( H ) Immunoprecipitation assays with anti - PD - L1 ( avelumab ) using OVCAR - 3 cell lines . Blue arrow indicates cleaved and activated ROCK1 .

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(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.


( J ) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors ( n = 3 ) .

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(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).


( K ) Tumor regression efficacy of MD5 - 1 ( 50μg ) , GSK269962A ( < 2mg / kg ) and MD5 - 1 + GSK269962A ( 50μg + 2mg / kg ) 4T1 tumors ( n = 4 - 7 ) . 6 - 8 weeks old BALB / c mice bearing 4T1 tumors ( ~ 100mm3 ) were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . GSK269962A ( ROCK1i ) was injected directly into the tumors . Tumor volumes were quantified at indicated days by caliper measurements .

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(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.


( B - D ) FACs plots confirming expression of human DR5 ( huDR5 ) and Chi - G4S - DR5 in mouse 4T1 cells . Chi - DR5 was not expressed on cell surface ( C ) .

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

(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).


( E ) Cell viability analysis of huDR5 and Chi - G4S - DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 ( n = 3 ) .

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(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).


( F ) Comparison of tumor growth of grafted huDR5 and Chi - G4S - DR5 stable 4T1 cells in Balb / c mice ( n = 3 ) .

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(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).


( G ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 150 - 200mm3 ) were treated with 4 doses ( 100μg ) of IgG1 , lexatumumab , lexatumumab + avelumab followed by tumor recovery and surface PD - L1 analysis using flow

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(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry


( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) .

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(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).


( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . average tumor weight is shown ( n = 4 ) .

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(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).


( J - K ) Confirmation of selective CD8 + T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti - PD - L1 avelumab alone or avelumab + anti - CD8a antibody . CD4 + T - cells remained unchanged .

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(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.


( L ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , ROCK1i , lexatumumab + ROCK1i and anti - CD8 + lexatumumab + ROCK1i ( n = 4 - 6 , 50μg Lexatumumab , 2mg / kg ROCK1i , 6 doses ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections . Various treatments were started when tumors were ~ 100mm3 size .

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(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.


( M ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , avelumab , lexatumumab + avelumab and anti - CD8 + lexatumumab + avelumab ( n = 4 - 6 ) .

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(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).


( N ) 6 - 8 weeks old C57BL / 6 bearing Chi - G4S - DR5 expressing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Various treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements .

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

(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( O ) KMTR2 ( 8 doses ) antibody used as the DR5 agonist for the experiment . ( P ) Average of tumor weights of data shown in ( O ) .

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

Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O).


( Q ) Kaplan - Meier plot depicting the survival of syngeneic Chi - G4S - DR5 4T1 tumor bearing animals injected i . p . with 100μg of indicated antibodies such as IgG1 , KMTR2 and avelumab . Animals were injected with GSK269962A 2mg / kg ( in PBS ) directly into tumors wherever ROCK1i is indicated .

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(Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.


( A ) Chi - G4S - DR5 stable 4T1 tumors harboring mice were treated lexatumumab , lexatumumab + ROCK1i , and avelumab + lexatumumab and other controls as indicated . Antibodies were treated i . p at 100μg dose ( 6 total ) , ROCK1i ( in PBS ) was injected directly into tumors at 2mg / kg dose ( 6 total ) . Various treatments were started when tumors were ~ 400mm3 size . Harvested tumors were grouped together and sized matched ( 3 independent sets : n = 2 - 6 tumors in each set ) followed by TIL isolation CD8 / CD45 and CD4 / CD45 expressing cells were measured by flow cytometry .

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

(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.


( B ) Plots showing % of total double positive TILs ( CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining 3 independent experiments . ( C ) Ratio of CD8 + CD45 + / CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3 ) .

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(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).


Chi - G4S - DR5 stable MC38 tumors harboring mice were treated ( 6 total doses ) lexatumumab , avelumab , ROCK1i , lexatumumab + ROCKi , and avelumab + lexatumumab and IgG1 control as indicated . Harvested tumors homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , Foxp3 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments .

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Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( H ) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5 - 1 and bispecific avelu - MD5 antibody .

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(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.


( I ) Balb / c mice harboring luciferase stable 4T1 tumors were i . p . injected with indicated antibodies . 50μg dose and mice were imaged after 5 doses .

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(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.


( J ) 6 - 8 weeks old C57BL / 6 mice bearing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Indicated treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements .

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(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.


( K ) ~ 150 - 200mm3 size MC38 tumor bearing C57LB / 6 mice were treated with indicated MD5 - 1 , avelumab and bispecific antibodies along with control IgG1 , 6 total doses . Harvested tumors were homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments .

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

(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.


( L ) 6 - 8 weeks old C57BL / 6 mice were injected with MC38 cells . When tumors reached ~ 150 - 200mm3 , animals were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day . On day 18 , tumors were harvested , sized matched and pooled by treatment group , exposed to collagenase / DNase and were single cell suspensions enriched for CD8 + cells . Enriched CD8 + T - cells from various treatments were restimulated with anti - CD3 ( OKT3 ) antibody for 4 additional hours . CD8 gated cells were next analyzed for IFN - γ intracellular expression using flow cytometry .

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(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.


( M ) Percentage of IFN - γ + CD8 + double positive cells from 3 independent experiments .

(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.


( E and F ) RT - PCR and western blot showing the effect of RNAi - mediated B52 depletion in S2R + cells ( E ) and in larval wing discs ( F ) .

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(E and F) RT-PCR and western blot showing the effect of RNAi-mediated B52 depletion in S2R+ cells (E) and in larval wing discs (F).


( D ) Quantification of Posterior / Total wing area in male flies of the genotypes illustrated in panel C . UAS - GFP was used as a control . Each point represents a single wing , bars represent mean with standard deviation . * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) . Flies were grown at 18 ° C .

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(D) Quantification of Posterior/Total wing area in male flies of the genotypes illustrated in panel C. UAS-GFP was used as a control. Each point represents a single wing, bars represent mean with standard deviation. *** p-value<0.001, **** p-value<0.0001 (unpaired two-tailed t-tests). Flies were grown at 18°C.


( C ) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain ( hh - Gal4 driver ) . For each target gene , relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti - ßgal antibody ( for ex - lacZ and diap1 - lacZ ) or direct visualization of GFP ( ban - sensor , in this case posterior domain was visualized by immunostaining against V5 - tag present in UAS - Yki transgenes ) . In the scatter dot plot , each symbol ( circle , square and triangle ) represents a single wing disc , bars represent mean with SEM . * * p - value < 0 . 01 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) .

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(C) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).


( C ) Comparison of Yki isoforms binding capabilities by dual - luciferase co - IP . Graphs show the normalized co - IP ( NcoIP ) results for 13 proteins tested against the two Yki isoforms and the negative control ( Flag - Firefly ) .

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(C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).


( D ) Interaction between Sd and Tgi in absence or presence of Yki isoforms . ( E ) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms . ( F ) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms . In C , D , E and F , bars represent mean and error bars represent SD , n = 4 . p - values : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( unpaired two - tailed t - tests ) .

(D) Interaction between Sd and Tgi in absence or presence of Yki isoforms. (E) Interaction between Sd and Wbp2 in absence or presence of Yki isoforms. (F) Interaction between Tgi and Wbp2 in absence or presence of Yki isoforms. In C, D, E and F, bars represent mean and error bars represent SD, n=4. p-values: *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001 (unpaired two-tailed t-tests).


( G ) Luciferase assay using 3xSd - Luciferase reporter in S2 cells , transfected with single or combination of Yki isoforms . Error bars represent SD , n = 6 . * p - value < 0 . 05 ; * * * * p - value < 0 . 0001 ( unpaired two - tailed t - tests ) .

(G) Luciferase assay using 3xSd-Luciferase reporter in S2 cells, transfected with single or combination of Yki isoforms. Error bars represent SD, n=6. * p-value<0.05; **** p-value<0.0001 (unpaired two-tailed t-tests).


( H ) Co - overexpression of Yki isoforms using GMR - Gal4 driver provides evidence of competition between Yki isoforms .

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(H) Co-overexpression of Yki isoforms using GMR-Gal4 driver provides evidence of competition between Yki isoforms.


( C ) Comparison of posterior / total area ratio of females wings upon depletion of B52 in a yki + ( wild type ) or yki2 - only background . Each point corresponds to a single wing , bars represent mean with SD ( unpaired two - tailed t - tests : * * * * p - value < 0 . 0001 ) .

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(C) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001).


( E and F ) Quantification of sibling clones size in the wing pouch . In ( D ) , sibling wild - type clones are labelled with 2xGFP and 2xRFP . In ( E ) , unlabelled clones are homozygous for the yki2 - only - A allele whereas sibling clones ( RFP / RFP ) are wild type . In both graphs , clones pairs are sorted according to RFP / RFP clones size .

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

(E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.


( C ) Distribution of wing size and wing asymmetry among the flies analyzed in panel B . Each circle represents the area of a single wing . Each wing was measured in duplicate . The difference between Right ( orange ) and Left ( blue ) sides of a single fly is schematized by a black line .

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(C) Distribution of wing size and wing asymmetry among the flies analyzed in panel B. Each circle represents the area of a single wing. Each wing was measured in duplicate. The difference between Right (orange) and Left (blue) sides of a single fly is schematized by a black line.


( A - C ) qRT - PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages ( A ) , THP1 cells ( B ) and human peripheral blood mononuclear cells ( PBMC ) ( C ) that were left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib ( 0 . 1 , 1 , 10 μM ) . The data are presented as fold induction of Ifnb1 / Gapdh transcripts . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by one - way ANOVA followed by Dunnett ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 .

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

(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.


( D ) MTS detection of the viability of mouse peritoneal macrophages ( PM ) , THP1 and PBMC cells left untreated ( DMSO ) or treated with Palbociclib ( 1 μM , 10 μM ) for 4 h . The data shown are mean ± SEM from n = 3 biological replicates . Statistical significance was determined by one - way ANOVA . ns , not significant .

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

(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.


( E - F ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( E ) and THP1 cells ( F ) stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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

(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( G - H ) qRT - PCR measurement of Il1b ( G ) and Il6 ( H ) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant . ( I - J ) qRT - PCR measurement of Il1b ( I ) and Il6 ( J ) transcripts in THP1 cells left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(G-H) qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant. (I-J) qRT-PCR measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.


( K - L ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( K ) and THP1 ( L ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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

(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( M - N ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( M ) and THP1 ( N ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( A ) A real - time OR - ID assay showing the association - dissociation curves of surface - immobilized Palbociclib with recombinant STING protein . The data shown are representative of n = 3 biological replicates .

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

(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with Flag - STING ( C ) The data shown are representative of n = 3 biological replicates .

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

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) mouse peritoneal macrophages ( D ) The data shown are representative of n = 3 biological replicates .

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

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) THP1 cells ( E ) . The data shown are representative of n = 3 biological replicates .

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates.


( F ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - c - diAMP ( 2 μM ) and recombinant SUNO - tagged STING protein ( 10 μg / mL ) in the presence of DMSO and increasing concentrations of Palbociclib ( 0 , 1 , 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(F) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - STING in the presence of DMSO and indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

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

(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( H ) . Scale bar , 25 μm .

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

Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm.


STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( I ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * * , P < 0 . 0001 .

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

STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001.


( J ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STEEP and HA - STING in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( K ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - TBK1 in the presence of DMSO or indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

(K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


( C ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with corresponding Flag - STING mutants . The data shown are representative of n = 3 biological replicates .

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

(C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING mutants. The data shown are representative of n=3 biological replicates.


( D ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING or Flag - STING Y167A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( E ) . Scale bars , 25 μm .

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

Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm.


STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( F ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - TBK1 and Flag - STING or Flag - STING Y167A . The data shown are representative of n = 3 biological replicates .

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

(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-TBK1 and Flag-STING or Flag-STING Y167A. The data shown are representative of n=3 biological replicates.


( H ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING C91A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( B ) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; ns , not significant .

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

(B) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; ns, not significant.


The representative image of colon tissue harvested from mice of indicated groups is shown in ( C ) .

The representative image of colon tissue harvested from mice of indicated groups is shown in (C).


The length of colon is analyzed in ( D ) . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

The length of colon is analyzed in (D). Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( E - F ) Representative H & E staining of the histological changes in colon tissues harvested from mice of indicated groups ( E ) . Scale bar , 200 μm . The quantification of histological score was shown in ( F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(E-F) Representative H&E staining of the histological changes in colon tissues harvested from mice of indicated groups (E). Scale bar, 200 μm. The quantification of histological score was shown in (F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Il6 ( H ) and Il1b ( I ) in mouse colon tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(G-I) qRT-PCR measurement of transcripts of Ifnb1 (G), Il6 (H) and Il1b (I) in mouse colon tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not significant.


( B - F ) Representative H & E staining of the histological changes in heart , stomach , tongue and thigh muscle tissues harvested from mice of indicated groups ( B ) . Scale bar , 200 μm . The quantification of histological score was shown in ( C - F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * , P < 0 . 05 ; * * , P < 0 . 01 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative H&E staining of the histological changes in heart, stomach, tongue and thigh muscle tissues harvested from mice of indicated groups (B). Scale bar, 200 μm. The quantification of histological score was shown in (C-F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. *, P<0.05; **, P<0.01; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Cxcl10 ( H ) and Il6 ( I ) in mouse stomach tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(G-I) qRT-PCR measurement of transcripts of Ifnb1(G), Cxcl10 (H) and Il6 (I) in mouse stomach tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( A ) BMDMs were infected with L . pneumophila JR32 or the legA9 mutant with MOI of 0 . 5 . CFUs were scored at 1 , 24 , 48 , and 72 h . ( B ) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL , or legA9 + legA9 pBC ‐ KS + PL . ( A , B ) Data are presented as means ± SD of at least three independent experiments . Asterisks indicate significant differences , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) .

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

(A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48, and 72 h. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL, or legA9+ legA9 pBC‐KS+ PL. (A, B) Data are presented as means ± SD of at least three independent experiments. Asterisks indicate significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed t‐test).


Four female C57BL / 6 mice / group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally . Lungs were homogenized and plated for CFUs counting at ( C ) 4 or ( D ) 48 h postinfection . ( C , D ) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 two ‐ tailed t ‐ test )

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

Four female C57BL/6 mice/group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 h postinfection. (C, D) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments. Asterisks indicate significant differences (**p < 0.01 two‐tailed t‐test)


( A ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . Nuclei were stained blue with DAPI and L . pneumophila stained green with L . pneumophila antibody . Lyso − tracker red was used to stain acidified lysosomes . White arrows show L . pneumophila colocalization with lysotracker .

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(A) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso−tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker.


( B ) Percent colocalization of L . pneumophila with lyso ‐ tracker .

(B) Percent colocalization of L. pneumophila with lyso‐tracker.


( C ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . LC3 is stained red with LC3 antibody . White arrows show L . pneumophila colocalization with LC3 .

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(C) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. LC3 is stained red with LC3 antibody. White arrows show L. pneumophila colocalization with LC3.


( D ) Percent colocalization of L . pneumophila with LC3 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) .

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(D) Percent colocalization of L. pneumophila with LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01, ***p < 0.001; two‐tailed t‐test).


Induction of autophagy in macrophages restricts legA9 mutant replication , and rescues colocalization with LC3 and fusion with lysosomes . BMDMs were untreated or pretreated with rapamycin ( Rap ) for 1 h and kept throughout the infection . BMDMs were infected with L . pneumophila JR32 or the leg A9 mutant for ( A ) 1 or ( B ) 24 h with an MOI of 0 . 5 . CFUs were scored at these time points .

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Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points.


( C , D ) Percent colocalization of L . pneumophila with ( C ) lyso ‐ tracker and ( D ) LC3 . Hundred bacteria were scored from each coverslips . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

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(C, D) Percent colocalization of L. pneumophila with (C) lyso‐tracker and (D) LC3. Hundred bacteria were scored from each coverslips. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01; two‐tailed t‐test).


( A ) BMDMs derived from WT and Atg5 − / − mice were infected with L . pneumophila JR32 or legA9 mutant at an MOI of 0 . 5 . Data are shown as mean ± SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 ; two − tailed t − test .

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(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test.


( B ) Western blot showing the absence of Atg 5 in the knockout . Data are representative of three independent experiments .

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(B) Western blot showing the absence of Atg 5 in the knockout. Data are representative of three independent experiments.


( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . Polyubiquitin is stained red . White arrows show L . pneumophila colocalization with polyubiquitin .

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(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. Polyubiquitin is stained red. White arrows show L. pneumophila colocalization with polyubiquitin.


( B ) Percent colocalization of L . pneumophila with polyubiquitin . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . Data are presented as means + SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

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(B) Percent colocalization of L. pneumophila with polyubiquitin. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. Data are presented as means + SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.01; two‐tailed t‐test).


( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . P62 is stained red with p62 antibody . White arrows show L . pneumophila colocalization with p62 .

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(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62.


( B ) Percent colocalization of L . pneumophila with p62 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm .

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(B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm.


( C ) BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA ( performed in triplicate and the SD are too small to detect ) .

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(C)BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the SD are too small to detect).


BMDMs were nucleofected with scrambled control ( sictrl ) or siRNA against p62 ( sip 62 ) . Twenty ‐ four hours after nucleofection , murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0 . 5 for 1 h ( D and F ) or 24 h ( E and G ) .

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BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip 62). Twenty‐four hours after nucleofection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 h (D and F) or 24 h (E and G).


( H ) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection . ( A - B and D - H ) Data are shown as mean + SD of n = 3 and are representative of three independent experiments . ( C ) Data are representative of one experiment n = 3 . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 ; two − tailed t − test ) .

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(H) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection. (A-B and D-H) Data are shown as mean + SD of n = 3 and are representative of three independent experiments. (C) Data are representative of one experiment n = 3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01; two−tailed t−test).


( A ) Caspase − 1 KO ( casp − 1 − / − ) macrophages were infected with L . pneumophila JR32 or legA9 with an MOI of 0 . 5 for 1 , 24 , 48 , and 72 h .

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(A) Caspase−1 KO (casp−1−/−) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48, and 72 h.


( B ) CFUs were measured at the indicated time points . Levels of IL ‐ 1β were detected in supernatants of WT or casp ‐ 1 − / − BMDMs infected with JR32 or the legA9 mutant after 24 h . WT BMDMs were either not treated ( NT ) or infected with L . pneumophila JR32 or legA9 mutant bacteria for 2 h .

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(B) CFUs were measured at the indicated time points. Levels of IL‐1β were detected in supernatants of WT or casp‐1−/− BMDMs infected with JR32 or the legA9 mutant after 24 h. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 h.


Activation of casp ‐ 1 or casp ‐ 7 was detected in cell extracts using ( C ) casp ‐ 1 or ( D ) casp ‐ 7 antibodies , respectively . ( A - D ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

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Activation of casp‐1 or casp‐7 was detected in cell extracts using (C) casp‐1 or (D) casp‐7 antibodies, respectively. (A-D) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


( A ) BMDMs were not infected ( NT ) or infected with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 , 1 or 5 for 24 h . Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic ( apoptosis ) histone − associated − DNA fragments ( ELISA ) .

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(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA).


( B ) BMDMs were either not treated ( NT ) or treated with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 or 5 for 24 h . The fold change in LDH release was measured from the overall population of macrophages . ( A - B ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

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(B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A-B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


( A ) Unlabeled or Cy5 - labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase ( dsRNA - RL ) or the GFP ( dsRNA - GFP ) coding sequences visualised on gel stained with ethidium bromide ( EtBr - stained , top ) or by in - gel fluorescence ( bottom ) .

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(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).


( B ) Gating strategy used in the flow cytometry - based assay . First , cells were selected ( side scatter ( SSC ) vs . forward scatter ( FSC ) ) and gated for singlets ( SSC - Width ( W ) vs . FSC ) to hence exclude doublets or cell aggregates . The single cell gate was then further analysed for DAPI , gating on the live cells ( DAPI negative ) . DsRNA - transfected cells ( Cy5 - positive ) were then gated and the level of GFP expression analysed within that population . Flow cytometry counting beads ( gated on SSC vs . FSC plot ) were added to quantify cell number in each sample .

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(B) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample.


( C ) mESCs stably expressing GFP ( mESCs - GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and the level of GFP in Cy5 + cells was analysed 3 days later and is represented as histograms ( left ) . Right panel , bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control ( D ) mESCs stably expressing d2GFP ( mESCs - d2GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA . The level of d2GFP was analysed at the indicated timepoints post - transfection and displayed as in ( C ) .

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(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).


( E ) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and d2GFP level was measured as in ( D ) .

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(E) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).


( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs ( top panels ) or siRNAs ( bottom panels ) . GFP level was measured in Cy5 + cells by flow cytometry 72 hours ( hrs ) post - transfection .

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(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.


( B ) Mavs + / - and Mavs - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs . The level of d2GFP in Cy5 + cells was measured at various timepoints post - transfection as indicated . Histograms plots show d2GFP level 48 hrs post - transfection . Bar graphs show 3 timepoints as indicated .

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(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.


( C ) WT , Ifnar1 + / - or Ifnar1 - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry at 48 hrs post - transfection .

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(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.


( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR ( α - IFNAR Ab ) , an isotype control antibody ( IgG1 ) or no antibody ( no Ab ) . Cells were then were transfected or not ( mock ) with Cy5 - labeled dsRNA as indicated and GFP level monitored in Cy5 + cells 72 hrs post - transfection .

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(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.


( B ) Mavs - / - MEFs stably expressing d2GFP were treated with recombinant IFN ( 200 U / ml ) for 24 hrs prior to transfection or not ( mock ) with Cy5 - labeled dsRNA , as indicated . IFN treatment was maintained for 2 days post - transfection and then GFP level monitored in Cy5 + cells .

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(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.


( A ) Northern blot analysis of Ifnar1 - / - MEFs at 24 h . p . t with the indicated dsRNA or transfection reagent alone ( mock ) using a probe specific for dsRNA - GFP ( top ) or dsRNA - RL ( bottom ) . Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG - human Dicer were loaded in parallel . Two lanes ( delimited by dashed lines ) were left empty between the Ifnar1 - / - MEFs transfected samples and the in vitro dicing assay reactions . A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17 , 21 and 25 - nt in length was run in parallel . Endogenous U6 was used as a loading control . Signals corresponding to the dsRNA - derived siRNAs are indicated with an arrow . The membrane was first probed for dsRNA - GFP ( top ) , then probed for the miRNA marker ( top left ) and subsequently stripped and reprobed for dsRNA - RL ( middle ) and finally stripped and reprobed for both U6 ( bottom ) and for the miRNA marker again ( middle left ) .

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(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).


( B ) Western blot of Dicer in d2GFP - expressing Mavs - / - MEFs either not transduced ( NT ) or transduced to express a non - targeting shRNA control ( CTL shRNA ) or two different Dicer - specific targeting shRNAs ( DICER shRNA 1 and 2 ) . P97 served as loading control .

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(B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control.


( C ) Cells described in ( B ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later . Please note that the biphasic pattern of d2GFP level observed in dsRNA - GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of

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(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP


( D ) Western blots of Ago2 in parental Mavs - / - MEFs stably expressing d2GFP and three Mavs - / - Ago2 - / - clones generated by CRISPR / CAS9 - mediated genome engineering . P97 served as loading control .

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(D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control.


( E ) Mavs - / - ( parental line ) and Mavs - / - Ago2 - / - MEFs ( clone 5 . 1 ) stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

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(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Western blots for Ago2 ( top ) and HA tag ( bottom ) of parental Mavs - / - MEFs and two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ; see Fig 4A ) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA - tagged wild - type ( HA - mAgo2 WT ) or catalytic mutant ( HA - mAgo2 D597A ) versions of mouse Ago2 . β - actin served as loading control . The membrane was first analysed for Ago2 and β - actin , stripped and then analysed for HA tag .

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(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag.


( B ) Parental non - transduced ( NT ) Mavs - / - MEFs and complemented Mavs - / - Ago2 - / - MEFs as described in ( A ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Ifnar1 - / - MEFs were transfected with the indicated Cy5 - labeled dsRNA and the next day were infected with SFV - Rluc at the indicated multiplicities of infection ( MOI ) . Renilla luciferase activity was measured 24 hours later . Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold - reduction in viral activity achieved with dsRNA - RL vs . dsRNA - GFP treatment .

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(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.


( B ) As in ( a ) but SFV - Rluc virus titre was measured by plaque assay and expressed as plaque forming units ( PFU ) per ml of supernatant at 24 h post - infection ( MOI = 0 . 1 ) .

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(B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1).


( C ) As in ( a ) but with Ifnar1 - / - or Mavs - / - MEFs and a single dose of SFV - Rluc ( MOI = 0 . 1 ) .

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(C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1).


( D ) As in ( a ) but with two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ) individually complemented to express wild - type ( HA - mAgo2 WT ) or a catalytic mutant ( HA - mAgo2 D597A ) version of mAgo2 or transduced with a control vector ( as described in Fig 5 ) . SFV - Rluc was used at an MOI = 0 . 1 .

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(D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.


A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole ‐ cell lysates of MDA ‐ MB ‐ 231 cells followed by immunoblotting to examine the co ‐ immunoprecipitatedPIPKIγi5 and LAPTM4B .

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A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole‐cell lysates of MDA‐MB‐231 cells followed by immunoblotting to examine the co‐immunoprecipitatedPIPKIγi5 and LAPTM4B.


B PIPKIγi5 specifically interacts with LAPTM4B , but not LAPTM4A or LAPTM5 . Top : schematic diagram of all three LAPTM members . Bottom : each Flag ‐ tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc ‐ tagged PIPKIγi5 and empty vector or Flag ‐ tagged LAPTM , and the co ‐ immunoprecipitatedPIPKIγi5 was examined by immunoblotting .

B PIPKIγi5 specifically interacts with LAPTM4B, but not LAPTM4A or LAPTM5. Top: schematic diagram of all three LAPTM members. Bottom: each Flag‐tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc‐tagged PIPKIγi5 and empty vector or Flag‐tagged LAPTM, and the co‐immunoprecipitatedPIPKIγi5 was examined by immunoblotting.


C LAPTM4B selectively associates with PIPKIγi5 . Top : schematic diagram of four PIPKI isoforms . Bottom : Myc ‐ tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag ‐ tagged LAPTM4B and each isoform of PIPKI , followed by immunoblotting to examine the co ‐ immunoprecipitatedLAPTM4B .

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C LAPTM4B selectively associates with PIPKIγi5. Top: schematic diagram of four PIPKI isoforms. Bottom: Myc‐tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag‐tagged LAPTM4B and each isoform of PIPKI, followed by immunoblotting to examine the co‐immunoprecipitatedLAPTM4B.


D Kinase activity of PIPKIγi5 is required for LAPTM4B association . HEK293 cells expressing LAPTM4B and wild ‐ type ( WT ) or kinase ‐ dead ( KD ) PIPKIγi5 were starved overnight , stimulated or not with 100 ng / ml EGF for 15 min , and harvested for immunoprecipitation with anti ‐ myc . The co ‐ immunoprecipitatedLAPTM4B was detected by immunoblotting .

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D Kinase activity of PIPKIγi5 is required for LAPTM4B association. HEK293 cells expressing LAPTM4B and wild‐type (WT) or kinase‐dead (KD) PIPKIγi5 were starved overnight, stimulated or not with 100 ng/ml EGF for 15 min, and harvested for immunoprecipitation with anti‐myc. The co‐immunoprecipitatedLAPTM4B was detected by immunoblotting.


E Endogenous LAPTM4B is targeted to both early and late endosomes . MDA ‐ MB ‐ 231 cells were fixed and costained for endogenous LAPTM4B ( red ) and EEA1 or LAMP1 ( green ) . Boxes are selected regions for magnified view . Note : non ‐ specific nuclear staining by the LAPTM4B anti ‐ sera . Scale bar : 10 μm . F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 ( mean + SD ; n ≥ 4 ) .

E Endogenous LAPTM4B is targeted to both early and late endosomes. MDA‐MB‐231 cells were fixed and costained for endogenous LAPTM4B (red) and EEA1 or LAMP1 (green). Boxes are selected regions for magnified view. Note: non‐specific nuclear staining by the LAPTM4B anti‐sera. Scale bar: 10 μm.F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 (mean + SD; n ≥ 4).


H LAPTM4B partially colocalizes with PIPKIγi5 . MDA ‐ MB ‐ 231 cells expressing Myc ‐ tagged PIPKIγi5 were stained with LAPTM4B anti ‐ sera ( red ) , anti ‐ Myc ( green ) , and DAPI . Box is selected region for magnified view . Scale bar : 10 μm .

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H LAPTM4B partially colocalizes with PIPKIγi5. MDA‐MB‐231 cells expressing Myc‐tagged PIPKIγi5 were stained with LAPTM4B anti‐sera (red), anti‐Myc (green), and DAPI. Box is selected region for magnified view. Scale bar: 10 μm.


I MDA ‐ MB ‐ 231 cells stably expressing Flag ‐ LAPTM4B were stained with LAPTM4B anti ‐ sera followed by silver ‐ enhanced immuno ‐ electron microscopy . The early and late MVEs were defined by the number of intraluminal vesicles . N , nucleus ; M , mitochondria ; MVE , multi ‐ vesicular endosome ; and PM , plasma membrane . Scale bars : 2 μm ( left ) ; 200 nm ( middle and right ) .

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I MDA‐MB‐231 cells stably expressing Flag‐LAPTM4B were stained with LAPTM4B anti‐sera followed by silver‐enhanced immuno‐electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi‐vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right).


A MDA ‐ MB ‐ 231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng / ml EGF for indicated time periods , followed by whole ‐ cell lysate harvest for immunoblotting analysis of EGFR levels . B - D Quantification of the levels of EGFR ( B ) , pEGFR ( Y1068 ) ( C ) , and pAKT ( S473 ) ( D ) from the analysis in ( A ) ( mean ± SD ; n = 3 ) .

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A MDA‐MB‐231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng/ml EGF for indicated time periods, followed by whole‐cell lysate harvest for immunoblotting analysis of EGFR levels.B-D Quantification of the levels of EGFR (B), pEGFR (Y1068)(C), and pAKT (S473)(D) from the analysis in (A) (mean ± SD; n = 3).


E Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pulsed with 25 ng / ml Alexa ‐ 555 ‐ EGF for 3 min , washed , and chased for indicated time periods followed by fixation , DAPI staining , and fluorescence microscopy . Scale bar : 10 μm . F Quantification of the relative amounts of Alexa ‐ 555 ‐ EGF internalized in the indicated conditions ( mean + SD , n = 3 ) . G Quantification of the Alexa ‐ 555 ‐ EGF degradation in control and LAPTM4B knockdown cells in ( E ) ( mean ± SD , n = 3 ) .

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E Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pulsed with 25 ng/ml Alexa‐555‐EGF for 3 min, washed, and chased for indicated time periods followed by fixation, DAPI staining, and fluorescence microscopy. Scale bar: 10 μm.F Quantification of the relative amounts of Alexa‐555‐EGF internalized in the indicated conditions (mean + SD, n = 3).G Quantification of the Alexa‐555‐EGF degradation in control and LAPTM4B knockdown cells in (E) (mean ± SD, n = 3).


H Control or LAPTM4B ‐ overexpressing MDA ‐ MB ‐ 231 cells were starved and then stimulated with 100 ng / ml EGF for 1 - 4 h . EGFR degradation and signaling were analyzed by Western blot . Specific antibodies recognizing pEGFR ( Y1068 ) and pAKT ( S473 ) were used . I - K Quantification for the levels of EGFR ( I ) and pEGFR ( J ) normalized to actin and pAKT ( K ) normalized to AKT in control or LAPTM4B ‐ overexpressing cells ( mean ± SD , n = 3 ) .

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H Control or LAPTM4B‐overexpressing MDA‐MB‐231 cells were starved and then stimulated with 100 ng/ml EGF for 1-4 h. EGFR degradation and signaling were analyzed by Western blot. Specific antibodies recognizing pEGFR (Y1068) and pAKT (S473) were used.I-K Quantification for the levels of EGFR (I) and pEGFR (J) normalized to actin and pAKT (K) normalized to AKT in control or LAPTM4B‐overexpressing cells (mean ± SD, n = 3).


A - D Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 cells were starved , pretreated or not with chloroquine for 2 h , stimulated with 100 ng / ml EGF for 15 min , washed , and chased for indicated time periods before fixation for costaining of EGFR ( red ) with EEA1 ( A , green ) or LAMP1 ( C , green ) . Quantification of the average percentages of EGFR signals colocalized with EEA1 ( B ) and LAMP1 ( D ) at indicated time points ; mean + SD ; n = 3 ; * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one ‐ tailed t ‐ test .

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A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test.


E MDA ‐ MB ‐ 231 cells were starved , stimulated with 100 ng / ml EGF for 15 or 120 min , fixed , and costained for EGFR and LAPTM4B , followed by fluorescence microscopy . Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads , respectively . F The amounts of total EGFR staining in individual cells in ( E ) were plotted against LAPTM4B levels at 15 min and 120 min , respectively . Note : for LAPTM4B quantification , the non ‐ specific nuclear staining was not included . Trend lines and Pearson ' s correlation coefficients are shown . AU , arbitrary unit .

E MDA‐MB‐231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively.F The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non‐specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit.


MDA ‐ MB ‐ 231 cells expressing GFP ‐ Rab5Q79L were starved and stimulated with 100 ng / ml Alexa ‐ 555 ‐ EGF for 90 min followed by fixation and immunostaining for LAPTM4B ( blue ) . The percentages of luminal Alexa ‐ 555 ‐ EGF in individual endosomes were plotted against endosomal LAPTM4B levels . Trend lines and Pearson ' s correlation coefficients are shown . Data are representative for three independent experiments .

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MDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa‐555‐EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments.


C Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved overnight , cell surface EGFR was labeled with immuno ‐ gold on ice . Cells were then stimulated with EGF for 1 h at 37 ° C and fixed for the EM study . Scale bar , 200 nm . See Materials and Methods for details . D Relative amounts of immuno ‐ gold ‐ labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified . Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification ( mean + SD ; * * * P 0 . 001 , one ‐ tailed t ‐ test ) .

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C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test).


MDA ‐ MB ‐ 231 cells were transfected with GFP ‐ Rab5Q79L and Flag ‐ LAPTM4B , starved , and stimulated with Alexa ‐ 555 ‐ EGF for 90 min and followed by intraluminal sorting analysis . See Materials and Methods for details . Quantification of EGF localization in LAPTM4B ‐ positive and LAPTM4B ‐ negative endosomes in ( E ) ( mean + SD ; n = 3 ; * * * P 0 . 0007 , one ‐ tailed t ‐ test ) .

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MDA‐MB‐231 cells were transfected with GFP‐Rab5Q79L and Flag‐LAPTM4B, starved, and stimulated with Alexa‐555‐EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B‐positive and LAPTM4B‐negative endosomes in (E) (mean + SD; n = 3; ***P 0.0007, one‐tailed t‐test).


A Co ‐ immunoprecipitation ( co ‐ IP ) of full ‐ length or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 cells .

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A Co‐immunoprecipitation (co‐IP) of full‐length or N‐terminus‐deleted LAPTM4B with PIPKIγi5 in HEK293 cells.


B GST ‐ tagged LAPTM4B N ‐ terminus ( 93 amino acids ) and T7 ‐ tagged PIPKIγi5 C ‐ terminus ( 223 amino acids ) were purified from E . coli for in vitro GSTpull ‐ down assay .

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B GST‐tagged LAPTM4B N‐terminus (93 amino acids) and T7‐tagged PIPKIγi5 C‐terminus (223 amino acids) were purified from E. coli for in vitro GSTpull‐down assay.


C Co ‐ IP of full ‐ length or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells . ∆ 25 : amino acids 1 - 25 deleted .

C Co‐IP of full‐length or N‐terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells. ∆25: amino acids 1-25 deleted.


E Coomassie Brilliant Blue staining of purified wild ‐ type and mutated LAPTM4B N ‐ termini .

E Coomassie Brilliant Blue staining of purified wild‐type and mutated LAPTM4B N‐termini.


F Purified LAPTM4B N ‐ termini from ( E ) were used in PIP strips assay . LPA , lysophosphatidic acid ; LPC , lysophosphocholine ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine 1 ‐ phosphate ; PI3 , 4 , 5P3 , PtdIns ( 3 , 4 , 5 ) P3 , phosphatidylinositol ( 3 , 4 , 5 ) ‐ trisphosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

F Purified LAPTM4B N‐termini from (E) were used in PIP strips assay. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1‐phosphate; PI3,4,5P3, PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)‐trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.


GST pull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ termini with PIPKIγi5 C ‐ terminus .

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GST pull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐termini with PIPKIγi5 C‐terminus.


H Co ‐ IP of wild ‐ type or PMB ‐ mutated LAPTM4B with wild ‐ type or kinase dead PIPKIγi5 in HEK293 cells .

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H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.


I Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with increased concentration of PtdIns ( 4 , 5 ) P2 addition . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean ± SD ; n = 4 ) .

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I Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with increased concentration of PtdIns(4,5)P2 addition. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean ± SD; n = 4).


J LAPTM4B N ‐ terminus binds multiple phosphoinositides including PtdIns ( 4 , 5 ) P2 in liposome ‐ binding assay .

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J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.


K Top : GSTpull ‐ down assay of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of different phosphoinositides . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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K Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of different phosphoinositides. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


L Top : GSTpull ‐ down assay of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ terminus and PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of PtdIns ( 4 , 5 ) P2 . Bottom : Quantification of the relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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L Top: GSTpull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of PtdIns(4,5)P2. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


A Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were starved and stimulated with 100 ng / ml EGF for 30 min , and whole ‐ cell lysates were subject to co ‐ immunoprecipitation ( co ‐ IP ) assay .

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A Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay.


B Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin , starved , and stimulated with 100 ng / ml EGF for 30 min . Total ubiquitinated proteins were purified from whole ‐ cell lysates by Ni ‐ NTA agarose and analyzed by Western blot .

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B Control or LAPTM4B knockdown MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin, starved, and stimulated with 100 ng/ml EGF for 30 min. Total ubiquitinated proteins were purified from whole‐cell lysates by Ni‐NTA agarose and analyzed by Western blot.