Figure 3 ( B Proportions and numbers of ( B - Kb - SIINFEKL + CD8 + T cells were enumerated , or ( E - IFN - γ - secreting CD8 + T cells following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T cells co - stained with CD11a and ( B ) Kb - SIINFEKL or ( E ) IFN - γ .

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Figure 3(B Proportions and numbers of (B- Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E- IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ.


Figure 4 ( B - C57BL / 6 mice received 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites , either intravenously ( n = 6 - 8 mice per group ) or ( E - intradermally ( n = 4 mice per group ) . Additional control mice did not receive sporozoites ( n = 6 - 8 intravenously , n = 4 intradermally ) . Spleens and livers were harvested either at day 14 ( n = 5 livers and n = 6 spleens for intravenous immunisation ) or day 42 ( n = 8 for intravenous immunisation ) , and IFN - γ - secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T cells co - stained with IFN - γ and CD11a ( B , E ) .

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Figure 4(B- C57BL/6 mice received 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, either intravenously (n=6-8 mice per group) or (E- intradermally (n=4 mice per group). Additional control mice did not receive sporozoites (n=6-8 intravenously, n=4 intradermally). Spleens and livers were harvested either at day 14 (n=5 livers and n=6 spleens for intravenous immunisation) or day 42 (n=8 for intravenous immunisation), and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with IFN-γ and CD11a (B, E).


Figure 5 ( C ) The upper panel of bar charts show the percentage of CD11ahi IFN - γ + CD8 + T cells and the lower panel the absolute cell counts . Bar charts show mean values ( ± SEM ) from representative experiments ( * * * , p < 0 . 001 ; one - way ANOVA with Tukey ' s multiple comparison test ) . See Appendix Table S4 for exact p - values .

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Figure 5(C) The upper panel of bar charts show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.


Figure 6 ( B , C ) Flow cytometry ( B ) and scatter plots ( C ) represent CD8 + T cells derived from peripheral blood co - stained with IFN - γ and CD11a , following ex vivo restimulation with SIINFEKL . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± SEM ) . ( D ) Protective efficacy as measured by quantitative real - time PCR . Groups of mice were vaccinated as described and challenged 19 days later with 10 , 000 WT ( n = 7 non - vaccinated , n = 9 vaccinated ) , CSPSIINFEKL ( n = 6 non - vaccinated , n = 4 vaccinated ) or UIS4SIINFEKL sporozoites ( n = 3 non - vaccinated , n = 11 vaccinated ) . 42 hours later livers were removed , and parasite load was assessed by qPCR . Plots show the relative parasite load of mice in each condition ( * * , p < 0 . 01 ; Mann - Whitney U test ) . See Appendix Table S4 for exact p - values . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± SEM ) .

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

Figure 6(B, C) Flow cytometry (B) and scatter plots (C) represent CD8+ T cells derived from peripheral blood co-stained with IFN-γ and CD11a, following ex vivo restimulation with SIINFEKL. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).(D) Protective efficacy as measured by quantitative real-time PCR. Groups of mice were vaccinated as described and challenged 19 days later with 10,000 WT (n=7 non-vaccinated, n=9 vaccinated), CSPSIINFEKL (n=6 non-vaccinated, n=4 vaccinated) or UIS4SIINFEKL sporozoites (n=3 non-vaccinated, n=11 vaccinated). 42 hours later livers were removed, and parasite load was assessed by qPCR. Plots show the relative parasite load of mice in each condition (**, p<0.01; Mann-Whitney U test). See Appendix Table S4 for exact p-values. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).


Figure 1 ( A ) Antibacterial activity of NGJ1 , T6SS - 1 - and T6SS - 2 - strains against target bacteria .

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Figure 1(A) Antibacterial activity of NGJ1, T6SS-1- and T6SS-2- strains against target bacteria.


Figure 2 ( A ) Growth curve of recombinant BL21 ( DE3 ) cells upon IPTG mediated induction of proteins , represented as colored line graphs . ( B ) Bacterial two - hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons . Interaction between T25 - zip and T18 - zip was used as positive control while pKNT25 and pUT18C ( empty vectors ) was used as negative control . Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction . ( C ) Yeast two - hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR ( PAARTseTBg ) proteins encoded in their operon . Interaction between P53 and SV40 large T - antigen ( T ) proteins was used as positive control . The pGBKT7 and pGADT7 ( empty vectors ) were used as negative control .

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Figure 2(A) Growth curve of recombinant BL21 (DE3) cells upon IPTG mediated induction of proteins, represented as colored line graphs.(B) Bacterial two-hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons. Interaction between T25-zip and T18-zip was used as positive control while pKNT25 and pUT18C (empty vectors) was used as negative control. Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction.(C) Yeast two-hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR (PAARTseTBg) proteins encoded in their operon. Interaction between P53 and SV40 large T-antigen (T) proteins was used as positive control. The pGBKT7 and pGADT7 (empty vectors) were used as negative control.


Figure 3 ( A , B ) Effect of purified TseTBg ( TseTBg1 / TseTBg2 ) and one of the variants ( TseTBg2D116A , K129A ) proteins on degradation of bacterial genomic DNA . ( C , D ) RNase activity of TseTBg effector proteins , in presence or absence of cognate TsiTBg immunity proteins . ( E ) Effect of TseTBg1 , TseTBg2 , TseTBg2D116A , K129A proteins on degradation of pHM1 , a broad host - range plasmid isolated from E . coli and NGJ1 . ( F ) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1 , reintroduced into E . coli from NGJ1 . ( G ) Effect of ectopic expression of DNA adenine methylase ( Dam ) protein on TseTBg proteins mediated degradation of plasmid DNA .

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Figure 3(A, B) Effect of purified TseTBg (TseTBg1/TseTBg2) and one of the variants (TseTBg2D116A,K129A) proteins on degradation of bacterial genomic DNA.(C, D) RNase activity of TseTBg effector proteins, in presence or absence of cognate TsiTBg immunity proteins.(E) Effect of TseTBg1, TseTBg2, TseTBg2D116A,K129A proteins on degradation of pHM1, a broad host-range plasmid isolated from E. coli and NGJ1. (F) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1, reintroduced into E. coli from NGJ1. (G) Effect of ectopic expression of DNA adenine methylase (Dam) protein on TseTBg proteins mediated degradation of plasmid DNA.


Figure 4 ( E , F ) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression ( quantified using 4 - methylumbelliferyl ß - D - glucuronide , MUG , as substrate ) under the external ( pExtTseTBg1 / pExtTseTBg2 ) and internal ( pIntTseTBg1 / pIntTseTBg2 ) promoters in E . coli .

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Figure 4(E, F) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression (quantified using 4-methylumbelliferyl ß-D-glucuronide, MUG, as substrate) under the external (pExtTseTBg1/pExtTseTBg2) and internal (pIntTseTBg1/pIntTseTBg2) promoters in E. coli.


Figure 5 ( A ) Interaction of external ( pExtTseTBg1 ) and internal ( pIntTseTBg1 ) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein . ( B ) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein ( TsiTBg1ΔHTH ) . ( C ) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein . ( D ) Interaction of control ( random ) DNA with the TsiTBg1 protein .

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Figure 5(A) Interaction of external (pExtTseTBg1) and internal (pIntTseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein. (B) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein (TsiTBg1ΔHTH). (C) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein. (D) Interaction of control (random) DNA with the TsiTBg1 protein.


Figure 1Figure 1 . Cytoplasmic conical structures in Thermococcus kodakaraensis . ( A ) A tomographic slice shows a side view of a conical structure ( c ) in the cytoplasm , rotated and enlarged in ( B ) . ( C ) A tomographic slice shows a side view of the cone in another cell , highlighting the subunit texture along the edge of the cone ( arrowheads ) . ( D , E ) Top views of a cone at different heights show the inner ring ( r ; enlarged in inset to highlight 19 - subunit structure ) and outer cone . ( F - H ) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone . ( I , J ) Different views of a 3D segmentation of the cone shown in ( A ) , embedded in a tomographic slice . s , S - layer ; m , membrane ; a , archaella ; rib , ribosomes ; rez , ribosome - excluding zone . Scale bars 100 nm ; scale bar in ( D ) applies to ( D , E ) ; scale bar in ( F ) applies to ( F - H ) ; segmentation not to scale .

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Figure 1Figure 1. Cytoplasmic conical structures in Thermococcus kodakaraensis. (A) A tomographic slice shows a side view of a conical structure (c) in the cytoplasm, rotated and enlarged in (B). (C) A tomographic slice shows a side view of the cone in another cell, highlighting the subunit texture along the edge of the cone (arrowheads). (D, E) Top views of a cone at different heights show the inner ring (r; enlarged in inset to highlight 19-subunit structure) and outer cone. (F-H) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone. (I, J) Different views of a 3D segmentation of the cone shown in (A), embedded in a tomographic slice. s, S-layer; m, membrane; a, archaella; rib, ribosomes; rez, ribosome-excluding zone. Scale bars 100 nm; scale bar in (D) applies to (D, E); scale bar in (F) applies to (F-H); segmentation not to scale.


Figure 2Figure 2 . Cones are associated with chemosensory arrays , ribosome - excluding zones and filament bundles . Tomographic slices show side ( A ) and top ( B , C ) views of cones ( c ) in three cells , highlighting associated chemosensory arrays ( ca ) , ribosome - excluding zones ( rez ) and filament bundles ( f ) . For a slice - by - slice view through the tomogram shown in ( A ) , see Movie S1 . ( D ) and ( E ) show different views of a 3D segmentation of the structures shown in ( C ) , with the conical structure in blue and the filament bundle in red . s , S - layer ; m , membrane ; a , archaellum ; o , other filaments . Scale bar 100 nm ( applies to A - C ) ; segmentation not to scale .

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Figure 2Figure 2. Cones are associated with chemosensory arrays, ribosome-excluding zones and filament bundles. Tomographic slices show side (A) and top (B, C) views of cones (c) in three cells, highlighting associated chemosensory arrays (ca), ribosome-excluding zones (rez) and filament bundles (f). For a slice-by-slice view through the tomogram shown in (A), see Movie S1. (D) and (E) show different views of a 3D segmentation of the structures shown in (C), with the conical structure in blue and the filament bundle in red. s, S-layer; m, membrane; a, archaellum; o, other filaments. Scale bar 100 nm (applies to A-C); segmentation not to scale.


Figure 3Figure 3 . Archaellum orientation with respect to the cell envelope . ( A - D ) show tomographic slices through side views of cones . In the bottom row , white lines show the angle of the archaellum with respect to the surface layer , and red dashed lines show the conserved distance from the archaellum at the membrane to the cone . Scale bar 100 nm ( applies to A - D ) .

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Figure 3Figure 3. Archaellum orientation with respect to the cell envelope. (A-D) show tomographic slices through side views of cones. In the bottom row, white lines show the angle of the archaellum with respect to the surface layer, and red dashed lines show the conserved distance from the archaellum at the membrane to the cone. Scale bar 100 nm (applies to A-D).


Figure 4Figure 4 . Structure of the T . kodakaraensis archaellum . ( A ) A sub - tomogram average of the archaellum reveals structural features , including four layers of density in the cytoplasm ( L1 - L3 , cone ) . CM , cytoplasmic membrane . The speculated identity of densities in the archaellum is proposed : archaellum fiber = FlaA / B flagellins ; integral membrane density = FlaJ ; L1 = FlaI ; L2 / L3 / cone = FlaH / FlaC / D / E . Scale bar 10 nm ( applies to A , B ) . Figure 4 . Structure of the T . kodakaraensis archaellum . ( B ) For comparison , a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown ( adapted with permission from [ 8 ] ) . Arrows indicate components with recognized homology . OM , outer membrane ; IM , inner membrane . Scale bar 10 nm ( applies to A , B ) .

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Figure 4Figure 4. Structure of the T. kodakaraensis archaellum. (A) A sub-tomogram average of the archaellum reveals structural features, including four layers of density in the cytoplasm (L1-L3, cone). CM, cytoplasmic membrane. The speculated identity of densities in the archaellum is proposed: archaellum fiber = FlaA/B flagellins; integral membrane density = FlaJ; L1 = FlaI; L2/L3/cone = FlaH/FlaC/D/E.Scale bar 10 nm (applies to A, B).Figure 4. Structure of the T. kodakaraensis archaellum.(B) For comparison, a subtomogram average of the type IVa pilus machine from Myxococcus xanthus is shown (adapted with permission from [8]). Arrows indicate components with recognized homology. OM, outer membrane; IM, inner membrane. Scale bar 10 nm (applies to A, B).


Figure 1 ( B ) flg22 - induced ROS production in Col - 0 and fer - 4 mutant roots under Pi - starvation conditions . Seedlings grown in either HP or LP media were treated with 1 μM flg22 , RALF23 or both flg22 and RALF23 for 15 mins . Roots were harvested and stained with H2DCFDA . One representative root was shown . ( C ) Average H2DCFDA signal intensity in roots shown in ( B ) . The fluorescence intensity was quantified with ImageJ . The data shown indicate the means ± SDs ( n = 30 , n refers to the number of roots per group ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results . ( F ) RALF23 inhibits flg22 - induced MAMP - responsive FRK1 gene expression in a Pi content - dependent manner . Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested . The relative expression of FRK1 was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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Figure 1(B) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. (F) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


Figure 2 ( A ) The PSR induces the cleavage of PRORALF23 - GFP into mature RALF23 - GFP . The 35S promoter - driven RALF23 - GFP line was grown in either HP or LP media for 5 days and roots were harvested . Premature and mature RALF23 levels were determined with anti - GFP . β - actin was used as a loading control . Data information : All experiments were repeated three times with similar results . ( E ) The PSR activates RALF expression in a PHR1 - dependent manner . Col - 0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested . The relative expression of RALFs was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results . ( G ) PHR1 directly binds to RALF promoters , as shown via ChIP - PCR . The Myc - labelled PHR1 overexpression line was immunoprecipitated with anti - Myc . ChIP analysis was performed on Col - 0 , CRY1 - Myc and PHR1 - Myc transformed plants , with Col - 0 and CRY1 - Myc transformed plants as negative controls . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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

Figure 2(A) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results.(E) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.(G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


Figure 3 ( B ) PHR1 negatively regulates FLS2 - BAK1 complex formation under Pi - starvation conditions . Col - 0 , phr1 mutant seedlings were grown in either HP or LP media for 5 days , then lysing roots into protoplasts , transformed with water or PHR1 - Myc plasmid and treated with flg22 for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti - FLS2 and anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as negative control . Data information : All experiments were repeated three times with similar results . ( D ) Application of RALF23 / 34 disrupted FLS2 - BAK1 complex formation in phr1 mutants . phr1 mutants grown in LP media were treated with water , 1 μM flg22 , 1 μM RALF23 , 1 μM RALF34 or both flg22 and RALF for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti - FLS2 and anti - BAK1 antibodies , respectively . IgG was used as a negative control . Data information : All experiments were repeated three times with similar results . ( E ) RALF23 / 34 promoted the growth of Pto DC3000 in phr1 mutants . Col - 0 and phr1 mutant seedlings grown in LP media were root inoculated ( by soaking ) with a bacterial suspension ( OD600 = 0 . 2 ) for 2 min , and the number of bacteria from individual root was quantified at 5 dpi . The data shown indicate the means ± SDs ( n = 18 , n refers to the number of roots per group ) . The different lowercase letters indicate statistical significance ( one - way ANOVA ) . Data information : All experiments were repeated three times with similar results . ( F ) FER negatively regulates FLS2 - BAK1 complex formation under Pi - starvation conditions . Col - 0 , fer - 4 mutant and FER / fer - 4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours . The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti - FLS2 and anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as a negative control . Data information : All experiments were repeated three times with similar results .

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

Figure 3(B) PHR1 negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, phr1 mutant seedlings were grown in either HP or LP media for 5 days, then lysing roots into protoplasts, transformed with water or PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control. Data information: All experiments were repeated three times with similar results.(D) Application of RALF23/34 disrupted FLS2-BAK1 complex formation in phr1 mutants. phr1 mutants grown in LP media were treated with water, 1 μM flg22, 1 μM RALF23, 1 μM RALF34 or both flg22 and RALF for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. IgG was used as a negative control. Data information: All experiments were repeated three times with similar results.(E) RALF23/34 promoted the growth of Pto DC3000 in phr1 mutants. Col-0 and phr1 mutant seedlings grown in LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA). Data information: All experiments were repeated three times with similar results.(F) FER negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, fer-4 mutant and FER/fer-4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control. Data information: All experiments were repeated three times with similar results.


Figure 4 ( A - B ) Relative Pseudomonas abundance in rhizosphere samples after ( A ) , Pi - stress ( LP ) or ( B ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t test ) . ( C - D ) Relative Bacillus abundance in rhizosphere samples after ( C ) , Pi - stress ( LP ) or ( D ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . ( E - F ) Relative Geodermatophilus abundance in rhizosphere samples after ( E ) , Pi - stress ( LP ) or ( F ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . ( G - H ) Relative Methylobacillus abundance in rhizosphere samples after ( G ) , Pi - stress ( LP ) or ( H ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . The data shown indicate the means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t test ) .

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Figure 4(A-B) Relative Pseudomonas abundance in rhizosphere samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test). (C-D) Relative Bacillus abundance in rhizosphere samples after (C), Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (E-F) Relative Geodermatophilus abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (G-H) Relative Methylobacillus abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).


Figure 5Bacterial inoculation promotes plant growth and alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days . The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B . subtilis . ( A ) , Images were taken 4 weeks after inoculation . Data information : All experiments were repeated three times with similar results . ( C ) Pto DC3000 and B . subtilis upregulate phosphate absorption gene expression . Col - 0 , phr1 and fer - 4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B . subtilis in Pi - sufficient or Pi - deficient condition , and the relative expression of Pi - absorbing genes was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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

Figure 5Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.(C) Pto DC3000 and B. subtilis upregulate phosphate absorption gene expression. Col-0, phr1 and fer-4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis in Pi-sufficient or Pi-deficient condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


Figure 6The fer - 4 rhizosphere microbiome promotes plant growth and alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days . The seedlings were then transferred to vermiculite and inoculated with Col - 0 or fer - 4 rhizosphere microbiomes . ( B ) , Images were taken 4 weeks after inoculation . Data information : All experiments were repeated three times with similar results . ( D ) The fer - 4 rhizosphere microbiome upregulated phosphate absorption gene expression . The roots of Col - 0 were harvested after 4 weeks of inoculation with the Col - 0 or fer - 4 rhizosphere microbiome under LP condition , and the relative expression of Pi - absorbing genes was quantified via RT - qPCR . The data shown indicate the means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t test ) . Data information : All experiments were repeated three times with similar results .

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

Figure 6The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.(D) The fer-4 rhizosphere microbiome upregulated phosphate absorption gene expression. The roots of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or fer-4 rhizosphere microbiome under LP condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


Figure 1D . Expression distribution of lncORF in both groups by Ribo - seq . H . Scatter diagram of lncRNA - seq in HCT116 / L - OHP and HCT116 cells .

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Figure 1D. Expression distribution of lncORF in both groups by Ribo-seq.H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.


Figure 2A . Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation , and PCR verification of the 12 screened lncRNA in both RNC - RNA and total RNA from two groups of CRC cells , among which the translation level of lnc - AP ( marked with a green box ) was the most notably dysregulated in both groups of drug - resistant cells ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . B . PCR analysis of lnc - AP in serum samples from oxaliplatin - resistant ( PD , n = 15 ) and - sensitive ( PR , n = 9 ) CRC patients ( mean ± SEM , t test ) . C . In the public database lncCAR , high expression level of lnc - AP was correlated with long overall survival in CRC patients . E . PCR and WB analysis of HEK293T cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . F . PCR and WB analysis of HCT116 / L - OHP and SW480 / L - OHP cells transfected with either lnc - AP - Flag OE plasmid ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector . J . IF detection of pep - AP in HCT116 / L - OHP and SW480 / L - OHP cells transfected with either HA - lnc - AP OE plasmid . Scale bar , 10 µm .

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

Figure 2A. Schematic diagram of sucrose density gradient ultracentrifugation for RNC isolation, and PCR verification of the 12 screened lncRNA in both RNC-RNA and total RNA from two groups of CRC cells, among which the translation level of lnc-AP (marked with a green box) was the most notably dysregulated in both groups of drug-resistant cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance.B. PCR analysis of lnc-AP in serum samples from oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).C. In the public database lncCAR, high expression level of lnc-AP was correlated with long overall survival in CRC patients.E. PCR and WB analysis of HEK293T cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR and WB analysis of HCT116/L-OHP and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.J. IF detection of pep-AP in HCT116/L-OHP and SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.


Figure 3B . CCK - 8 assay of inhibition ratio by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA . C . IF detection of H2AFX for DNA damage by L - OHP in two groups of CRC cells with down - or up - regulation of lnc - AP , and nuclei were stained with DAPI ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . G . Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01H . PCR analysis of lnc - AP attenuation and restoration in HCT116 and SW480 cells ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA . J . CCK - 8 assay of inhibition ratio by L - OHP in HCT116 and SW480 cells with lnc - AP / pep - AP attenuation and restoration ( n = 3 , biological replicates , mean ± SEM ) . Data information : * si - NC was the abbreviation of commercial normal control of siRNA .

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

Figure 3B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.


Figure 4D . Survival analysis correlated with TALDO1 using TCGA data . Upper : the Kaplan curve of overall survival probability for patients with high or low level of TALDO1 . Lower : the expression graph of TALDO1 in patients who were dead or not . F . IF detection of the colocalization of pep - AP - Flag and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells . Scale bar , 10 µm . G . WB confirmation of the interaction between HA - pep - AP and TALDO1 in HCT116 / L - OHP and SW480 / L - OHP cells .

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Figure 4D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not.F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.


Figure 5A . WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells , which was quantified by gray analysis on the right ( n = 3 , biological replicates , mean ± SEM , t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA . D . Cellular ROS was measured by flow cytometry and shown in both curve and bar charts ( n = 3 , biological replicates , mean ± SEM , t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA . E . MMP detection . Scale bar , 10 µm . F . IF detection of H2AFX for DNA damage by L - OHP . Scale bar , 10 µm .

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Figure 5A. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm.


Figure 6E . Cellular ROS was measured by flow cytometry and shown in both curve ( left ) and bar ( right ) charts ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA . F . MMP detection with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells . Scale bar , 10 µm .

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Figure 6E. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.


Figure 7C . Flow cytometry of the cell apoptosis ratio under L - OHP with down - or up - regulation of lnc - AP / pep - AP in two groups of CRC cells , which was quantified in bar charts on the right ( n = 3 , biological replicates , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector and si - NC was the abbreviation of commercial normal control of siRNA . E . CCK - 8 assay of the inhibition ratio by L - OHP with inhibitors of apoptosis Z - VAD - FMK or ferroptosis Fer - 1 in HCT116 / L - OHP and SW480 / L - OHP cells ( n = 3 , biological replicates , mean ± SEM ) . Data information : * Control referred to the corresponding plasmid

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Figure 7C. Flow cytometry of the cell apoptosis ratio under L-OHP with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.E. CCK-8 assay of the inhibition ratio by L-OHP with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector


Figure 8B . Alterations of tumor volume in different groups with or without pep - AP OE ( n = 6 , biological replicates , mean ± SEM ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector . F WB analysis of pep - AP , TALDO1 , PARP , cleaved - PARP and cleaved - caspase - 3 within the tumors in C ( a ) . G . Relative NADPH / NADP + ratio within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . H . GSH contents within the tumors in C ( a ) ( n = 6 , mean ± SEM , Dunnett - t test ) . Data information : * for P < 0 . 05 ; * * for P < 0 . 01 ; NS for no significance . Control referred to the corresponding plasmid vector . I . IF detection of pep - AP , TALDO1 and H2AFX , TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C ( a ) .

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Figure 8B. Alterations of tumor volume in different groups with or without pep-AP OE (n = 6, biological replicates, mean±SEM). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.F WB analysis of pep-AP, TALDO1, PARP, cleaved-PARP and cleaved-caspase-3 within the tumors in C(a).G. Relative NADPH/NADP+ ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.I. IF detection of pep-AP, TALDO1 and H2AFX, TUNEL of DNA fragmentation and IHC of Ki67 within the tumors in C(a).


Figure 1Spearman correlations between protein expression and sensitivity to Plk1 inhibitors ( BI2536 , GSK461364 , BRD - K70511574 , and GW - 843682X ) , based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database ( Li et al , 2017 ) . Proteins with expression levels that correlate with area under the curve ( AUC ) values for at least two drugs are included in the graph . The color and size of the data point represent the direction and significance ( p value ) of the correlation . Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity ( i . e . , proteins in red are more highly expressed in resistant cell lines ) . Spearman correlations between protein expression and sensitivity to Plk1 inhibitors ( BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins ( cMet , E - cadherin , β - catenin , and Snail ) ; each data point represents an individual non - small cell lung cancer cell line . E - cadherin , β catenin , and cMet protein expression correlated with drug resistance .

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Figure 1Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536, GSK461364, BRD-K70511574, and GW-843682X), based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database (Li et al, 2017). Proteins with expression levels that correlate with area under the curve (AUC) values for at least two drugs are included in the graph. The color and size of the data point represent the direction and significance (p value) of the correlation. Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity (i.e., proteins in red are more highly expressed in resistant cell lines).Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins (cMet, E-cadherin, β-catenin, and Snail); each data point represents an individual non-small cell lung cancer cell line. E-cadherin, β catenin, and cMet protein expression correlated with drug resistance.


Figure 2Three epithelial / resistant non - small cell lung cancer cell lines were treated with 5 ng / ml TGF - β for 14 days to induce a mesenchymal phenotype , which was confirmed with Western blot ( left ) and qPCR ( right ) analysis of epithelial - to - mesenchymal transition ( EMT ) markers ( A ) . Parental and TGF - β isogenic cell lines were treated with 50nM volasertib for 72 hours . Cells were then harvested , and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β - actin ( B ) . Apoptosis was measured by the Apo - BrdU assay ( C ) DNA damage was measured by the Comet assay ( D ) .

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Figure 2Three epithelial/resistant non-small cell lung cancer cell lines were treated with 5 ng/ml TGF-β for 14 days to induce a mesenchymal phenotype, which was confirmed with Western blot (left) and qPCR (right) analysis of epithelial-to-mesenchymal transition (EMT) markers (A).Parental and TGF-β isogenic cell lines were treated with 50nM volasertib for 72 hours. Cells were then harvested, and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β-actin (B).Apoptosis was measured by the Apo-BrdU assay (C)DNA damage was measured by the Comet assay (D).


Figure 3Thirty - three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib , including those involved in the cMet / FAK / Src signaling axis ( blue text ) and those involved in the PI3K / Akt signaling axis ( orange text ) . The same cell lines treated identically were subjected to immunoblotting for the indicated proteins ( upper ) with densitometric quantification normalized with β - actin ( lower ) . Epithelial ( red text ) and mesenchymal ( blue text ) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control and subjected to Western blotting with the indicated antibodies . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - sided Student t test are indicated by * p < 0 . 05 . H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Figure 3Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text).The same cell lines treated identically were subjected to immunoblotting for the indicated proteins (upper) with densitometric quantification normalized with β-actin (lower).Epithelial (red text) and mesenchymal (blue text) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control and subjected to Western blotting with the indicated antibodies. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-sided Student t test are indicated by *p < 0.05.H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Figure 4Cell viability was measured using CellTiter - Glo in NSCLC cell lines treated with volasertib , tepotinib , or a 1 : 2 ratio of both for 72 hours . Left : representative cell viability graph of cells treated with the indicated drugs . Right : heatmap depicting the calculated combination indices at Fa = 0 . 25 and Fa = 0 . 5 . Apoptosis was measured using the Apo - BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours . Immunoblots from cells treated with indicated drugs for 24 hours ( left ) with densitometric quantification normalized with β - actin ( right ) . All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ . Apoptosis was measured using the Apo - BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control . Immunoblots from cells transfected with siRNA as indicated for 48 hours ( left ) with densitometric quantification normalized with β - actin ( right ) . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - way analysis of variance with Bonferroni or Benjamini - Hochberg ( BH ) correction for multiple comparison are indicated ( * p < 0 . 01 ) . Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text , respectively .

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Figure 4Cell viability was measured using CellTiter-Glo in NSCLC cell lines treated with volasertib, tepotinib, or a 1:2 ratio of both for 72 hours. Left: representative cell viability graph of cells treated with the indicated drugs. Right: heatmap depicting the calculated combination indices at Fa = 0.25 and Fa = 0.5.Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours.Immunoblots from cells treated with indicated drugs for 24 hours (left) with densitometric quantification normalized with β-actin (right).All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control.Immunoblots from cells transfected with siRNA as indicated for 48 hours (left) with densitometric quantification normalized with β-actin (right). Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg (BH) correction for multiple comparison are indicated (*p < 0.01). Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text, respectively.


Figure 5Mice bearing epithelial ( red text ) and mesenchymal ( blue text ) non - small cell lung cancer tumors were treated with vehicle control , volasertib ( 30 mg / kg per week intravenously ) , tepotinib ( 25 mg / kg per day orally ) , or the combination for 5 weeks to generate tumor growth curves of patient - derived xenografts ( PDX ; A ) and cell line xenografts The percent change in tumor volume at the end of treatment ( normalized to day zero ) was calculated with each data point representing an individual mouse , and the bar is the mean value ± standard error of 10 mice for each groupTumor growth curve of mesenchymal PDX TC424 ( top ) and cell line xenograft Calu6 ( bottom ) upon treatment for 5 weeks and after cessation of treatment ( recovery phase ) . Data are means ± standard error of the mean from five mice for each group . Kaplan - Meier survival curves for mesenchymal PDX TC424 ( top ) and cell line xenograft Calu6 ( bottom ) with death due to excessive tumor burden as the endpoint . Quantification of TUNEL - positive cells in paraffin - embedded xenograft tissue sections .

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Figure 5Mice bearing epithelial (red text) and mesenchymal (blue text) non-small cell lung cancer tumors were treated with vehicle control, volasertib (30 mg/kg per week intravenously), tepotinib (25 mg/kg per day orally), or the combination for 5 weeks to generate tumor growth curves of patient-derived xenografts (PDX; A) and cell line xenografts The percent change in tumor volume at the end of treatment (normalized to day zero) was calculated with each data point representing an individual mouse, and the bar is the mean value ± standard error of 10 mice for each groupTumor growth curve of mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) upon treatment for 5 weeks and after cessation of treatment (recovery phase). Data are means ± standard error of the mean from five mice for each group.Kaplan-Meier survival curves for mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) with death due to excessive tumor burden as the endpoint.Quantification of TUNEL-positive cells in paraffin-embedded xenograft tissue sections.


Figure 6Calu6 , H157 , and H1355 TPR - Met - expressing or vector control ( pBABE ) - expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo - BrdU assay . Parental and TPR - Met - expressing cell lines were treated with the indicated drugs for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . β - actin was used as a loading control . Parental and TPR - Met - expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - way analysis of variance with Bonferroni or Benjamini - Hochberg correction for multiple comparisons are indicated . Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies . * p < 0 . 05 . * * p < 0 . 01 .

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Figure 6Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay.Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control.Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated.Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.


Figure 7Cell viability measured by CellTiter - Glo in Calu6 parental and Calu6 - VAR cells treated with different concentrations of volasertib for 72 hours . Micrograph shows acquisition of a mesenchymal - to - epithelial transition phenotype in VAR cells . Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting ( upper ) and reverse - transcriptase PCR ( lower ) , with GAPDH expression used for normalization . Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo - BrdU assay . Parental and VAR cells were treated with the indicated drug concentrations for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ .

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Figure 7Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours.Micrograph shows acquisition of a mesenchymal-to-epithelial transition phenotype in VAR cells.Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization.Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo-BrdU assay.Parental and VAR cells were treated with the indicated drug concentrations for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.


Figure 8Epithelial and mesenchymal non - small cell lung cancer ( NSCLC ) cell lines after treatment with 50nM volasertib for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Calu6 ( mesenchymal ) and H1975 ( epithelial ) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Calu6 ( mesenchymal ) and H1975 ( epithelial ) NSCLC cell lines after treatment with fibronectin for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours . Cells were then harvested , and lysates were immunoprecipitated ( IP ) using β1 - integrin , vimentin , and Plk1 antibodies , then immunoblotted for the indicated proteins .

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Figure 8Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after treatment with fibronectin for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins.


figf1 ( a - d ) ELISA for IL - 1β ( a - c ) , IL - 18 ( d ) and IL - 6 ( e ) in supernatants of resting or LPS - primed bone marrow - derived macrophages ( BMMs ) stimulated with palmitate conjugated to BSA ( PA - BSA ) or BSA control . ( e , f ) ELISA for IL - 1β ( e ) and IL - 18 ( f ) in supernatants . Resting or LPS - primed BMMs generated from wild - type ( WT ) , Nlrp3 − / − , Pycard − / − or Nlrc4 − / − mice were stimulated with PA - BSA . ( g ) IL - 1β ELISA of supernatants from LPS - primed BMMs stimulated with PA - BSA in the absence or presence of the pan - caspase inhibitor zVAD or caspase - 1 inhibitor zYVAD . ( h ) ELISA for IL - 1β in supernatants . Resting or LPS - primed BMMs generated from WT or Casp1 - / - mice were stimulated with PA - BSA , BSA control , ATP or nigericin . ( i ) ELISA for TNF in supernatants . Resting or LPS - primed BMMs generated from WT , Nlrp3 - / - , Pycard - / - or Casp1 - / - mice were stimulated with PA - BSA as indicated . Values are expressed as mean ± s . d . , and the results are representative of three independent experiments .

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figf1(a-d) ELISA for IL-1β (a-c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control.(e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA.(g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD.(h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1-/- mice were stimulated with PA-BSA, BSA control, ATP or nigericin.(i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3-/-, Pycard-/- or Casp1-/- mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.


figf2Immunoblotting for procaspase - 1 and activated caspase - 1 ( p10 ) , pro - IL - 1β and cleaved IL - 1β ( p17 ) in supernatants ( Sup ) and cell lysates ( Lys ) from resting or LPS - primed BMMs generated from WT , Nlrp3 − / − , Pycard − / − ( a ) or Nlrc4 − / − ( b ) mice stimulated with PA - BSA ( 0 . 2 or 0 . 5 mM ) as indicated at top . Data are representative of two independent experiments .

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figf2Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.


figf3 ( a ) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 ( DHR ) in LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of ROS inhibitor APDC ( 50 μM ) . MFI , mean fluorescence intensity . ( b ) ELISA for IL - 1β on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC . ( c ) immunoblotting for caspase - 1 p10 and IL - 1β p17 on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC . ( d ) ELISA for IL - 1β . Resting or LPS - primed BMMs were stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of the ROS inhibitors APDC ( 10 or 50 μM ) and N - acetyl - cysteine ( NAC , 5 or 25 mM ) or the NAPDH oxidase inhibitor diphenyleneiodonium ( DPI , 5 or 25 μM ) . Values are expressed as mean ± s . d . In a , one of two independent experiments is shown . In each of b - d , the results are representative of three independent experiments .

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figf3(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity.(b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.(c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.(d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.


figf4 ( a ) Thr172 - phosphorylated ( p - ) and total AMPK - α subunit determined by immunoblotting . Resting or LPS - primed BMMs were untreated or stimulated with PA - BSA ( 0 . 5 mM ) for 24 h . ( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( b ) ROS production determined using the fluoroprobe DHR . ( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( c , d ) IL - 1β ( c ) and IL - 6 ( d ) in supernatants , measured by ELISA . ( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( e ) Immunoblotting for caspase - 1 , IL - 1β and AMPK signaling molecules . ( f ) Transfection of BMMs with empty vector ( EV ) or constitutively active ( CA ) AMPK - α1 subunit . N , non - transfected . Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK - α1 and pmaxGFP . Values are percentages of GFP - positive cells in the ranges indicated by the bars . ( g ) ELISA for IL - 1β in supernatants of cells transfected as in f and stimulated with PA - BSA ( 0 . 5 mM ) for 24 h . In each of a , b and f , one of two independent experiments is shown . In each of c - e and g , the results are representative of three independent experiments . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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

figf4(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h.(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR.(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA.(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules.(f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.


figf5 ( a ) Immunoblotting for AMPK activation ( Thr172 p - AMPK - α ) and LC3B ( top ) , and densitometric analysis to quantify ratio of LC3B - II to actin ( bottom ) . BMMs were pretreated with LPS ( 200 ng / ml ) for 3 h , followed by PA - BSA ( 0 . 5 mM ) treatment for 24 h in the absence or presence of chloroquine ( 50 μM ) . Med , medium control . ( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) . ( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) . Cells were fixed and examined by transmission electron microscopy for autophagosomes ( c ) . Quantitation ( c , right ) is based on counting autophagosomes in ten cells per treatment . ( d ) Immunoblotting ( left ) and densitometric analysis to quantify ratio of LC3B - II to actin and ULK1 species ( right ) in treated BMMs . ( e ) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator . In each of a and e , one of three independent experiments is shown . In each of b and d , one of two independent experiments is shown . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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

figf5(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control.(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right).(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (c). Quantitation (c, right) is based on counting autophagosomes in ten cells per treatment.(d) Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs.(e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.


figf6 ( a , b ) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL - 1β ( 2 ng / ml ) ( a ) or TNF ( 2 ng / ml ) ( b ) for 24 h , then stimulated with insulin ( 200 nM ) for 10 min . p - Akt ( Ser473 ) was determined by flow cytometry ( right ) with quantification shown in the graph at left ( a ) . p - Akt ( Ser473 ) was determined by immunoblotting ( b ) . ( c ) p - IRS1 ( Ser307 ) , determined by immunoblotting , in FL83B mouse liver cells treated with either IL - 1β or TNF for 24 h . ( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without stimulation . ( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without stimulation ( g ) Akt serine 473 phosphorylation measured by flow cytometry . FL83B cells were pretreated with WT or Pycard − / − CM in the absence or presence of the IL - 1 receptor antagonist anakinra ( 1 μg / ml ) for 24 h , then stimulated with insulin . ( h ) p - Akt ( Ser473 ) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF . The results shown are representative of three independent experiments and are expressed as mean ± s . d .

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

figf6(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).(c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h.(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation.(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.(h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.


figf7 ( a ) ITT performed 2 h after IL - 1β injection . Eight - week old male C57BL / 6 mice were intraperitoneally injected with saline or mouse recombinant IL - 1β ( 1 μg / kg ) . ( b ) ITT in WT mice on regular diet ( RD ) or HFD for 12 weeks , or Il1b - / - mice on HFD for 12 weeks . ( c , d ) ITT ( c ) and serum IL - 1β ( d , left ) and TNF ( d , right ) determined by ELISA , 2 h after IL - 1β injection . Recombinant mouse IL - 1β was administered to WT and Tnfa − / − mice ( n = 5 for each group ) . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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figf7(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg).(b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b-/- mice on HFD for 12 weeks.(c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


figf8 ( a ) Blood glucose and insulin levels measured in WT ( n = 6 ) , Nlrp3 − / − ( n = 5 ) or Pycard − / − ( n = 6 ) mice under fasting or refed conditions after 12 weeks of HFD . ( b - e ) Glucose tolerance test ( GTT ) ( b , d ) and insulin tolerance test ( ITT ) ( c , e ) performed for WT , Nlrp3 − / − ( b , c ) and Pycard − / − ( d , e ) mice on HFD for 12 weeks . ( f ) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks . ( g , h ) Insulin - stimulated phosphorylation of IRβ , IRS1 and Akt ( Ser473 ) in liver tissues of individual WT and Pycard − / − mice ( g ) , and p - Akt ( Ser473 ) in liver , white adipose ( WAT ) and muscle tissues of individual WT and Nlrp3 − / − mice ( h ) on HFD for 12 weeks , after insulin ( 2 IU / kg ) infusion . Graphs at right of blots show the quantitation of each molecule . pY , phosphotyrosine . ( i , j ) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3 − / − mice ( i ) and Pycard − / − mice ( j ) on regular diet ( RD ) or HFD for 12 weeks . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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figf8(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.(b-e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks.(f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks.(g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine.(i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


Figure 2B , C Cell death measured by lactase dehydrogenase ( LDH ) ( B ) and cell viability measured by 3 - ( 4 , 5 - dimethyl - thiazol - . 2 - yl ) - 2 , 5 - diphenyl tetrazolium bromide ( MTT ) ( C ) in T98 cells 42 hrs post - transfection of pure and interrupted SCA8 repeat tracts ; LDH n = 8 , MTT n = 12 , n = independent experiments , * p < 0 . 05 , NT not transfected , EV : empty vector ; unpaired t test ; mean ± SEM . D , E qRT - PCR showing transcript levels of Pure 96 and Int . 95 ( D , n = 6 ; p = 0 . 0187 ) , and Pure 104 and Int . 102 ( E , n = 6 ; * * * * p < 0 . 0001 ) repeats ; n = independent experiments ; unpaired t test ; mean ± SEM .

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Figure 2B, C Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted SCA8 repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM.D, E qRT-PCR showing transcript levels of Pure 96 and Int.95 (D, n=6; p=0.0187), and Pure 104 and Int.102 (E, n=6; **** p<0.0001) repeats; n=independent experiments; unpaired t test; mean ± SEM.