 bacteria. Data are the cumulative results from two experiments, each involving 8 animals per group. **P < 0.01, ****P < 0.0001 vs WT mice, as determined by Kaplan-Meier analysis. Data information: means + SDs of three duplicate determinations, each conducted in a different animal.


( F - G ) Neutrophil influx ( F ) and bacterial burden ( G ) in peritoneal lavage fluid samples collected from Fpr2 - / - mice at 3 h after i . p . challenge with live GBS ( 2x107 CFU ) . At 1h post - challenge mice were inoculated i . p . with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle . Data information : , individual data points and means . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle treated mice determined by unpaired t test or Mann - Whitney U

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(F-G) Neutrophil influx (F) and bacterial burden (G) in peritoneal lavage fluid samples collected from Fpr2-/- mice at 3 h after i.p. challenge with live GBS (2x107 CFU). At 1h post-challenge mice were inoculated i.p. with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle. Data information: , individual data points and means. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle treated mice determined by unpaired t test or Mann-Whitney U test


( C and D ) , Cxcl2 release in neutrophil cultures stimulated with combinations of HK - bacteria , GBS fMet peptides or TLR agonists . Neutrophils were exposed to a 50 µM concentration of Fpr agonists ( fMIFL , WKYMVM or fPep8 and fPep10 ) for 1 h before the addition of the indicated TLR agonists , which included HK - GBS ( 10 μg / ml ) , LPS ( 1 µg / ml ) and RNA ( 1 µg / ml ) . In some cases , neutrophils were exposed to graded doses ( 10 , 20 and 50 µM ) of the indicated Fpr agonists alone . Data information : Data are expressed as means + SD from three independent experiments , each conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(C and D), Cxcl2 release in neutrophil cultures stimulated with combinations of HK-bacteria, GBS fMet peptides or TLR agonists. Neutrophils were exposed to a 50 µM concentration of Fpr agonists (fMIFL, WKYMVM or fPep8 and fPep10) for 1 h before the addition of the indicated TLR agonists, which included HK-GBS (10 μg/ml), LPS (1 µg/ml) and RNA (1 µg /ml). In some cases, neutrophils were exposed to graded doses (10, 20 and 50 µM) of the indicated Fpr agonists alone. Data information: Data are expressed as means + SD from three independent experiments, each conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A ) , Effect of actinonin on Cxcl2 production by neutrophils . Neutrophils were stimulated with live GBS ( MOI 5 ) in the presence or absence of the formyl peptidase inhibitor actinonin ( 32µg / ml ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(A), Effect of actinonin on Cxcl2 production by neutrophils. Neutrophils were stimulated with live GBS (MOI 5) in the presence or absence of the formyl peptidase inhibitor actinonin (32µg/ml). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of treatment with ROS scavengers / inhibitors on Cxcl2 production . Neutrophils were treated with the singlet oxygen scavenger MnTBAP ( 100 μM ) , the hydrogen peroxide scavenger DMTU ( 10 mM ) , the NADPH oxidase inhibitor DPI ( 20 μM ) or the ASK1 inhibitor GS - 444217 ( 1 μM ) for 1 h before the addition of live GBS ( MOI of 5 ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(D), Effect of treatment with ROS scavengers/inhibitors on Cxcl2 production. Neutrophils were treated with the singlet oxygen scavenger MnTBAP (100 μM), the hydrogen peroxide scavenger DMTU (10 mM), the NADPH oxidase inhibitor DPI (20 μM) or the ASK1 inhibitor GS-444217 (1 μM) for 1 h before the addition of live GBS (MOI of 5). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A - B ) Bone - marrow - derived neutrophils were exposed to different concentrations of signaling molecule ( A ) or transcription factors inhibitors ( B ) for 1 hour and then stimulated with live GBS ( MOI 5 ) . Target molecules are indicated in parentheses . Data are means + SD from three independent experiments conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(A-B) Bone-marrow-derived neutrophils were exposed to different concentrations of signaling molecule (A) or transcription factors inhibitors (B) for 1 hour and then stimulated with live GBS (MOI 5). Target molecules are indicated in parentheses. Data are means + SD from three independent experiments conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( C ) , Effect of p38MAPK inhibition on the activation of AP - 1 family members in neutrophils stimulated with live GBS . Neutrophils were pretreated with SB202190 ( 5μM ) , stimulated for 90min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(C), Effect of p38MAPK inhibition on the activation of AP-1 family members in neutrophils stimulated with live GBS. Neutrophils were pretreated with SB202190 (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on the activation of AP - 1 family members in neutrophils . Neutrophils from WT , Fpr - or MyD88 - deficient mice were stimulated for 90 min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 vs WT mice , as determined by unpaired t - test .

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(D), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on the activation of AP-1 family members in neutrophils. Neutrophils from WT, Fpr- or MyD88-deficient mice were stimulated for 90 min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001 vs WT mice, as determined by unpaired t-test.


( E ) , Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) .

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(E), Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS (MOI 5).


( F ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) . Shown is a blot representative of three separate experiments .

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(F), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS (MOI 5). Shown is a blot representative of three separate experiments.


A ) ELISA quantification of PD - L1 , pTau 181 and Aβx - 42 in the CSF of control and AD patients ( n = 10 + / - SEM , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , PD - L1 : t = 2 . 47 df = 18 , Aβx - 42 : t = 4 . 83 df = 18 , pTau 181 : t = 5 , 768 df = 18 ) .

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A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).


B , C ) ( B ) Colocalization of PD - L1 and GFAP in AD and ( C ) in control patients ( MX - O4 = amyloid deposits , upper picture : bar = 100 µm , lower picture : bar = 30 µm ) . D ) Immunohistochemical detection of PD - L1 in 9 - month - old female APP / PS1 mice ( bar = 50 µM ) . E ) Immunohistochemical detection of PD - L1 in 4 - month - old female APP / PS1 mice ( bar = 20 µm ) .

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B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).


A ) C6 cells expressing PD - L1 - myc treated with the γ - secretase inhibitor DAPT for 18 h . PD - L1 was detected using a myc - antibody .

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A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.


B ) Immunoblot of HEK293 cells expressing PD - L1 - myc and presenilin 1 ( hPS1 ) or a presenilin 1 dominant negative mutant ( hPS1 D246A ) .

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B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).


D ) 18 h 1 μM PMA treatment of C6 cells expressing PD - L1 - myc . Secretion of PD - L1 in the conditioned medium ( CM ) was detected using antibody AF1019 .

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D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.


E ) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg / ml TNFα / 100 U / μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019 .

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E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.


A ) Colocalization of PD - 1 and CD11b ( MX - 04 = methoxy - XO4 , stains amyloid deposits ) in AD by immunohistochemistry ( bar = 50 µm ) . B ) Colocalization of PD - 1 and Iba1 or PD - 1 and Aβ in a female APP / PS1 mouse at 9 months of age by immunohistochemistry ( bar = 20 µm ) .

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A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm).


E ) ELISA analysis of TNFα secretion by wild type ( wt ) and PD - 1 - / - microglia induced by 0 . 5 µM Aβ1 - 42 for 6 h in vitro ( n = 3 biological replicates , one - way ANOVA ( DF = 1 , F = 8 . 29 , p = 0 . 021 ) , Tukey ' s HSD , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


F - H ) Microglia from methoxy - XO4 - injected , 8 - month - old female , wild type ( wt ) and APP / PS1 mice were analyzed ( F ) for PD - 1 ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 13 . 99 , p = 0 . 0013 ) , Tukey ' s HSD , * * p < 0 . 01 ) , ( G ) for CD11b ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 30 . 06 , p = 5 . 47x10 - 05 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and ( H ) CD45 by flow cytometry ( n = 5 biological replicates per group , one - way ANOVA ( DF = 1 , F = 24 . 96 , p = 0 . 00013 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and normalized to the expression of wild type mice ( represented by dashed lines ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


A , B ) ELISA of the ( A ) RIPA soluble fraction of female APP / PS1 and APP / PS1 PD - 1 - / - mice for Aβx - 40 ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 6 , p = 0 . 45 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( B ) of the SDS soluble fraction ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 32 . 8 , p = 2x10 - 5 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).


D , E ) Mice were analyzed for ( D ) percentage of the covered areas ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 905 , p = 0 . 90 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( E ) the number of plaques per mm2 ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 4 . 08 , p = 0 . 054 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).


G ) Time needed to reach the hidden platform ( latency in seconds ) in the Morris water maze test ( mean + / - SEM of biological replicates with n = 12 for wt , n = 5 for PD - 1 - / - , n = 7 for APP / PS1 , and n = 8 for APP / PS1 PD - 1 - / - , one - way ANOVA ( DF = 3 , F = 17 . 72 , p = 1x10 - 9 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).


A ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type or PD - 1 - / - microglia for up to 6 h ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) . B ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type microglia cultured in astrocyte - conditioned medium ( ACM ) from wild type or PD - L1 - / - astroglial cultures ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate )

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A) In vitro phagocytosis of FAM-Aβ1-42 by wild type or PD-1-/- microglia for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis of FAM-Aβ1-42 by wild type microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate)


D ) Quantification of microglial in vivo phagocytosis in female APP / PS1 and APP / PS1 PD - 1 - / - mice at 8 . 5 month of age after intraperitoneal injection of methoxy - XO4 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , Student ' s t - test ( t = 2 . 1808 , df = 6 . 9829 ) , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Quantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


E - G ) Relative expression of the cell surface marker ( E ) CD36 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , ANOVA ( DF = 1 , F = 0 . 002 , p = 0 . 94 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) , ( F ) CD11b ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) and ( G ) CD45 on methoxy - XO4 + versus methoxy - XO4 - microglia in APP / PS1 and APP / PS1 PD - 1 - / - mice normalized to methoxy - X04 - negative cells from APP / PS1 mice ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


B ) Different expression of inflammasome - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia . C ) Different expression of complement system - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.


( a - d ) ELISA for IL - 1β ( a - c ) , IL - 18 ( d ) and IL - 6 ( e ) in supernatants of resting or LPS - primed bone marrow - derived macrophages ( BMMs ) stimulated with palmitate conjugated to BSA ( PA - BSA ) or BSA control .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-d) ELISA for IL-1β (a-c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control.


( e , f ) ELISA for IL - 1β ( e ) and IL - 18 ( f ) in supernatants . Resting or LPS - primed BMMs generated from wild - type ( WT ) , Nlrp3 − / − , Pycard − / − or Nlrc4 − / − mice were stimulated with PA - BSA .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA.


( g ) IL - 1β ELISA of supernatants from LPS - primed BMMs stimulated with PA - BSA in the absence or presence of the pan - caspase inhibitor zVAD or caspase - 1 inhibitor zYVAD .

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(g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD.


( h ) ELISA for IL - 1β in supernatants . Resting or LPS - primed BMMs generated from WT or Casp1 - / - mice were stimulated with PA - BSA , BSA control , ATP or nigericin .

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(h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1-/- mice were stimulated with PA-BSA, BSA control, ATP or nigericin.


( i ) ELISA for TNF in supernatants . Resting or LPS - primed BMMs generated from WT , Nlrp3 - / - , Pycard - / - or Casp1 - / - mice were stimulated with PA - BSA as indicated . Values are expressed as mean ± s . d . , and the results are representative of three independent experiments .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3-/-, Pycard-/- or Casp1-/- mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.


Immunoblotting for procaspase - 1 and activated caspase - 1 ( p10 ) , pro - IL - 1β and cleaved IL - 1β ( p17 ) in supernatants ( Sup ) and cell lysates ( Lys ) from resting or LPS - primed BMMs generated from WT , Nlrp3 − / − , Pycard − / − ( a ) or Nlrc4 − / − ( b ) mice stimulated with PA - BSA ( 0 . 2 or 0 . 5 mM ) as indicated at top . Data are representative of two independent experiments .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.


( a ) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 ( DHR ) in LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of ROS inhibitor APDC ( 50 μM ) . MFI , mean fluorescence intensity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity.


( b ) ELISA for IL - 1β on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC .

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(b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( c ) immunoblotting for caspase - 1 p10 and IL - 1β p17 on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( d ) ELISA for IL - 1β . Resting or LPS - primed BMMs were stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of the ROS inhibitors APDC ( 10 or 50 μM ) and N - acetyl - cysteine ( NAC , 5 or 25 mM ) or the NAPDH oxidase inhibitor diphenyleneiodonium ( DPI , 5 or 25 μM ) . Values are expressed as mean ± s . d . In a , one of two independent experiments is shown . In each of b - d , the results are representative of three independent experiments .

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(d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.


( a ) Thr172 - phosphorylated ( p - ) and total AMPK - α subunit determined by immunoblotting . Resting or LPS - primed BMMs were untreated or stimulated with PA - BSA ( 0 . 5 mM ) for 24 h .

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(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( b ) ROS production determined using the fluoroprobe DHR .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( c , d ) IL - 1β ( c ) and IL - 6 ( d ) in supernatants , measured by ELISA .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( e ) Immunoblotting for caspase - 1 , IL - 1β and AMPK signaling molecules .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules.


( f ) Transfection of BMMs with empty vector ( EV ) or constitutively active ( CA ) AMPK - α1 subunit . N , non - transfected . Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK - α1 and pmaxGFP . Values are percentages of GFP - positive cells in the ranges indicated by the bars . ( g ) ELISA for IL - 1β in supernatants of cells transfected as in f and stimulated with PA - BSA ( 0 . 5 mM ) for 24 h . In each of a , b and f , one of two independent experiments is shown . In each of c - e and g , the results are representative of three independent experiments . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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(f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a ) Immunoblotting for AMPK activation ( Thr172 p - AMPK - α ) and LC3B ( top ) , and densitometric analysis to quantify ratio of LC3B - II to actin ( bottom ) . BMMs were pretreated with LPS ( 200 ng / ml ) for 3 h , followed by PA - BSA ( 0 . 5 mM ) treatment for 24 h in the absence or presence of chloroquine ( 50 μM ) . Med , medium control .

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(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control.


( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) .

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(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right).


( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) . Cells were fixed and examined by transmission electron microscopy for autophagosomes ( c ) . Quantitation ( c , right ) is based on counting autophagosomes in ten cells per treatment .

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(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (c). Quantitation (c, right) is based on counting autophagosomes in ten cells per treatment.


( d ) Immunoblotting ( left ) and densitometric analysis to quantify ratio of LC3B - II to actin and ULK1 species ( right ) in treated BMMs .

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(d) Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs.


( e ) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator . In each of a and e , one of three independent experiments is shown . In each of b and d , one of two independent experiments is shown . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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(e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a , b ) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL - 1β ( 2 ng / ml ) ( a ) or TNF ( 2 ng / ml ) ( b ) for 24 h , then stimulated with insulin ( 200 nM ) for 10 min . p - Akt ( Ser473 ) was determined by flow cytometry ( right ) with quantification shown in the graph at left ( a ) . p - Akt ( Ser473 ) was determined by immunoblotting ( b ) .

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(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).


( c ) p - IRS1 ( Ser307 ) , determined by immunoblotting , in FL83B mouse liver cells treated with either IL - 1β or TNF for 24 h .

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(c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without stimulation .

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(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without

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(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation


( g ) Akt serine 473 phosphorylation measured by flow cytometry . FL83B cells were pretreated with WT or Pycard − / − CM in the absence or presence of the IL - 1 receptor antagonist anakinra ( 1 μg / ml ) for 24 h , then stimulated with insulin .

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(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.


( h ) p - Akt ( Ser473 ) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF . The results shown are representative of three independent experiments and are expressed as mean ± s . d .

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(h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.


( a ) ITT performed 2 h after IL - 1β injection . Eight - week old male C57BL / 6 mice were intraperitoneally injected with saline or mouse recombinant IL - 1β ( 1 μg / kg ) .

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(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg).


( b ) ITT in WT mice on regular diet ( RD ) or HFD for 12 weeks , or Il1b - / - mice on HFD for 12 weeks .

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(b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b-/- mice on HFD for 12 weeks.


( c , d ) ITT ( c ) and serum IL - 1β ( d , left ) and TNF ( d , right ) determined by ELISA , 2 h after IL - 1β injection . Recombinant mouse IL - 1β was administered to WT and Tnfa − / − mice ( n = 5 for each group ) . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


( a ) Blood glucose and insulin levels measured in WT ( n = 6 ) , Nlrp3 − / − ( n = 5 ) or Pycard − / − ( n = 6 ) mice under fasting or refed conditions after 12 weeks of HFD .

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(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.


( b - e ) Glucose tolerance test ( GTT ) ( b , d ) and insulin tolerance test ( ITT ) ( c , e ) performed for WT , Nlrp3 − / − ( b , c ) and Pycard − / − ( d , e ) mice on HFD for 12 weeks .

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(b-e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks.


( f ) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks .

(f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks.


( g , h ) Insulin - stimulated phosphorylation of IRβ , IRS1 and Akt ( Ser473 ) in liver tissues of individual WT and Pycard − / − mice ( g ) , and p - Akt ( Ser473 ) in liver , white adipose ( WAT ) and muscle tissues of individual WT and Nlrp3 − / − mice ( h ) on HFD for 12 weeks , after insulin ( 2 IU / kg ) infusion . Graphs at right of blots show the quantitation of each molecule . pY , phosphotyrosine .

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(g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine.


( i , j ) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3 − / − mice ( i ) and Pycard − / − mice ( j ) on regular diet ( RD ) or HFD for 12 weeks . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


B . Phenotype of the patients as indicated . ( a ) Sparse woolly hair . ( b ) Protrusion of upper lip due to deformity of teeth . ( c ) Ichthyosis - like fine scale and erythema .

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B. Phenotype of the patients as indicated. (a) Sparse woolly hair. (b) Protrusion of upper lip due to deformity of teeth. (c) Ichthyosis-like fine scale and erythema.


C . Images of the heart of the patients as indicated . ( a ) M - mode echocardiography taken from the parasternal long axis view of the left ventricle ( LV ) illustrating poor LV function with severe involvement of the interventricular septum ( IVS ) and reduced motion of the left ventricular posterior wall ( LVPW ) . ( b ) bi - dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function . The SMOG is enhanced in the right atrium . ( c ) bi - dimensional apical four chamber view with free tricuspid valve regurgitation ( TR ) due to lack of cooptation of the tricuspid valve leaflets during systole ( marked by a red bar ) as a result of critical reduction of right ventricular function and right ventricular enlargement . ( d ) Right and left ventricular thrombi seen in the short axis apical parasternal view . T1 - large right ventricular thrombus , T2 & amp ; amp ; T3 - left ventricular thrombi . ( e ) The color - flow Doppler picture : TR . Free regurgitation of the tricuspid valve shown in modified four chambers apical view . RA - right atrium , RV - right ventricle , LA - left atrium , LV - left ventricle , TR - tricuspid regurgitation color - flow Doppler .

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C. Images of the heart of the patients as indicated. (a) M-mode echocardiography taken from the parasternal long axis view of the left ventricle (LV) illustrating poor LV function with severe involvement of the interventricular septum (IVS) and reduced motion of the left ventricular posterior wall (LVPW). (b) bi-dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function. The SMOG is enhanced in the right atrium. (c) bi-dimensional apical four chamber view with free tricuspid valve regurgitation (TR) due to lack of cooptation of the tricuspid valve leaflets during systole (marked by a red bar) as a result of critical reduction of right ventricular function and right ventricular enlargement. (d) Right and left ventricular thrombi seen in the short axis apical parasternal view. T1-large right ventricular thrombus, T2&amp;T3-left ventricular thrombi. (e) The color-flow Doppler picture: TR. Free regurgitation of the tricuspid valve shown in modified four chambers apical view. RA-right atrium, RV-right ventricle, LA-left atrium, LV-left ventricle, TR-tricuspid regurgitation color-flow Doppler.


D . Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10 . a c - post - mortem heart sections ( 2 . 7Y ) . ( a ) Prominent fibrosis and lymphocytic interstitial inflammation ( Hematoxylin Eosin x50 ) . ( c ) Sub - epicardial inflammation and mild interstitial " myocarditis " ( Hematoxylin Eosin x100 ) . ( b d ) - Fetal heart ( VI7 ) homozygous for p . Tyr747Ter . No inflammatory infiltrate or other histological abnormalities are seen . ( b ) Normal arrayed myocardium ( Hematoxylin Eosin x50 ) . ( d ) Sub - epicardial region with no histopathological changes . ( Hematoxylin Eosin x100 ) .

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D. Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10. a c- post-mortem heart sections (2.7Y). (a) Prominent fibrosis and lymphocytic interstitial inflammation (Hematoxylin Eosin x50). (c) Sub-epicardial inflammation and mild interstitial "myocarditis" (Hematoxylin Eosin x100). (b d)- Fetal heart (VI7) homozygous for p.Tyr747Ter. No inflammatory infiltrate or other histological abnormalities are seen. (b) Normal arrayed myocardium (Hematoxylin Eosin x50). (d) Sub-epicardial region with no histopathological changes. (Hematoxylin Eosin x100).


F . Segregation of c . 2241C > G , p . Y747X in PPP1R13L in the studied family : The affected girl VI10 , is homozygous for the causative SV p . Y747X , as are the four fetuses that were aborted . The parents are obligate carriers as shown and , of the two healthy sisters , one carries the wild allele only , and the other is heterozygous for the causative SV .

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F. Segregation of c.2241C> G, p.Y747X in PPP1R13L in the studied family: The affected girl VI10, is homozygous for the causative SV p.Y747X, as are the four fetuses that were aborted. The parents are obligate carriers as shown and, of the two healthy sisters, one carries the wild allele only, and the other is heterozygous for the causative SV.


A . PPP1R13L mRNA expression level is lower in VI10 PDFs ( set as 1 ) than in CFs .

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A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs.


B . and C . Western blots ( WBs ) analyses demonstrated the absence of iASPP in VI10 , VI4 and VII1 PDFs .

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B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs.


D . WB analysis demonstrated that PPP1R13L - shRNA downregulated the putative iASPP band .

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D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.


A . Higher induction of inflammatory cytokine mRNA levels in LPS - stimulated VI10 PDFs than in CFs . The expression level in resting conditions of the PDFs was set as 1 . Differences between knockdown and control with p values ≤ 0 . 05 ( Student ' s t test ) are indicated with an asterisk . B . as in A . for patient VII1 .

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A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1.


C . knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks in the graph .

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C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks in the graph.


A . WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs .

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A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs.


B . ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS - stimulated PDFs than in LPS - stimulated - CFs . The binding activity at the IL1B promoter was set as 1 . Differences between knockdown and control with p value = 0 . 00436 ( Student ' s t test ) are indicated with an asterisk .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk.


C . NF - kB inhibitor BOT - 64 blocked the inducible mRNA expression of the inflammatory cytokines . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with an asterisks and exact values .

O O O O O O O O O O O O O O O O O O O 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. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.


A . WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes .

O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O

A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes.


B . qPCR showed that ppp1r13l - knocked down murine cardiomyocytes ( using two alternative shRNA sequences ) expressed higher level of pro - inflammatory cytokine mRNAs after 4 hrs of LPS stimulation . The expression in ppp1r13l - knocked down murine cardiomyocytes was set as 1 . Genome wide expression analysis is presented in Figure 1S . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks and exact values .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.


A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C ( Mybpc3 ) , phospholamban ( Pln ) , ryanodine receptor 2 ( Ryr2 ) , and sarcoglycan gamma ( Sgcg ) . Hearts removed from perfused mice and mRNA was subjected for q PCR . Difference between knockdown and control with p values = 0 . 03 ( Student ' s t test ) is indicated with an asterisk .

O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student's t test) is indicated with an asterisk.


B . A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn , 7 - week - and 12 - week - hearts ( p < 0 . 05 threshold , calculated by Deseq algorithm " R " ) . The expression level of s100a9 was higher in wa3 mice than in WT by 2 . 8 times . For the full list of genes see Tables S1 and S2 , and functional analyses in Figures S3 - S6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm "R"). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6.


C . Hierarchical clustering heat map of differentially expressed genes in the hearts of 7 - and 12 - week old wa3 mice in comparison to WT .

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C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.


A . LPS ( 5mg / Kg for 2 hrs ) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts . The results are presented as fold induction of untreated controls . The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts , while of Mybpc3 and Sgcg was unchanged . For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7 . Differences between knockdown and control with p values = 0 . 04998 ( Student ' s t test ) are indicated with an asterisk .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk.


B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS ( 1mg / Kg ) .

O O O O O 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 shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).


A - D SQST ‐ 1 : : GFP is very weakly expressed and diffusely localized in the cytoplasm in wild ‐ type larval intestine , but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae . ( A ) and ( C ) represent DIC images of the animals in ( B ) and ( D ) , respectively .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.


E Compared to wild ‐ type , SQST ‐ 1 : : GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay . SQST ‐ 1 : : GFP levels in bp399 mutants are greatly reduced after 12 h of starvation . 200 young adult animals for each genotype were collected for analysis .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.


H , I SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants are separable from LAAT ‐ 1 : : Cherry ‐ labeled lysosomes under normal conditions , but are delivered to lysosomes upon starvation . Some aggregates are enclosed by lysosomes ( inserts ) . Scale bar : 20 μm .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.


J - Q RNAi inactivation of let ‐ 363 , rpn ‐ 2 , cogc ‐ 1 , or hgrs ‐ 1 suppresses the accumulation of SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants . ( J ) , ( L ) , ( N ) , and ( P ) : DIC images of the animals in ( K ) , ( M ) , ( O ) , and ( Q ) . Scale bar : 20 μm .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.


R SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are greatly reduced by inactivation of rpt ‐ 3 and rpn ‐ 2 . 200 young adult animals for each genotype were collected for analysis .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.


A , B SQST ‐ 1 : : GFP aggregates are absent in the rpl ‐ 43 ( bp399 ) ; sma ‐ 3 ( wk20 ) mutant intestine .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.


C Percentage of the indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : many SQST ‐ 1 : : GFP aggregates in all intestinal cells , as in rpl ‐ 43 ( bp399 ) mutants ; M : fewer SQST ‐ 1 : : GFP aggregates in some but not all intestinal cells ; N : no or very few SQST ‐ 1 : : GFP aggregates in intestinal cells . For each genotype , > 30 animals were examined .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.


D SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are dramatically decreased in a Western blot when sma ‐ 3 , lin ‐ 35 , or daf ‐ 2 is simultaneously depleted . Loss of function of daf ‐ 16 partially restores SQST ‐ 1 levels in rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutants .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.


E Endogenous LGG ‐ 1 is elevated in a Western blot in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants .

E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.


F mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


G , H No SQST ‐ 1 : : GFP aggregates are observed in the lin ‐ 35 ( n745 ) ; rpl ‐ 43 ( bp399 ) mutant intestine .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.


I , J Numerous SQST ‐ 1 : : GFP aggregates are formed in rpl ‐ 43 ( bp399 ) ; lin ‐ 15A ( n767 ) mutants .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.


K Endogenous LGG ‐ 1 levels are increased in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants .

K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.


L mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


M , N rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutant larvae contain no intestinal SQST ‐ 1 : : GFP aggregates .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.


O , P SQST ‐ 1 : : GFP aggregates are partially restored in daf ‐ 16 ( mu86 ) ; rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) animals .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.


A , B hsp ‐ 4 : : GFP is very weakly expressed in wild ‐ type animals .

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A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.


C Percentage of indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined for each genotype .

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C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.


D , E Phsp ‐ 4 : : GFP expression is much higher in rpt ‐ 3 ( RNAi ) animals .

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D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.


F , G Upregulation of Phsp ‐ 4 : : GFP expression in rpt ‐ 3 ( RNAi ) animals is suppressed by the xbp ‐ 1 ( zc12 ) mutation .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.


H , I SQST ‐ 1 : : GFP aggregates are absent in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.


J , K Loss of function of xbp ‐ 1 restores SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.


M The increase in bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 mRNA levels in npl ‐ 4 . 1 , rpt ‐ 3 , and pep ‐ 2 RNAi animals is dependent on xbp ‐ 1 . * P 0 . 05 , * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


A Phsp ‐ 60 : : GFP is weakly expressed in wild ‐ type animals .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

A Phsp‐60::GFP is weakly expressed in wild‐type animals.


B egl ‐ 46 ( RNAi ) causes upregulation of Phsp ‐ 60 : : GFP .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O

B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.


C Percentage of indicated mutant animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined in each group .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.


D Levels of SQST ‐ 1 : : GFP in various genetic mutants . 200 young adult animals for each genotype were collected for analysis .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.


E , F Loss of function of atfs ‐ 1 suppresses the upregulation of Phsp ‐ 60 : : GFP in egl ‐ 46 ( n1126 ) mutants . ( E ) : DIC image of ( F ) .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).


G , H The dramatic decrease in the number of SQST ‐ 1 : : GFP aggregates in the intestine in rpl ‐ 43 ( bp399 ) ; egl ‐ 46 ( RNAi ) mutants ( G ) is restored by simultaneous loss of function of atfs ‐ 1 ( gk3094 ) ( H ) .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).


I , J GFP : : LGG ‐ 1 forms numerous puncta in the intestine in egl ‐ 46 ( RNAi ) ( I ) , gfi ‐ 1 ( RNAi ) ( J ) animals .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.


( a ) Western blot analysis of myosin - VI - depleted HeLa cells untreated or treated with 100 nM bafilomycin A1 . ( b ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 , * * * P0 .

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(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001


( c ) Confocal immunofluorescence microscopy of LC3 punctae ( green ) in HeLa cells following knockdown of myosin VI . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images ( d ) Quantification of LC3 - and p62 - positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ; n = 3 ) from > 1 , 500 cells per experiment .

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(c) Confocal immunofluorescence microscopy of LC3 punctae (green) in HeLa cells following knockdown of myosin VI. Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images (d) Quantification of LC3- and p62-positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell (±s.d.; n = 3) from >1,500 cells per experiment.


( e ) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA - resistant GFP - myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132 .

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(e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132.


( f ) Western blot analysis of MEFs cultured from wild - type and Snell ' s waltzer ( SV ) mice treated with 1 μM MG132 or 100 nM bafilomycin A1 .

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(f) Western blot analysis of MEFs cultured from wild-type and Snell's waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1.


( g ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 . ( h ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control . ( ± s . d . ; n = 3 ) .

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(g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3).


( i ) Western blot analysis of cortical neurons from wild - type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1 . ( j ) Quantification of western blot LC3 - II intensity was performed ( n = 2 ) . ( k ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control ( n = 2 ) .

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(i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2).


( l ) Western blot ( top ) and confocal immunofluorescence microscopy ( bottom ) of mock - or myosin - VI - siRNA - treated HeLa cells stably expressing the RFP - GFP - LC3 reporter . Hoechst labels the nuclei ( blue ) . ( m ) Quantitative data of RFP and GFP signal overlap from confocal images of mock - or myosin - VI - siRNA - treated HeLa cells expressing RFP - GFP - LC3 . The data are represented as the Pearson ' s coefficient of the RFP and GFP signal correlation from > 100cells per experiment ( ± s . d . ; n = 3 ) .

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(l) Western blot (top) and confocal immunofluorescence microscopy (bottom) of mock- or myosin-VI-siRNA-treated HeLa cells stably expressing the RFP-GFP-LC3 reporter. Hoechst labels the nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock- or myosin-VI-siRNA-treated HeLa cells expressing RFP-GFP-LC3. The data are represented as the Pearson's coefficient of the RFP and GFP signal correlation from >100cells per experiment (±s.d.; n = 3).