Figure 7Anti - nuclear - IgM ( ANA ) of control - immunized ( CI , n = 3 ) and complex InsA peptide ( cInsA ) immunized mice on day 7 ( n = 3 ) and day 85 ( n = 3 ) analyzed by indirect immunofluorescence . We used ( E ) total serum ( dilutions : 1 : 20 ) or ( G ) Insulin - specific IgM ( concentration : 500 ng / mL ) purified from serum at indicated days on commercial HEp - 2 slides . Scale bar : 10 µm . Green fluorescence indicates IgM bound to nuclear structures . Data are representative for three independent experiments with isotype control : n = 3 , day 7 : n = 3 , day 85 : n = 3 . Coomassie stained SDS - page showing primary ( cInsA d7 ) and memory ( cInsA d85 ) Insulin - specific purified IgM pre - incubated with Insulin and calf thymus dsDNA . Samples were loaded onto the gel after size exclusion with a cut - off at 10 . 000 kD ( fractions referring to > / < 104 kD ) . IgM heavy chain ( HC ) : 69 kD , IgM light chain ( LC ) : 25 kD , J - chain : 15 kD . Data shown are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Anti-nuclear-IgM (ANA) of control-immunized (CI, n=3) and complex InsA peptide (cInsA) immunized mice on day 7 (n=3) and day 85 (n=3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) Insulin-specific IgM (concentration: 500 ng/mL) purified from serum at indicated days on commercial HEp-2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n=3, day 7: n=3, day 85: n=3.Coomassie stained SDS-page showing primary (cInsA d7) and memory (cInsA d85) Insulin-specific purified IgM pre-incubated with Insulin and calf thymus dsDNA. Samples were loaded onto the gel after size exclusion with a cut-off at 10.000 kD (fractions referring to >/< 104 kD). IgM heavy chain (HC): 69 kD, IgM light chain (LC): 25 kD, J-chain: 15 kD. Data shown are representative of three independent experiments.


Figure 2 Total carbonyl - bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E . coli and D . radiodurans with and without irradiation . The left axis is the number of sequence - unique proteins detected as carbonylated . The right axis is the number of sites in total detected as carbonylated ( red ) or not oxidized ( black ) in peptides bearing at least one carbonyl . Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples . Volcano plots for relative protein abundance changes measured by mass spectrometry in E . coli ( left ) and D . radiodurans ( right ) after irradiation using the same biological replicates as in Fig 2A . Black - circled points are those proteins with significant changes ( paired , 2 - sided t - test p - value < 0 . 05 ) of > 2 - fold or < 0 . 5 - fold . Red points are proteins with at least one carbonylated peptide detected . Fold change and p - value cutoffs considered for significance are indicated by dashed lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples. Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.


Figure 3Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism . Ratios are given above each pair of bars . All proportions are significantly different between each RKPT and their respective carbonylation state by two - tailed z - test of two proportions ( p - values < 0 . 01 ; see Materials and Methods ) , meaning carbonylated proportions are not determined simply by relative prevalence of RKPT . See also Appendix Fig S1 . Prevalence of all canonical amino acids before irradiation of E . coli and D . radiodurans , combining all 3 biological replicates for each condition . Ratios are given above each pair of bars . All proportions are significantly different between species by two - tailed z - test of two proportions ( p - values < 0 . 01 ) . See also Fig EV1 and EV2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1.Prevalence of all canonical amino acids before irradiation of E. coli and D. radiodurans, combining all 3 biological replicates for each condition. Ratios are given above each pair of bars. All proportions are significantly different between species by two-tailed z-test of two proportions (p-values < 0.01). See also Fig EV1 and EV2.


Figure 1 ( A ) In vitro pull - downs between strep ( II ) - tagged DDB1 - DCAF12 and untagged wild type ( WT ) CCT5 or CCT5 ( 1 - 529 ) ( Δ ( Ct ) ) seen on a Coomassie - stained SDS - PAGE gel . ( B ) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1 - DCAF12 or terbium - coupled streptavidin ( Tb - SA ) ( n = 3 ) . Signal originating in the absence of TbDDB1 - DCAF12 is unspecific and was subtracted in subsequent experiments . ( C ) TR - FRET counter - titration of unlabeled wild type ( WT ) CCT5 or an unlabeled CCT520 peptide into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) . ( D ) TR - FRET counter - titrations of label - free CCT5 C - terminal peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of the peptides and IC50 values for the titrations are listed on the table . Peptides are labeled for their degron position and corresponding CCT5 amino acid number . ( E ) Counter - titration of unlabeled CCT510 mutant peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of the peptides and IC50 values for the titrations are listed on the table . Peptides are labeled for their degron position .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number. (E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position. D


Figure 3 ( D ) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type ( WT ) or mutant TbDDB1 - DCAF12 complexes ( n = 3 ) . Unspecific signal arising from the Tb - SA label was subtracted . ( E ) In vitro ubiquitination of CCT5 by wild type ( WT ) or mutant CRL4DCAF12 complexes in the presence of ubiquitin , ATP , E1 and E2 enzymes .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.


Figure 5 ( A ) Counter - titration of unlabeled CCT5 or unlabeled TRiC into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) . ( B ) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin , ATP , E1 and E2 enzymes . CCT5 , but not TRiC , is modified with poly - ubiquitin ( Ubn ) chains by CRL4DCAF12 . Free CCT5 protein carries an N - terminal strep ( II ) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC .

O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.


Figure 1 ( A ) Left , western blotting of PDI protein in replicative senescent hMSCs at early - passage ( EP ) and late - passage ( LP ) . GAPDH was used as the loading control . Right , statistical analysis of relative PDI protein expression levels . n = 3 independent experiments . ( B ) Left , western blotting of PDI protein in wild - type ( WT ) and Werner syndrome ( WS ) hMSCs . GAPDH was used as the loading control . Right , statistical analysis of relative PDI protein expression levels . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( C ) Left , western blotting of PDI protein in the liver of mice at young ( 2 months ) and old ( 22 months ) ages , GAPDH was used as the loading control . Right , statistical analysis of relative PDI protein expression levels . n = 6 mice . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( G ) Left , Ki67 staining of PDI + / + and PDI - / - hESCs . Scale bar , 25 µm . Right , statistical analysis of Ki67 - positive cells . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Left, western blotting of PDI protein in replicative senescent hMSCs at early-passage (EP) and late-passage (LP). GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 3 independent experiments. (B) Left, western blotting of PDI protein in wild-type (WT) and Werner syndrome (WS) hMSCs. GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(C) Left, western blotting of PDI protein in the liver of mice at young (2 months) and old (22 months) ages, GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 6 mice. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(G) Left, Ki67 staining of PDI+/+ and PDI-/- hESCs. Scale bar, 25 µm. Right, statistical analysis of Ki67-positive cells. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


Figure 2 ( C ) Immunostaining of PDI and calreticulin ( CRT ) in PDI + / + and PDI - / - hMSCs , CRT was used as ER marker . Scale bar , 25 µm . ( D ) Western blotting analysis of PDI family members expression in PDI + / + and PDI - / - hMSCs at early - passage ( P3 ) and late - passage ( P8 ) . GAPDH was used as the loading control . ( G ) Left , Ki67 staining of PDI + / + and PDI - / - hMSCs at P8 . Scale bar , 20 µm . Right , statistical analysis of Ki67 - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( H ) Left , SA - β - gal staining of PDI + / + and PDI - / - hMSCs at P8 . Scale bar , 200 µm . Right , statistical analysis of SA - β - gal - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( K ) Left , western blotting showing the P16 expression in PDI + / + and PDI - / - hMSCs at P8 . GAPDH was used as the loading control . Right , statistical analysis of relative P16 protein expression levels . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( M ) Left , Immunostaining of 53BP1 and γH2AX in PDI + / + and PDI - / - hMSCs at P8 . White arrowheads indicate 53BP1 and γH2AX double positive cells . Right , statistical analysis of 53BP1 and γH2AX double positive cells . Scale bar , 10 µm . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( P ) Left , Ki67 staining of PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI . Scale bar , 10 µm . Right , statistical analysis of Ki67 - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( Q ) Left , SA - β - gal staining of PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI . Scale bar , 200 µm . Right , statistical analysis of SA - β - gal - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Immunostaining of PDI and calreticulin (CRT) in PDI+/+ and PDI-/- hMSCs, CRT was used as ER marker. Scale bar, 25 µm.(D) Western blotting analysis of PDI family members expression in PDI+/+ and PDI-/- hMSCs at early-passage (P3) and late-passage (P8). GAPDH was used as the loading control.(G) Left, Ki67 staining of PDI+/+ and PDI-/- hMSCs at P8. Scale bar, 20 µm. Right, statistical analysis of Ki67-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(H) Left, SA-β-gal staining of PDI+/+ and PDI-/- hMSCs at P8. Scale bar, 200 µm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(K) Left, western blotting showing the P16 expression in PDI+/+ and PDI-/- hMSCs at P8. GAPDH was used as the loading control. Right, statistical analysis of relative P16 protein expression levels. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(M) Left, Immunostaining of 53BP1 and γH2AX in PDI+/+ and PDI-/- hMSCs at P8. White arrowheads indicate 53BP1 and γH2AX double positive cells. Right, statistical analysis of 53BP1 and γH2AX double positive cells. Scale bar, 10 µm. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(P) Left, Ki67 staining of PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI. Scale bar, 10 µm. Right, statistical analysis of Ki67-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(Q) Left, SA-β-gal staining of PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI. Scale bar, 200 µm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


Figure 3 ( A ) Oxidative folding of Myc - tagged J - Chain ( JcM ) . PDI + / + and PDI - / - hMSCs were transfected with JcM for 48 hr , then were pulsed with DTT and chased at indicated time after DTT removal . The lysates were analyzed using non - reducing SDS - 15 % PAGE and α - Myc western blotting . The mobility of reduced JcM monomers ( Red ) , oxidized monomers ( Oxi ) , homodimers ( Dim ) , and high - molecular - weight ( HMW ) species are indicated . ( D ) Time - dependent changes of H2O2 levels in the ER of PDI + / + and PDI - / - hMSCs at P8 . Left , the fluorescence intensity ratio of HyPer - ER at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min , followed by addition of 0 . 5 mM DTT ( yellow bar ) for another 15 min . Right , statistical analysis of the fluorescence intensity ratio at 488 / 405 nm excitation of HyPer - ER probe at 15 min post DTT addition . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( E ) Analysis of the H2O2 levels in PDI + / + and PDI - / - hMSCs at P8 using Amplex Red assays . n = 3 biological repeats . ( F ) Analysis of the cellular total ROS levels in PDI + / + and PDI - / - hMSCs at P8 using H2DCFDA probe . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( M ) Confocal imaging of PDI + / + and PDI - / - hMSCs expressing Hyper7 - nucleus at steady state and upon addition of 2 mM DTT . The fluorescence intensity ratio at 488 / 405 nm obtained from individual cells was calculated at indicated times with a representative false color image . Scale bar , 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Oxidative folding of Myc-tagged J-Chain (JcM). PDI+/+ and PDI-/- hMSCs were transfected with JcM for 48 hr, then were pulsed with DTT and chased at indicated time after DTT removal. The lysates were analyzed using non-reducing SDS-15% PAGE and α-Myc western blotting. The mobility of reduced JcM monomers (Red), oxidized monomers (Oxi), homodimers (Dim), and high-molecular-weight (HMW) species are indicated.(D) Time-dependent changes of H2O2 levels in the ER of PDI+/+ and PDI-/- hMSCs at P8. Left, the fluorescence intensity ratio of HyPer-ER at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min, followed by addition of 0.5 mM DTT (yellow bar) for another 15 min. Right, statistical analysis of the fluorescence intensity ratio at 488/405 nm excitation of HyPer-ER probe at 15 min post DTT addition. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(E) Analysis of the H2O2 levels in PDI+/+ and PDI-/- hMSCs at P8 using Amplex Red assays. n = 3 biological repeats. (F) Analysis of the cellular total ROS levels in PDI+/+ and PDI-/- hMSCs at P8 using H2DCFDA probe. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(M) Confocal imaging of PDI+/+ and PDI-/- hMSCs expressing Hyper7-nucleus at steady state and upon addition of 2 mM DTT. The fluorescence intensity ratio at 488/405 nm obtained from individual cells was calculated at indicated times with a representative false color image. Scale bar, 10 μm.


Figure 4 ( C , D ) Density plot showing that upregulated ( C ) or downregulated ( D ) genes in the LP hMSCs ( PDI + / + , LP vs . PDI + / + , EP ) that were restored after PDI deletion ( PDI - / - , LP vs . PDI + / + , LP ) . Comparisons were performed with two - sided Wilcoxon signed - rank test . ( K ) Conjoint analysis showing the secretion levels of PDI and those in ( I ) and various SASPs from multiple senescence inducers and in plasma proteome from 3 , 087 healthy individuals RS , replicative senescence ( PDI + / + , LP vs . PDI + / + , EP ) . ATV , atazanavir treatment ; RAS , inducible RAS overexpression ; IR , X - irradiation . The color keys that change from dark blue to dark purple represent the fold change of gene and protein , change from white to red represent the change in protein levels per unit increase in the covariate in plasma proteomic .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(C, D) Density plot showing that upregulated (C) or downregulated (D) genes in the LP hMSCs (PDI+/+, LP vs. PDI+/+, EP) that were restored after PDI deletion (PDI-/-, LP vs. PDI+/+, LP). Comparisons were performed with two-sided Wilcoxon signed-rank test.(K) Conjoint analysis showing the secretion levels of PDI and those in (I) and various SASPs from multiple senescence inducers and in plasma proteome from 3,087 healthy individuals RS, replicative senescence (PDI+/+, LP vs. PDI+/+, EP). ATV, atazanavir treatment; RAS, inducible RAS overexpression; IR, X-irradiation. The color keys that change from dark blue to dark purple represent the fold change of gene and protein, change from white to red represent the change in protein levels per unit increase in the covariate in plasma proteomic.


Figure 5 ( A ) qPCR analysis of SERPINE1 expression in PDI + / + and PDI - / - hMSCs at EP and LP . n = 3 independent experiments . Data information : In ( A , data are presented as mean ± SEM , two - tailed Student ' s t - test . ( B ) Western blotting analysis of SERPINE1 protein in PDI + / + and PDI - / - hMSCs at EP and LP . GAPDH was used as the loading control of cell lysates . Ponceau was used as the loading control of conditional medium . ( F ) Left , Ki67 staining in PDI - / - hMSCs transduced with sgNTC or SERPINE1 - targeting activation sgRNAs . Scale bar , 20 μm . Right , statistical analysis of Ki67 - positive cells . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( G ) Left , SA - β - gal staining in PDI - / - hMSCs transduced with sgNTC or SERPINE1 - targeting activation sgRNAs . Scale bar , 200 μm . Right , statistical analysis of SA - β - gal - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( H ) qPCR analysis of the SERPINE1 mRNA expression in PDI + / + and PDI - / - hMSCs treated with the indicated concentration H2O2 for 4 days . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( J ) H2O2 levels in PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer - ER at 488 / 405 nm excitation after the addition of 0 . 5 mM DTT for 15 min . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( K ) H2O2 levels in PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer7 - nucleus at 500 / 400 nm excitation after the addition of 2 mM DTT for 20 min . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) qPCR analysis of SERPINE1 expression in PDI+/+ and PDI-/- hMSCs at EP and LP. n = 3 independent experiments. Data information: In (A, data are presented as mean ± SEM, two-tailed Student's t-test.(B) Western blotting analysis of SERPINE1 protein in PDI+/+ and PDI-/- hMSCs at EP and LP. GAPDH was used as the loading control of cell lysates. Ponceau was used as the loading control of conditional medium.(F) Left, Ki67 staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 20 μm. Right, statistical analysis of Ki67-positive cells. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(G) Left, SA-β-gal staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 200 μm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(H) qPCR analysis of the SERPINE1 mRNA expression in PDI+/+ and PDI-/- hMSCs treated with the indicated concentration H2O2 for 4 days. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(J) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer-ER at 488/405 nm excitation after the addition of 0.5 mM DTT for 15 min. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(K) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer7-nucleus at 500/400 nm excitation after the addition of 2 mM DTT for 20 min. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


Figure 6 ( A ) Western blotting of PDI expression in replicative senescent ( RS ) hMSCs , Hutchinson - Gilford Progeria Syndrome ( HGPS ) hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . GAPDH was used as the loading control . ( B ) Single clonal formation assay in RS hMSCs , HGPS hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test . ( C ) SA - β - gal staining in RS hMSCs , HGPS hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . n = 3 biological repeats . Scale bar , 200 µm . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Western blotting of PDI expression in replicative senescent (RS) hMSCs, Hutchinson-Gilford Progeria Syndrome (HGPS) hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. GAPDH was used as the loading control.(B) Single clonal formation assay in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(C) SA-β-gal staining in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Scale bar, 200 µm. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


Figure 1 A1 ) Representative WBs showing DRD2 and RGS9 - 2 downregulation in adult ( P60 - P90 ) Tor1a + / - ( + / - ) striata , characterized by a reduced torsinA protein level with respect to Tor1a + / + ( + / + ) littermates . The striatal level of RGS7 is unchanged . The dot plot shows Tor1a + / - data , normalized to Tor1a + / + controls of the same experiment . TorsinA : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t test * * * P = 0 . 0003 ; DRD2 : Tor1a + / + N = 18 , Tor1a + / - N = 16 , t test * * P = 0 . 0029 ; RGS9 - 2 : Tor1a + / + N = 32 , Tor1a + / - N = 33 , t test * P = 0 . 0115 ) . RGS7 : Tor1a + / + N = 7 , Tor1a + / - N = 7 , t test P = 0 . 8721 ) . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type samples of the same experiment . A2 , A3 , A4 ) Time - course of changes in torsinA , DRD2 and RGS9 - 2 striatal levels along postnatal development . Summary plot data are normalized to the Tor1a + / + P60 sample of each independent experiment . A2 ) TorsinA level is significantly reduced in mutants throughout the developmental period considered ( P7 N = 3 ; P14 - P60 N = 4 ; one - way ANOVA P < 0 . 0001 and Bonferroni ' s Multiple Comparison Test * * P < 0 . 01 at P7 , P14 and P60 ) . A3 , A4 ) DRD2 and RGS9 - 2 levels show parallel courses , with a similar increase from P7 to P21 in Tor1a + / + as well as Tor1a + / - striatal lysates ( P7 : DRD2 N = 3 , RGS9 - 2 N = 4 ; P14 - P21 N = 4 ; one - way ANOVA with Bonferroni ' s Multiple Comparison Test P > 0 . 05 ) . At P60 , a simultaneous reduction of DRD2 and RGS9 - 2 levels is observed in Tor1a + / - striatal lysates ( N = 4 ; Tor1a + / - one sample t test DRD2 : * P = 0 . 0250 , RGS9 - 2 : * P = 0 . 0175 ) . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type P60 sample of the same experiment B ) Representative WB images and the dot plot show downregulation of torsinA , DRD2 and RGS9 - 2 striatal levels also in adult P60 - P90 Tor1a ∆ gag / + ( ∆ gag / + ) mice . TorsinA : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 12 , t test * * * P = 0 . 0006 ; DRD2 : Tor1a + / + N = 7 , Tor1a ∆ gag / + N = 9 , t test * P = 0 . 0183 ; RGS9 - 2 : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 9 , t test * P = 0 . 0192 . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type samples of the same experiment . Mean ± SEM is represented in the graphsC ) Left . Representative image of coronal striatal slices of fresh frozen brains of wild - type and Tor1a + / - mice probed with the DRD2 radioligand 3H - spiperone . Scale bar 1 . 5 mm . ( Right ) Graph showing DRD2 binding density in Tor1a + / - striatal sections , obtained from the densitometric quantification analysis , expressed as percentage variation compared to control

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment B) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphsC) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals


Figure 2A1 ) Representative WB of RGS9 - 2 , R7BP , and Gβ5 proteins on four Tor1a + / + and four Tor1a + / - striatal lysates . The bar histogram on the right shows the quantification of the three proteins in each sample . A2 ) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a + / - mice ( Tor1a + / + N = 11 , Tor1a + / - N = 10 , Mann Whitney test P = 0 . 2453 ) . A3 ) Conversely , the level of the R7 binding protein R7BP is increased in Tor1a + / - striatum ( Tor1a + / + N = 12 , Tor1a + / - N = 12 , t test * * P = 0 . 0014 ) . Values are represented as ratio of protein vs . loading control intensity level , normalized to the mean of Tor1a + / + control values of the same experimentB1 ) Representative WB of lysates of Tor1a + / + and Tor1a + / - dorsal striatum slices incubated for 5 hours in the presence ( w / leu ) or absence ( w / o leu ; contralateral striatum ) of the protease inhibitor leupeptin . Samples showing enhanced degradation of DRD2 and / or RGS9 - 2 proteins show additional bands at lower molecular weight . B2 ) Summary plot reporting mean ± SEM of RGS9 - 2 and DRD2 protein level values , expressed as the ratio of protein vs . loading control intensity level , normalized to the value of the Tor1a + / + w / leu sample measured in the same experiment . DRD2 : Tor1a + / + N = 4 , t test P = 0 . 4376 ; Tor1a + / - N = 4 , t test * P = 0 . 0427 ; RGS9 - 2 : Tor1a + / + N = 5 , t test * P = 0 . 0464 ; Tor1a + / - N = 5 , t test * P = 0 . 0190C1 , C2 , C3 , C4 ) Dot plots showing DRD2 , RGS9 - 2 , R7BP and Gβ5 protein levels measured in striatal detergent - resistant - membrane ( DRM ) preparations from Tor1a + / - ( + / - ) and WT ( + / + ) mice ( DRD2 : Tor1a + / + N = 16 , Tor1a + / - N = 13 , t test P = 0 . 0129 ; RGS9 - 2 : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t test * P = 0 . 0396 ; R7BP : Tor1a + / + N = 6 , Tor1a + / - N = 6 , t test * * * P = 0 . 0002 ; Gβ5 : Tor1a + / + N = 9 , Tor1a + / - N = 9 , t test P = 0 . 8461 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experimentB1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)


Figure 3WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a + / - mice . A ) The level of β - arrestin 2 ( β - Arr2 ) is not altered ( Tor1a + / + N = 11 , Tor1a + / - N = 12 , t test P = 0 . 6796 ) , whereas the level of spinophilin ( B ) is decreased ( Tor1a + / + N = 5 , Tor1a + / - N = 8 , t test * P = 0 . 0151 ) C ) A Sh - RGS9 - 2 - GFP viral preparation injected into the right striatum ( + ) of C57Bl / 6 mice effectively reduces both RGS9 - 2 and DRD2 protein levels , with respect to the contralateral striatum ( - ) . The summary plot reports mean ± SEM values ( RGS9 - 2 : N = 4 , paired t test * P = 0 . 0100 ; DRD2 : N = 4 , paired t test * P = 0 . 0432 ) D ) The endosomal marker Rab4 is upregulated in the Tor1a + / - striatum ( Tor1a + / + N = 13 , Tor1a + / - N = 14 , t test * * P = 0 . 0087 ) E ) Basal levels of markers of the degradative pathway , p62 and LC3 - II , show a trend towards upregulation in Tor1a + / - striatal lysates ( p62 : Tor1a + / + N = 11 , Tor1a + / - N = 11 , t test P = 0 . 2292 ; LC3 - II : Tor1a + / - N = 9 , Tor1a + / - N = 11 , t test P = 0 . 1574 ) F ) Treatment with bafilomycin A1 ( BafA1 ) induces a significantly more pronounced increase of LC3 - II in Tor1a + / - than in wild - type dorsal striatum slices . BafA1 - induced increase in LC3 - II level measured in Tor1a + / - slices was normalized to the increase observed in the Tor1a + / + samples of the same experiment ( N = 4 , one sample t test * * P = 0 . 0012 ) G ) LAMP - 2 , a lysosomal marker , shows a trend for upregulation ( Tor1a + / + N = 8 ; Tor1a + / - N = 6 , t test P = 0 . 2232 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151)C) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432)D) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087)E) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574)F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232)


Figure 4Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling ( red ) in choline acetyltransferase ( ChAT ) - positive ( cyano ) ChIs of Tor1a + / + and Tor1a + / - mice . A1 ) Two representative ChIs identified by the white box in the low magnification images ( bar = 20 µm ) are shown at higher magnification in ( A2 ) ( bar = 5 µm ) . A3 ) The graph reporting mean ± SEM values of p62 / ChAT signal overlap in ChIs , normalized to Tor1a + / + values of the same experiment , shows a significant increase of p62 in Tor1a + / - neurons ( Tor1a + / + n = 20 cells , N = 6 mice ; Tor1a + / - n = 20 cells , N = 6 mice , t test * * * P = 0 . 00008 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008)


Figure 5A ) Top . Low magnification merged confocal images of coronal striatal sections showing ChAT - labelled ( cyano ) ChIs in the area of mCherry fluorescence ( red ) indicating viral infection . Bar = 100 µm . Bottom . Higher magnification images of the ChIs identified by the white box in the upper images show viral - induced expression of mCherry and GFP signal . Bar = 20 µm . B ) The mCherry / ChAT overlap is increased in ChIs from Tor1a + / - mice ( Tor1a + / + n = 24 cells , Tor1a + / - n = 22 cells , N = 8 mice / genotype , t test * * * P = 0 . 0005 ) , but the mCherry / GFP fluorescence ratio indicated that the LC3 flux was unchanged ( Tor1a + / + n = 19 cells , Tor1a + / - n = 21 cells , N = 8 mice / genotype , Mann Whitney test P = 0 . 2334 ) C ) Merged and split channels confocal images ( bar = 10 µm ) of representative Tor1a + / + and Tor1a + / - ChIs showing red dots and yellow puncta ( indicated by white circles ) , quantified in ( D ) : red dots : Tor1a + / + n = 13 cells , Tor1a + / - n = 14 cells , t test * * * P = 0 . 0003 ; yellow dots : Tor1a + / + n = 13 cells , Tor1a + / - n = 14 cells , t test * * P = 0 . 0048 , N = 5 mice / genotype ) . Measurement of the mCherry / yellow dots ratio indicated that the autophagic flux was not altered ( Tor1a + / + n = 13 cells ; Tor1a + / - n = 14 cells , Mann Whitney test P = 0 . 8842 ) . Data in B , D ) are represented as mean ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)C) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM


Figure 6A ) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT ( cyano ) , DRD2 ( red ) , and the nuclear stain DAPI ( white ) . Bar = 200 μm . B1 , B2 ) Top . Representative ChAT - positive ( cyano ) cholinergic interneurons , labelled for DRD2 ( red ) , from a Tor1a + / + ( B1 ) or Tor1a + / - ( B2 ) striatal section . The colocalization mask of the two signals is shown in white ( right ) . Bar = 10 μm . Bottom . Higher magnification images ( bar = 5 μm ) of representative Tor1a + / + ( B1 ) and Tor1a + / - ( B2 ) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI ( green ) . A detail at higher magnification is shown in the

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets


Figure 7A ) Middle . Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum , reporting the coordinates relative to Bregma . HSV - LacZ ( left , grey ) and HSV - RGS9 - 2 ( right , red ) were injected into the left and right striatum , respectively , and mice sacrificed after 3 - 5 days . Left . Serial confocal images showing the extent of viral infection , as indicated by the robust dark β - gal staining . Right . The RGS9 - 2 immunolabelling ( red ) is particularly intense near the injection site , indicating enhanced protein expression with respect to the endogenous level . Bar = 200 µmB ) A representative WB shows correlated viral - mediated increases in RGS9 - 2 and DRD2 protein levels in Tor1a + / - dorsal striata ( lanes 4 and 5 ) . while in a sample where RGS9 - 2 level was not increased by viral infection ( lane 6 ) DRD2 level remained low as in the sham - injected striatum ( lane 7 ) . C , D ) The summary plots report mean ± SEM values of the ratio of protein vs . loading control intensity level measured from the injected ( HSV / LV ) or sham contralateral Tor1a + / - ( + / - ) striata , normalized to the Tor1a + / + ( + / + ) samples of the same experiment . C ) RGS9 - 2 : Tor1a + / + ( N = 10 ) ; Tor1a + / - injected ( N = 7 ) vs . contralateral ( N = 7 ) : paired t test * * P = 0 . 0011 ; D ) DRD2 : Tor1a + / + ( N = 9 ) ; Tor1a + / - injected ( N = 6 ) vs . contralateral ( N = 6 ) : paired t test * P = 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µmB) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380


Figure 8A ) As depicted in the cartoon and shown by representative confocal images , patch - clamp recordings were performed in the dorsal striatum , in the area of HSV - LacZ ( blue , left ) and HSV - RGS9 - 2 ( red , right ) viral infection , the latter showing an increased RGS9 - 2 immunolabelling with respect to the sham contralateral striatum of the same mouse . Representative confocal images at higher magnification show colocalization of RGS9 - 2 immunolabeling with Lucifer Yellow ( LY ) fluorescence in two ChIs loaded intracellularly with LY during whole - cell recordings in the infected areaB ) Representative patch - clamp recordings of ChIs from hMT ( top , perforated configuration ) and Tor1aΔgag / + ( bottom , cell - attached configuration ) mice , showing that viral - mediated delivery of RGS9 - 2 rescues the physiological response to the DRD2 agonist quinpirole ( 10 µM , 2 min ) . The dot plot reports the firing frequency changes induced by quinpirole in hMT striata ( N = 6 , HSV - RGS9 - 2 n = 9 , HSV - LacZ n = 14 , t test P < 0 . 0001 ) C ) Representative traces show a typical response of Tor1a + / + ChIs to quinpirole , characterized by a reduction in the frequency of the ongoing firing activity of the cell , as opposed to an aberrant increase in firing frequency in Tor1a + / - ChIs , quantified in the plot ( right , Tor1a + / + N = 6 , n = 14 ; Tor1a + / - N = 10 , n = 27 ; Tor1a + / + vs . Tor1a + / - in quinpirole t test * * * P = 0 . 0005 ; Tor1a + / - basal vs . quinpirole paired t test * * * P = 0 . 0009 ) D ) LV - mediated delivery of RGS9 - 2 rescues the physiological response to quinpirole in Tor1a + / - slices . Dot plot reporting firing frequency changes induced by quinpirole in Tor1a + / + ( N = 3 ) and Tor1a + / - ( N = 6 ) striatal ChIs infected with LV / HSV - RGS9 - 2 or control LV - GFP and HSV - LacZ ChIs ( Tor1a + / + RGS9 - 2 n = 7 ; Tor1a + / - RGS9 - 2 n = 9 ; Tor1a + / - LacZ / GFP n = 8 ; Tor1a + / - : LacZ / GFP vs . RGS9 - 2 t test * P = 0 . 0136 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected areaB) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001)C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009)D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)


Figure 1A Effects of myeloid - specific Lamtor1 deficiency on peritoneal IL - 1β production , CD11b + Ly6C + monocyte recruitment , and CD11b + Ly6G + neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution ( 20 mg / mL , 6 h ) , Data are shown as means ± SEM . * indicates P < 0 . 05 and * * * indicates P < 0 . 001 by Student ' s t - test , n = 6 mice ( peritoneal IL - 1β production ) and 4 mice ( FACS analysis ) . B Effects of myeloid - specific Lamtor1 deficiency in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) or the PBS control were injected intra - articularly into the tibia - tarsal joint of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre mice . Mice were assessed for joint swelling using electronic calipers . Images show photomicrographs ( upper panel ) and hematoxylin and eosin - stained sections ( middle and lower panels ) of ankle joints obtained at 24 h . Data are shown as the means ± SEM . * * * indicates P < 0 . 001 by Student ' s t - test , n = 7 mice each . C Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation . ELISA assay shows IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) , followed by nigericin ( 15 μM , 1 h ) , ATP ( 10 mM , 1 h ) , or GPN ( 100 nM , 6 h ) . D Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO different single clones , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . E , F Effects of Lamtor1 deficiency on IL - 1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS ( hydrogen peroxide ) . ELISA assay shows IL - 1β secretion in supernatants after treatment with LPS ( 200 ng / mL ) , Rotenone ( 10 μM ) , or Antimycin ( 10 μg / mL ) for 6 h or followed by hydrogen peroxide ( 3 . 86 mM ) . G Effects of Lamtor1 reconstitution in Lamtor1 - deficient macrophages on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in BMDM supernatants from Lamtor1flox / flox cells , Lamtor1flox / flox LysM - Cre cells , and Lamtor1flox / flox LysM - Cre cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with LPS ( 200 ng / mL , 4 h ) . H Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . I Effects of Lamtor1 deficiency on IL - 18 secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 18 secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . J Effects of Lamtor1 deficiency on IL - 1β secretion after Pyrin inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with TcdB ( 0 . 5 μg / mL , 3 h ) after LPS ( 200 ng / mL , 4 h ) stimulation . K Effects of Lamtor1 deficiency on IL - 1β secretion after AIM2 inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with Poly ( dA : dT ) ( 5 μg / mL , overnight ) and Lipofectamine 3000 after LPS ( 200 ng / mL , 4 h ) stimulation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Effects of myeloid-specific Lamtor1 deficiency on peritoneal IL-1β production, CD11b+ Ly6C+ monocyte recruitment, and CD11b+ Ly6G+ neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution (20 mg/mL, 6 h), Data are shown as means ± SEM. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 6 mice (peritoneal IL-1β production) and 4 mice (FACS analysis).B Effects of myeloid-specific Lamtor1 deficiency in an acute gouty arthritis model. MSU crystals (0.5 mg) or the PBS control were injected intra-articularly into the tibia-tarsal joint of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre mice. Mice were assessed for joint swelling using electronic calipers. Images show photomicrographs (upper panel) and hematoxylin and eosin-stained sections (middle and lower panels) of ankle joints obtained at 24 h. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 7 mice each.C Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation. ELISA assay shows IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h), followed by nigericin (15 μM, 1 h), ATP (10 mM, 1 h), or GPN (100 nM, 6 h).D Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO different single clones, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).E, F Effects of Lamtor1 deficiency on IL-1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS (hydrogen peroxide). ELISA assay shows IL-1β secretion in supernatants after treatment with LPS (200 ng/mL), Rotenone (10 μM), or Antimycin (10 μg/mL) for 6 h or followed by hydrogen peroxide (3.86 mM).G Effects of Lamtor1 reconstitution in Lamtor1-deficient macrophages on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in BMDM supernatants from Lamtor1flox/flox cells, Lamtor1flox/flox LysM-Cre cells, and Lamtor1flox/flox LysM-Cre cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with LPS (200 ng/mL, 4 h).H Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).I Effects of Lamtor1 deficiency on IL-18 secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-18 secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).J Effects of Lamtor1 deficiency on IL-1β secretion after Pyrin inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with TcdB (0.5 μg/mL, 3 h) after LPS (200 ng/mL, 4 h) stimulation.K Effects of Lamtor1 deficiency on IL-1β secretion after AIM2 inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with Poly(dA:dT) (5 μg/mL, overnight) and Lipofectamine 3000 after LPS (200 ng/mL, 4 h) stimulation.


Figure 2Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs ( A ) ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL , for 4 h ( BMDM ) stimulation . Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . WT and Lamtor1 KO THP - 1 cells ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL 2 h ( THP - 1 ) ] stimulation . C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation . Western blot shows GSDMD , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation . D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation and the percentage of propidium iodide positive cells were counted by flow - cytometry . E Effects of Lamtor1 deficiency on IL - 1β dynamics . Western blot shows IL - 1β and β - actin . WT cells and Lamtor1 KO cells were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) stimulation . F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with active form of GSDMD , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) . G Confirmation of the reconstitution of the active form Caspase - 1 ( p10 and p20 ) or GSDMD ( GSDMD - NT ) . H Effects of Lamtor1 deficiency on the priming phase . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with treated with LPS ( 200 ng / mL , 4 h ) . I Effects of IL - 1β and active form of Caspase - 1 reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with IL - 1β , active form of Caspase - 1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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Figure 2Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs (A) (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL, for 4 h (BMDM) stimulation.Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. WT and Lamtor1 KO THP-1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL 2 h (THP-1)] stimulation.C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation.D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation and the percentage of propidium iodide positive cells were counted by flow-cytometry.E Effects of Lamtor1 deficiency on IL-1β dynamics. Western blot shows IL-1β and β-actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).G Confirmation of the reconstitution of the active form Caspase-1 (p10 and p20) or GSDMD (GSDMD-NT).H Effects of Lamtor1 deficiency on the priming phase. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with treated with LPS (200 ng/mL, 4 h).I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


Figure 3A Representative images of mutant Lamtor1 . PMA - primed Lamtor1 KO THP - 1 macrophages reconstituted with full - length or truncated Lamtor1 . B Effects of the Lamtor1 point mutation on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion in THP - 1 cell supernatants from WT cells , Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full - length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine ( G2A ) ; all cell types were then treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) . C Effects of Lamtor1 deficiency on mTORC1 activity . Immunoblot analysis of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) . D Effect of the rapamycin treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from PMA ( 50 nM , overnight ) primed WT THP - 1 cells treated with rapamycin at indicated concentration for 2 h , then treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . E Effect of rapamycin treatment on Caspase - 1 processing . Western blot shows caspase - 1 / p20 . LPS ( 200 ng / mL , 4 h ) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin ( 15 μM , 1 h ) . F Effect of Torin - 1 treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from WT BMDMs pretreated with Torin - 1 ( 0 , 1 , 10 , 100 , or 250 nM ) for 2 h before LPS ( 200 ng / ml , 4 h ) and nigericin ( 15 μM , 1 h ) treatment ( left panel ) . Western blot shows phospho - p70 - S6K from BMDMs pretreated with Torin - 1 at the indicated concentrations for 2 h before LPS and nigericin treatment ( right panel ) .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1.B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h).E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h).F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel).


Figure 4A Effects of the HDAC6 inhibitor treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion and caspase - 1 processing ( p10 ) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin ( left to right : 10 and 20 mM ) or rocilinostat ( left to right : 30 and 60 mM ) . Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmidsCo - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmidsCo - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Lamtor1 - Flag co - immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1 - Flag knocked - in THP - 1 cells . ( D ) Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 . Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 . G PLA to confirm endogenous binding between Lamtor1 and HDAC6 . Lamtor1 - HDAC6 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over nine images in each experiment . H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells . HEK293T cells were transiently transfected with NanoLuc - fused Lamtor1 and Halo - tag - fused HDAC6 using Lipofectamine 2000 . Luminescence was measured 48 h after transfection following the addition of Nano - Glo Luciferase Assay Substrate ( Promega ) . I Immunoblot analysis of the truncated form of Flag - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - 144 ; ∆ 95 - 161 , Met1 - Val94 ) co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids . Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP - 1 cells . ELISA assay showing IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , Lamtor1 KO cells reconstituted with full - length Lamtor1 , and Met1 - Val94 Lamtor1 cells ; all cell types were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D)Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment.H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


Figure 5A Yeast two hybrid ( Y2H ) assays for Lamtor1 interactions with NLRP3 . Positive Y2H interaction between Lamtor1 as prey , and NLRP3 as bait . Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control . B PLA to confirm endogenous binding between Lamtor1 and NLRP3 . Lamtor1 - NLRP3 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over 10 images in each experiment . C , D Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids . Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids . Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids . G Effect of the HDAC6 knockdown on NLRP3 - Lamtor1 interactions . Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment.C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


Figure 6A , B Representative images of Lamtor1 and ASC . PMA - treated THP - 1 cells stably expressing GFP - tagged ASC and td - Tomato - tagged Lamtor1 were primed with LPS ( 200 ng / mL , 2 h ) and then stimulated with nigericin for the indicated duration . Time shows after nigericin stimulation ( min : s ) . C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation . Representative images of PMA - primed THP - 1 macrophages stably expressing GFP - tagged ASC treated with or without LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . Scale bars = 10 μm .

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Figure 6A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.