( C ) 8 - oxo - dG ( orange ) establishes a Hoogsteen base pair with dA ( cyan ) at primer terminus . Tyr505 Arg517 ( purple ) interact with minor groove of base pair . base pair geometry ( C1 ′ λ angles ) is indicated at bottom of figure . A simulated annealing Fo - Fc omit density map for pair is also shown ( contoured at 3σ ) .

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

(C) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of primer terminus active site with an undamaged reference structure ( 2PFO , black ) . catalytic aspartic are shown in yellow , incoming dUMPNPP in magenta , primer terminal dA in cyan . Expectedly , Mg2 + ( neon ) is occupying metal B site . However , metal A is absent in structure , resulting in a non - catalytic position of 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

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

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( A ) Glu529 ( purple ) forms a hydrogen bond with C2 - group of primer terminal templating 8 - oxo - dG ( orange ) in anti - conformation . ( B ) This interaction is abolished when 8 - oxo - dG ( orange ) adopts syn - conformation . An overlay with 8 - oxo - dG ( anti ) - containing structure reveals that Glu529 ( black ) would likely clash ( red - dotted ) with C8 - carbonyl of 8 - oxo - dG ( syn ) . Repositioning of Glu529 ( purple ) places side chain 4 . 5 Å away from C8 - carbonyl . ( C ) Comparison of catalytic efficiencies for extension past an 8 - oxo - dG ( anti ) : dC , 8 - oxo - dG ( syn ) : dA dG : dC base pairs at primer terminus using WT E529A Pol λ .

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

(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.


( A ) Undamaged structure containing a templating dG at primer terminus ( 2PFO ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at primer terminus . ( B ) Undamaged structure containing a templating dA at primer terminus ( 1XSN ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at primer terminus .

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

(A) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.


( B ) YTS KIR2DL1 were transfected with either N . S siRNA ( blue curve ) or with SHP - 1 Cbls siRNA ( red curve ) . After 48 hours , were loaded with calcium - sensitive Fluo - 3 - AM analyzed for basal intracellular calcium for 1 min . NK were then mixed with 721 . 221 Cw4 target at 37 ° C monitoring of calcium was continued . data shown are representative of three independent experiments .

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

(B) YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( C ) YTS KIR2DL1 were incubated with either 721 . 221 Cw4 or Cw7 target for 2 hours analyzed by flow cytometry to determine expression of CD107a . Expression of CD107a was measured by mean ( MFI ) . Relative expression of CD107a MFI was normalized to mock - transfected sample following Cw4 incubation . Data are means ± of five independent experiments . ( n = 5 ) . P values were calculated by One - sample t - One way ANOVA following Tukey ' s post hoc are indicated by asterisks * P < 0 . 05 , * * * * P < 0 . 0001 .

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(C) YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments.(n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P<0.0001 .


( E ) Killing . YTS KIR2DL1 were mock - transfected or transfected with N . S siRNA or SHP - 1 Cbls siRNA . After 48 hours , were incubated with [ 35S ] Met - loaded 721 . 221 Cw4 or Cw7 target . After 5 hours of co - culture , supernatant was collected , radioactive signal was measured . Data are means ± of three independent experiments . P values were calculated by One - way ANOVA following Tukey ' s post hoc are indicated by asterisks * P < 0 . 05 , * * P < 0 . 01 .

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(E) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .


( B ) Characterization of diameter zeta potential ( Z - potential ) with addition of each layer . Graph below presenting NP distribution .

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(B) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution.


( A ) NK92 - NKp46low , NK92 - NKp46high , YTS KIR2DL1 , K562 721 . 221 HLA - Cw4 were stained with anti - NKp46 monoclonal antibody followed by with Alexa568 - Fluor goat anti - mouse IgG secondary antibody . were then analyzed using flow cytometry . ( B ) NK92 - NKp46low , NK92 - NKp46 + , YTS KIR2DL1 , K562 , 721 . 221 HLA - Cw4 were incubated with 50 µg rhodamine - labeled NPs analyzed using flow cytometry to confirm internalization of coated NPs in a NKp46 - specific manner .

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

(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1, K562 and 721.221 HLA-Cw4 cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry. (B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner.


( C - F ) ( C ) NK92 - NKp46low , ( D ) NK92 - NKp46high , ( E ) YTS KIR2DL1 ( F ) K562 were incubated with 50 µg rhodamine - labeled NPs analyzed using confocal microscopy . Upper panel shows wide field . Lower panel shows single - . data shown are representative of three independent experiments .

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(C-F) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments.


( A ) YTS KIR2DL1 NK were incubated with NPs loaded with Cbl - b , c - Cbl SHP - 1 siRNA , or NPs loaded with 1500 pmol N . S siRNA . Following 48 hours of incubation , were lysed , was blotted with anti - Cbl - b , anti - c - Cbl or anti - SHP - 1 antibodies . GAPDH served as loading control . Densitometric of bands was performed using ImageJ normalized to GAPDH densitometry values . by ImageJ densitometry is summarized in graph below . Data are means ± of four independent experiments . ( n = 4 ) . P values were calculated by One - sample t - are indicated by asterisks . * P < 0 . 05 , * * P < 0 . 01 .

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(A) YTS KIR2DL1 and primary NK cells were incubated with NPs loaded with Cbl-b, c-Cbl and SHP-1 siRNA, or NPs loaded with 1500 pmol N.S siRNA. Following 48 hours of incubation, cells were lysed, and the membrane was blotted with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. GAPDH served as loading control. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of four independent experiments. (n=4). P values were calculated by One-sample t-tests and are indicated by asterisks. *P < 0.05, **P < 0.01.


( B ) YTS KIR2DL1 were incubated with SHP - 1 Cbls siRNAs - loaded NPs ( red curve ) , with NS siRNA loaded NPs ( green curve ) or with empty NPs ( blue curve ) . After 48 hours , were loaded with calcium - sensitive Fluo - 3 - AM analyzed for basal intracellular calcium for 1 min . NK were then mixed with 721 . 221 Cw4 target at 37 ° C , monitoring of calcium was continued . data shown are representative of three independent experiments .

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

(B) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNAs-loaded NPs (red curve), with NS siRNA loaded NPs (green curve) or with empty NPs (blue curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C, and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.


( D ) YTS KIR2DL1 were treated with SHP - 1 Cbls siRNAs - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , were incubated with either 721 . 221 Cw4 or Cw7 target , supernatant was then collected , granzyme B were evaluated using ELISA sandwich . of granzyme B were quantified on a standard curve . Data are means ± of five independent experiments . ( n = 5 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YTS KIR2DL1 cells were treated with SHP-1 and Cbls siRNAs-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected, and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified based on a standard curve. Data are means ± SEM of five independent experiments. (n=5). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. **P < 0.01.


( F ) pNK were pretreated with either SHP - 1 Cbls siRNA - loaded NPs or N . S siRNA - loaded NPs . After 48 hours , were incubated with either 721 . 221 Cw4 or Cw7 target for 2 hours analyzed by flow cytometry to determine expression of CD107a . Expression of CD107a was quantitated by mean ( MFI ) . data represent five independent experiments ( n = 5 ) Data are shown as mean ± . ) ( n = 5 ) . P values were calculated by One - sample t - One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * P ≤ 0 . 05 .

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

(F) pNK cells were pretreated with either SHP-1 and Cbls siRNA-loaded NPs or N.S siRNA-loaded NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was quantitated by mean fluorescence intensity (MFI). The data represent five independent experiments (n=5) Data are shown as mean ± SEM.) (n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.05.


( A ) YTS KIR2DL1 were incubated with SHP - 1 Cbls siRNA - loaded NPs ( red curve ) or empty NPs ( purple curve ) . After 48 hours , were loaded with Fluo - 3 - AM analyzed for intracellular calcium for 20 min at 37 ° C . YTS KIR2DL1 that were treated with PBS served as a negative control ( green curve ) . NK that were mixed with 721 . 221 Cw7 target served as a positive control ( blue curve ) . Calcium were analyzed by spectrofluorometry . ( n = 3 )

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

(A) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNA-loaded NPs (red curve) or empty NPs (purple curve). After 48 hours, cells were loaded with Fluo-3-AM and analyzed for intracellular calcium levels for 20 min at 37°C. YTS KIR2DL1 cells that were treated with PBS served as a negative control (green curve). NK cells that were mixed with 721.221 Cw7 target cells served as a positive control (blue curve). Calcium levels were analyzed by spectrofluorometry. (n=3)


( B ) Freshly isolated PBMCs were treated with SHP - 1 Cbls siRNA - loaded NPs , empty NPs , PBS as negative control or PHA as a positive control . Following incubation , supernatant from each sample was collected of human cytokines TNF - α , IL - 6 , IL - 10 IFN - γ were determined using ELISA . data represent four independent experiments ( n = 4 ) . P values were calculated by One way ANOVA following Tukey ' s post hoc , are indicated by asterisks . * P ≤ 0 . 008 . Data are shown as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Freshly isolated PBMCs were treated with SHP-1 and Cbls siRNA-loaded NPs, empty NPs, PBS as negative control or PHA as a positive control. Following incubation, supernatant from each sample was collected and the levels of human cytokines TNF-α, IL-6, IL-10 and IFN-γ were determined using ELISA. The data represent four independent experiments (n=4). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.008. Data are shown as mean ± SEM.


( C ) Average growth rates of tumors from first treatment to end point from same experimental groups of mice as indicated in B . P value was calculated by two - Student ' s t - is indicated within graph P = 0 . 001 . Data are shown as mean ± .

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(C) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B. P value was calculated by two-tailed Student's t-test and is indicated within the graph P =0.001. Data are shown as mean ± SEM.


( E ) Following 24 hours after last treatment , extracted from tumors of either SHP - 1 Cbls or N . S siRNA loaded NKp46 - NPs treated mice were analyzed by flow cytometry . Expression of CD107a was measured by mean ( MFI ) . Relative expression of CD107a MFI was normalized to sample treated with N . S siRNA loaded NPs . data represent three independent experiments . Data are shown as mean ± SEMn = 3 ; P value was calculated by one sample t - is indicated within graph P = 0 . 009 .

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(E) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice were analyzed by flow cytometry. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009.


( F ) Survival in mice treated with NK ' gene silenced for SHP - 1 Cbls using siRNA - loaded NPs ( n = 18 ) or N . S siRNA - loaded NPs ( n = 18 ) as demonstrated by Kaplan - Meier survival curve . P values were calculated using log - rank is indicated within graph P = 0 . 00009 .

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(F) Survival analysis in mice treated with NK cells' gene silenced for SHP-1 and Cbls using siRNA-loaded NPs (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009.


A ) Contribution of base change types to overall SNV composition in ABiM cohort for captured regions mRNA in captured regions , as well as SCAN - B whole mRNA .

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A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.


B ) Number of non - synonymous mutations per sample . Bars are colored by PAM50 subtypes Luminal A ( blue ) , Luminal B ( blue ) , HER2 - enriched ( pink ) , basal - like ( red ) , Normal - like ( green ) Unclassified ( gray ) .

B) Number of non-synonymous mutations per sample. Bars are colored by PAM50 subtypes Luminal A (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray).


C - F ) Lollipop plots showing location , abundance , impact of SNVs in C ) TP53 , D ) PIK3CA , E ) PTEN , F ) ERBB2 on respective encoded . change labels are shown for most mutated acid positions , with residues ordered left to right by mutation frequency within each label .

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C-F) Lollipop plots showing the location, abundance, and impact of SNVs in C) TP53, D) PIK3CA, E) PTEN, and F) ERBB2 on the respective encoded protein. Protein change labels are shown for the most mutated amino acid positions, with residues ordered left to right by mutation frequency within each label.


Waterfall plot of 20 most frequently mutated genes ( rows ) across 3 , 217 SCAN - B samples ( columns ) . Genes are ranked from top to bottom by mutation frequency . Samples are sorted by histological subtype alteration occurrence . Mutations are colored by predicted functional impact .

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Waterfall plot of the 20 most frequently mutated genes (rows) across 3,217 SCAN-B samples (columns). Genes are ranked from top to bottom by mutation frequency. Samples are sorted by histological subtype and alteration occurrence. Mutations are colored by predicted functional impact.


A - E ) Overall survival ( OS ) of with tumors containing mutations in genes A ) TP53 , B ) PIK3CA , C ) ERBB2 , D ) PTEN . E ) OS by PTEN - MutExp genotype ( " low " defined as PTEN mutation or PTEN expression in lower quartile across cohort , " normal " otherwise ) stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine chemotherapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV5 . In each Kaplan - Meier plot , wildtype ( wt ) normal cases are plotted in blue , mutated ( mut ) low cases are plotted in red , log - rank P value is given , hazard ( HR ) for mutation / low is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.


A - E ) Overall survival of with tumors containing mutations in pathways A ) WNT signaling , B ) Hedgehog signaling , C ) NOTCH2 signaling , D ) p53 independent damage repair , E ) TGFβ signaling , stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine chemo therapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV4 . In each Kaplan - Meier plot , wildtype ( wt ) cases are plotted in blue , mutated ( mut ) cases are plotted in red , log - rank P value is given , hazard ( HR ) for mutation is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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A-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV4. In each Kaplan-Meier plot, wildtype (wt) cases are plotted in blue, mutated (mut) cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.


Overall survival stratified by mutational burden ( TMB ) across treatment groups in 3 , 217 . Samples were classified as TMB - high if amount of non - synonymous mutations per expressed MB ( rnaMB ) was ≥ median number of non - synonymous mutations per rnaMB across whole SCAN - B cohort ( 0 . 082 mutations per rnaMB ) TMB - low otherwise . In each Kaplan - Meier plot , TMB - low cases are plotted in blue , TMB - high cases are plotted in red , log - rank P value is given , hazard ( HR ) for TMB - high is given with a 95 % CI after uni - variable multi - variable ( MV ) Cox regression adjustment . Covariables included in MV were at diagnosis , lymph node status , , variables denoted by following symbols : ¶ , ER , PgR , HER2 , NHG ; ¤ , ER , PgR , NHG ; $ , HER2 NHG ; # , NHG . Abbreviations : TMB , mutational burden ; ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade ; HoR , hormone receptor ; TNBC , triple - negative breast cancer .

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

Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.


E , Immunoblot of described in ( D ) , harvested at indicated time points after G1 / S release . Exemplary blot from three independent experiments .

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E, Immunoblot analysis of cells described in (D), harvested at the indicated time points after G1/S release. Exemplary blot from three independent experiments.


F , DUB activity for OTUD6B in asynchronous , G1 / S - or mitotically synchronized released A549 using HA - ubiquitin vinyl - sulfone to isolate active forms of DUBs . was performed by immunoblotting using indicated antibodies .

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

F, DUB activity assay for OTUD6B in asynchronous, G1/S- or mitotically synchronized and released A549 cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting using the indicated antibodies.


B , In - vivo ubiquitylation analyses of LIN28B in HEK293T in which OTUD6B was silenced by siRNA . were transfected with indicated siRNAs overexpression constructs , then treated with MG132 for 3 hrs . Lysis IP was done under denaturing conditions followed by WB . Data information : For B , exemplary blot of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, In-vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132 for 3 hrs. Lysis and IP was done under denaturing conditions followed by WB analysis. Data information: For B, exemplary blot of two independent experiments.


C , In - vivo ubiquitylation of LIN28B in OTUD6B a catalytically inactive variant of OTUD6B ( OTUD6B - C158A ) overexpressing . HEK293T were transfected with indicated combinations of FLAG - LIN28B , HA - Ubiquitin , OTUD6B , OTUD6B - C158A EV control treated with MG132 for 3 hrs 24 hrs later . Denatured WCE were subjected to FLAG - IP . WCE IP were analyzed by immunoblotting including a K48 - specific ubiquitin antibody . Exemplary blots from three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, In-vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B (OTUD6B-C158A) overexpressing cells. HEK293T cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132 for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP were analyzed by immunoblotting including a K48-specific ubiquitin antibody. Exemplary blots from three independent experiments are shown.


D , Pull - down of endogenous LIN28B from HEK293T WCE using bacterially purified GST - OTUD6B . Pulldowns WCE were analyzed by immunoblot using indicated antibodies . Data information : For D , exemplary blot of two independent experiments .

O O O O O O O O O O O O O O O O O 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, Pull-down of endogenous LIN28B from HEK293T WCE using bacterially purified GST-OTUD6B. Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies. Data information: For D, exemplary blot of two independent experiments.


E , Immunoblot of an in - vitro deubiquitylation using GST - purified OTUD6B from FLAG - purified ubiquitylated LIN28B from HEK293T . FLAG - IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG - LIN28B HA - Ubiquitin . Purified FLAG - LIN28B was eluted from beads incubated with GST or GST - OTUD6B followed by immunoblot . Data information : For E : exemplary blot of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunoblot analysis of an in-vitro deubiquitylation assay using GST-purified OTUD6B from bacteria and FLAG-purified ubiquitylated LIN28B from HEK293T. FLAG-IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG-LIN28B and HA-Ubiquitin. Purified FLAG-LIN28B was eluted from the beads and incubated with GST or GST-OTUD6B proteins followed by immunoblot analysis. Data information: For E: exemplary blot of two independent experiments.


A , Co - IP of FLAG - OTUD6B with endogenous LIN28B from MM1 . S synchronized in G1 / S 12 hrs post release . MM1 . S stably expressing FLAG - OTUD6B or FLAG - EV were synchronized at G1 / S harvested at indicated time points after release . IPs were preformed analyzed together with WCE by immunoblotting . Data information : blots are representations of three independent experiments .

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A, Co-IP of FLAG-OTUD6B with endogenous LIN28B from MM1.S cells synchronized in G1/S and 12 hrs post release. MM1.S cells stably expressing FLAG-OTUD6B or FLAG-EV were synchronized at G1/S and harvested at the indicated time points after release. IPs were preformed and analyzed together with WCE by immunoblotting. Data information: All blots are representations of three independent experiments.


B , Immunoblot of LIN28B OTUD6B throughout cycle . RPMI ( B ) were synchronized at G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized were released , harvested at indicated time points analyzed by immunoblot using indicated antibodies . Data information : blots are representations of three independent experiments .

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B, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. RPMI (B) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


C , Immunoblot of LIN28B OTUD6B throughout cycle . A549 ( C ) were synchronized at G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized were released , harvested at indicated time points analyzed by immunoblot using indicated antibodies . Data information : blots are representations of three independent experiments .

C, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. A549 (C) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


D , Immunoblot of LIN28B half - life in G1 / S - synchronized ( upper panel ) asynchronous ( lower panel ) A549 upon OTUD6B depletion . transfected with respective siRNAs were synchronized in G1 / S - phase or not treated with cycloheximide ( CHX ) as indicated . Data information : blots are representations of three independent experiments .

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D, Immunoblot analysis of LIN28B protein half-life in G1/S-synchronized (upper panel) and asynchronous (lower panel) A549 cells upon OTUD6B depletion. Cells transfected with the respective siRNAs were synchronized in G1/S-phase or not and treated with cycloheximide (CHX) as indicated. Data information: All blots are representations of three independent experiments.


D , Immunoblot of WCE obtained from a representative experiment depicted in ( C ) .

D, Immunoblot analysis of WCE obtained from a representative experiment depicted in (C).


C , Immunoblot of RPMI8226 , KMS12BM MM1 . S expressing indicated shRNAs . Depicted are examples of three independent experiments per . Specification of MYC rearrangements : RPMI8226 ( der ( 17 ) t ( ? 8 ; 17 ) ( q21 . 2 ; q25 ) ) , KMS12BM ( der ( 1 ; 8 ) ( q10 ; q10 ) x2 ) , MM1 . S ( der3t ( 3 ; 8 ) ) .

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C, Immunoblot analysis of RPMI8226, KMS12BM and MM1.S cells expressing the indicated shRNAs. Depicted are examples of three independent experiments per cell line. Specification of MYC rearrangements: RPMI8226 (der(17)t(?8;17)(q21.2;q25)), KMS12BM (der(1;8)(q10;q10)x2), MM1.S (der3t(3;8)).


A , Correlation between MYC OTUD6B expression in MM samples at diagnosis . mRNA expression in CD138 + MM was quantified by real - time qPCR ( n = 89 ) . Data are fit by linear regression ( black ) ; Pearson r , Pearson correlation coefficient ; P < 0 . 0001 ; by linear regression Pearson correlation .

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A, Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis. mRNA expression in primary CD138+ MM cells was quantified by real-time qPCR (n = 89 patients). Data are fit by linear regression (black line); Pearson r, Pearson correlation coefficient; P < 0.0001; by linear regression and Pearson correlation.


Addition of rhTNF at fracture site on days 0 1 led to augmented healing , indicated by increased % callus mineralization , at day 28 after operation in a dose - dependent manner . Data are presented as mean ± . Treatment with 1 ng TNF versus PBS control , * * * * P < 0 . 0001 by one - way ANOVA with Dunnett ' s multiple comparisons .

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Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose-dependent manner. Data are presented as mean ± SEM. Treatment with 1 ng TNF versus PBS control, ****P < 0.0001 by one-way ANOVA with Dunnett's multiple comparisons test.


Addition of rhTNF at fracture site must be given within first 24 h to augment healing , indicated by % callus mineralization . Data are presented as mean ± . 1 ng TNF versus PBS control treatment on days 0 1 , * * * P = 0 . 0009 by one - way ANOVA with Dunnett ' s multiple comparisons .

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

Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing, indicated by % callus mineralization. Data are presented as mean ± SEM. 1 ng TNF versus PBS control treatment on days 0 and 1, ***P = 0.0009 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with systemic anti - TNF or local rmIL - 10 led to impaired fracture repair at day 28 , indicated by % callus mineralization . Data are presented as mean ± . Treatment with PBS control versus anti - TNF , * P = 0 . 037 , PBS control versus rmIL - 10 , * P = 0 . 037 , by unpaired two - sided t - .

Treatment with systemic anti-TNF or local rmIL-10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti-TNF, *P = 0.037, PBS control versus rmIL-10, *P = 0.037, by unpaired two-sided t-tests.


Representative micro - CT at fracture site showing ( from left to right ) : lateral view of tibia , cross - section , cross longitudinal sections with color overlay 3D reconstruction . In color overlays , shade of blue corresponds to mineralization : blue denotes soft immature callus , whereas blue denotes hard mineralized callus . Scale bar , 2 mm .

Representative micro-CT images at the fracture site showing (from left to right): lateral view of tibia, cross-section, and cross and longitudinal sections with color overlay and 3D reconstruction. In the color overlays, the shade of blue corresponds to percentage mineralization: light blue denotes soft immature callus, whereas dark blue denotes hard mineralized callus. Scale bar, 2 mm.


Representative ISH image ( field ) showing TNF expression at murine fracture site at 24 h after fracture . Scale bar , 250 μm . Red box indicates region of interest .

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Representative ISH image (light field) showing TNF expression at the murine fracture site at 24 h after fracture. Scale bar, 250 μm. Red box indicates region of interest.


High - power of region of interest from ( E ) : at 24 h after fracture , mTNF expression ( signal on field , above ) co - localized with polymorphonuclear found on adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm . Neutrophils were identified by their polymorphonuclear as well as positive brown with anti - neutrophil elastase .

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High-power micrographs of region of interest from (E): at 24 h after fracture, mTNF expression (white signal on dark field, above) co-localized with polymorphonuclear cells found on the adjacent H&amp;E section (below). Scale bar, 25 μm. Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti-neutrophil elastase.


High - power : at 7 days , TNF expression ( signal on field , above ) co - localized with F4 / 80 - positive ( stained brown ) extravasating from a blood vessel on adjacent H & amp ; amp ; E section ( below ) . 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 O O O O O O O O O O O O O O O O O O O O O

High-power micrographs: at 7 days, TNF expression (white signal on dark field, above) co-localized with F4/80-positive cells (stained dark brown) extravasating from a blood vessel on the adjacent H&amp;E section (below). Scale bar, 25 μm.


Neutrophils were mobilized into murine systemic circulation within 30 min of injury in our murine fracture model . Compared to control number of blood neutrophils at 3 h * * * P = 0 . 0006 at 6 h * P = 0 . 042 , by one - way ANOVA with Dunnett correction .

Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model. Compared to control and number of blood neutrophils at 3 h ***P = 0.0006 and at 6 h *P = 0.042, by one-way ANOVA with Dunnett correction.


Depletion of neutrophils using anti - Ly6G . Intravenous anti - Ly6G treatment led to depletion of neutrophils in systemic circulation ( left ) as well local peri - fracture soft tissues ( right ) . Left : of neutrophils in blood harvested by cardiac puncture at 3 h post - injury . Data are presented as mean ± ( * * P = 0 . 0074 using unpaired one - t - ) . Right : of positively stained infiltrative neutrophils in adjacent muscle to fracture site comparing neutrophil depletion with anti - Ly6G antibody versus IgG control at day 1 day 7 post - fracture . At day 1 , number of neutrophils in control group as detected by Ly6G NE in control versus treatment : * * * * P = 0 . 0001 for both Ly6G NE , by unpaired two - t - . At day 7 , number of neutrophils in control group as detected by Ly6G NE in control versus treatment groups : * * * * P = 0 . 0001 for Ly6G * P = 0 . 020 for NE , by unpaired two - t - . Number of neutrophils at day 1 versus day 7 in control groups , * P = 0 . 037 for Ly6G * * * P = 0 . 0006 for NE , by unpaired two - t - .

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

Depletion of neutrophils using anti-Ly6G. Intravenous anti-Ly6G treatment led to depletion of neutrophils in the systemic circulation (left) as well the local peri-fracture soft tissues (right). Left: counts of neutrophils in the blood harvested by cardiac puncture at 3 h post-injury. Data are presented as mean ± SEM (**P = 0.0074 using unpaired one-tailed t-test). Right: counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti-Ly6G antibody versus IgG control at day 1 and day 7 post-fracture. At day 1, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment: ****P = 0.0001 for both Ly6G and NE, by unpaired two-tailed t-test. At day 7, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups: ****P = 0.0001 for Ly6G and *P = 0.020 for NE, by unpaired two-tailed t-test. Number of neutrophils at day 1 versus day 7 in control groups, *P = 0.037 for Ly6G and ***P = 0.0006 for NE, by unpaired two-tailed t-test.


Representative sections showing local infiltration of neutrophils in adjacent muscle stained using anti - Ly6G anti - neutrophil elastase antibodies at day 1 post - fracture following treatment with anti - Ly6G antibody or IgG control . Scale bar , 100 μm .

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Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti-Ly6G and anti-neutrophil elastase primary antibodies at day 1 post-fracture following treatment with anti-Ly6G antibody or IgG control. Scale bar, 100 μm.


Depletion of neutrophils using anti - Ly6G led to impaired fracture healing . Representative section stained with Masson ' s trichrome at day 14 comparing fracture healing in mouse treated with isotype control ( top ) versus anti - Ly6G ( bottom ) . Black in right column are identical to color in left column ; they have been labeled to clearly demonstrate anatomical structures . Control section shows advanced mineralized callus , while treatment section shows a large immature unmineralized callus . Muscle : red ; collagen , bone , mineralized callus : green . Scale bar , 1 mm .

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Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm.


Anti - Ly6G treatment led to impaired fracture healing as shown by reduced % callus mineralization at day 28 after surgery representative micro - CT . Anti - Ly6G treatment led to delayed mineralization remodeling of fracture callus compared to control . Data are presented as mean ± . * * * P = 0 . 0006 by unpaired two - sided t - . Scale Bar , 2 mm .

Anti-Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro-CT images. Anti-Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control. Data are presented as mean ± SEM. ***P = 0.0006 by unpaired two-sided t-test. Scale Bar, 2 mm.


Anti - Ly6G treatment depletes local monocytes / macrophages at fracture site . of positively stained infiltrative F4 / 80 + in adjacent muscle to fracture site following treatment with control or anti - Ly6G at day 1 day 7 . Data are presented as mean ± . * * P = 0 . 005 at day 1 * * P = 0 . 002 at day 7 , * P = 0 . 013 for control at day 1 versus control at day 7 , by unpaired two - sided t - .

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

Anti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.


Addition of 1 ng rhTNF to fracture supernatant in air pouch model led to increased numbers of neutrophils ( Ly6G + , CD11b + ) monocytes / macrophages ( Ly6G − , CD11b + , CD115 + ) in infiltrate . These effects were abrogated by addition of anti - CCL2 . Neutrophils : FS versus FS + TNF * P = 0 . 015 , FS + TNF versus FS + TNF + anti - CCL2 * P = 0 . 028 , FS + TNF + anti - CCL2 versus FS + TNF + IgG * P = 0 . 042 by one - way ANOVA followed by Bonferroni ' s multiple comparison . Monocytes / macrophages : FS versus FS + TNF * * P = 0 . 0063 , FS + TNF versus FS + TNF + anti - CCL2 * * * * P < 0 . 0001 , FS + TNF + anti - CCL2 v FS + TNF + IgG * P = 0 . 015 by one - way ANOVA followed by Bonferroni ' s multiple comparisons .

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

Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.


Addition of 1 ng rhTNF to fracture supernatant increased CCL2 in air pouch . " Neat " indicates level of CCL2 in fracture supernatant before injection into air pouch . Data are presented as mean ± . FS versus FS + TNF * * P = 0 . 0020 by one - way ANOVA with Bonferroni ' s multiple comparisons .

Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. "Neat" indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.


Addition of rhTNF to enriched bone marrow - derived murine neutrophils pre - exposed to fracture supernatant in vitro led to up - regulation of CCL2 production at 1 h . Data are presented as mean ± . No TNF versus TNF 0 . 01 ng / ml * P = 0 . 012 , TNF 0 . 1 ng / ml * * * P < 0 . 0001 , TNF 1 . 0 ng * * * P = 0 . 0001 , TNF 10 ng / ml * * * P < 0 . 0001 , TNF 100 ng / ml * P = 0 . 023 by one - way ANOVA with Dunnett ' s multiple comparisons .

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

Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with CCR2 antagonist , INCB3344 , led to impaired fracture healing compared to vehicle control as shown by reduced % callus mineralization at day 28 after operation . Data are presented as mean ± . * P = 0 . 011 unpaired two - sided t - . Representative micro - CT are shown . Scale bar , 2 mm .

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

Treatment with CCR2 antagonist, INCB3344, led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation. Data are presented as mean ± SEM. *P = 0.011 unpaired two-sided t-test. Representative micro-CT images are shown. Scale bar, 2 mm.


rhTNF treatment augmented early phase fracture healing in osteoporotic mice . Treatment with 1 ng rhTNF at fracture site on days 0 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but same final result at day 28 . Data are presented as mean ± . At day 14 , PBS control versus TNF , * * P = 0 . 0099 ; at day 28 , PBS control versus TNF , nsP = 0 . 37 , by unpaired two - sided t - . Representative micro - CT at day 14 are shown . rhTNF treatment led to mature callus bridging across fracture which was absent in PBS - treated control group . Scale bar , 2 mm .

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

rhTNF treatment augmented early phase fracture healing in osteoporotic mice. Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28. Data are presented as mean ± SEM. At day 14, PBS control versus TNF, **P = 0.0099; at day 28, PBS control versus TNF, nsP = 0.37, by unpaired two-sided t-test. Representative micro-CT images at day 14 are shown. rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS-treated control group. Scale bar, 2 mm.


( A - D ) A . castellanii was infected ( MOI 5 ) with L . pneumophila JR32 producing ( A ) GFP under control of PflaA ( Ptac - mCherry - PflaA - gfp ) mCherry constitutively , ( B ) GFP under control of PralF ( PralF - gfp ) stained with DAPI , ( C ) GFP under control of PsidC ( PsidC - gfp ) stained with DAPI , or ( D ) Timer constitutively ( pNP107 ) . infected amoebae were fixed at different time points post - infection , analyzed by confocal microscopy 3D reconstructed ( scale bars : 5 µm ) . Motile virulent ( GFP - positive ) emerge at colony cluster periphery only after replication has ceased ( red Timer ) .

(A-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).


( G ) Transmission electron of A . castellanii infected with L . pneumophila . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila wild - type JR32 subjected to high - pressure freezing electron microscopy . Many LCVs were still intact at 42 h post infection ( left panel ; arrow : intact LCV , arrowhead plasma ) , occasionally , were flagellated ( middle panel , arrow ) . Some LCVs were already ruptured , were released into host cytosol ( right panel ; arrows : ruptured LCV ) . Scale bars : 2 µm , 0 . 5 µm ( insets ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transmission electron micrographs of A. castellanii infected with L. pneumophila. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila wild-type strain JR32 and subjected to high-pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm, 0.5 µm (insets).


( A ) L . pneumophila subpopulation proteomics . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) . After lysis , released intracellular were sorted by flow cytometry in mCherry - positive / GFP - negative mCherry - positive / GFP - positive L . pneumophila subpopulations , their proteome was determined compared . abundance in each subset is depicted as a volcano plot . enriched in GFP - producing subpopulation have a positive log2 ( q - value ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value).


( D , E ) Expression of select L . pneumophila genes in amoebae . A . castellanii was infected ( MOI 5 ; 6 h 48 h ) with L . pneumophila JR32 harboring reporter constructs for ( D ) PfleQ - mCherry - PflaA - gfp or ( E ) PlegC8 - mCherry - PflaA - gfp , respectively . infected amoebae were fixed imaged by confocal microscopy ( scale bars : 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).


( A - E ) Representative live microscopy of D . discoideum infected with PflaA - GFP - producing L . pneumophila . D . discoideum Ax3 producing mCherry ( pDM1042 ) was infected ( MOI 5 ) with ( A , B ) wild - type L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) , ( C , D ) JR32 ( Ptac - mCerulean - PflaA - gfp ) or ( E ) ∆ flaA ( Ptac - mCerulean - PflaA - gfp ) embedded in agarose for 48 h . PflaA - GFP - positive L . pneumophila ( A ) escapes LCV ( 0 sec ) , followed by other , which are contained in cytosol localize to plasma , where a PflaA - GFP - positive L . pneumophila first leaves ruptured host ( 185 sec ) , or ( B ) triggers host ( loss of mCherry ) upon exit of amoeba . PflaA - GFP - positive L . pneumophila ( C ) escape LCV after 75 sec spread within cytosol for several minutes , ( D ) are first to exit host , leading to loss of mCherry host . ( E ) L . pneumophila ∆ flaA lyses LCV host less vigorously . Scale bars : 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.


( B ) L . pneumophila lacking flaA or lqsA are impaired for spreading from amoebae . A . castellanii amoebae were infected ( MOI 5 , 52 h ) with L . pneumophila JR32 , ∆ flaA or ∆ lqsA harboring Ptac - mCherry - PflaA - gfp embedded in 0 . 5 % agarose / PYG medium . are representative for L . pneumophila strains indicated : JR32 ( n = 25 ) , ∆ flaA ( n = 13 ) or ∆ lqsA ( n = 19 ) . Scale bars : 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.


( A , B ) Role of Lqs system for PflaA expression in intracellular L . pneumophila . A . castellanii was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , ( A ) fixed analyzed by confocal microscopy ( representative 3D reconstructions , scale bars : 10 µm ) , or ( B ) lysed , fixed analyzed by flow cytometry .

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

(A, B) Role of the Lqs system for PflaA expression in intracellular L. pneumophila. A. castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed and analyzed by flow cytometry.


( C - F ) Role of Lqs system for LCV escape localization of L . pneumophila . ( C , D ) A . castellanii or ( E , F ) D . discoideum was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , fixed treated with an anti ‐ ubiquitin antibody . Amoebae containing decorated with ubiquitin were quantified by confocal microscopy . Representative of ( C ) A . castellanii ( scale bars : 10 µm ) or ( E ) D . discoideum ( scale bars : 5 μm ) infected with L . pneumophila JR32 escaping or not from LCV are shown . of amoebae containing ubiquitinated was quantified by counting total number of infected number of ubiquitinated : ( D ) JR32 : n = 482 ; ΔlqsA : n = 834 ; ΔlqsR : n = 572 ; ΔlqsS : n = 729 ; ΔlqsT : n = 1469 ; ΔlqsS - ΔlqsT : n = 693 ; ( F ) : JR32 : n = 136 ; ΔlqsA : n = 201 ; ΔlqsR : n = 303 ; ΔlqsS : n = 183 ; ΔlqsT : n = 534 ; ΔlqsS - ΔlqsT : n = 180 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.


( G , H ) D . discoideum producing LCV / PtdIns ( 4 ) P probe P4C - GFP - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS - ∆ lqsT , or ∆ flaA harboring Ptac - mCerulean . Representative of ( G ) D . discoideum infected with L . - pneumophila JR32 in intact with intact LCV ( 18 . 5 % ) , lysed lysed LCVs ( 60 . 2 % ) , intact with lysed LCVs ( 15 . 5 % ) lysed with intact LCVS ( 5 . 8 % ) are shown ( scale bars : 5 µm ) . ( H ) of amoebae with intact LCVs was quantified by counting total number of infected number of with intact LCV : JR32 : n = 426 ; ΔlqsA : n = 468 ; ΔlqsR : n = 344 ; ΔlqsS : n = 325 ; ΔlqsT : n = 287 ; ΔlqsS - ΔlqsT : n = 289 ; ΔflaA : n = 248 .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.


D . discoideum producing LCV / PtdIns ( 4 ) P probe P4C - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila wild - type strains ( JR32 , Corby ) , or phospholipase triple mutants ( ∆ plcABC , ∆ plaACD ) harboring Ptac - mCerulean - PflaA - gfp reporter construct , fixed treated with an anti ‐ ubiquitin antibody , LCV escape localization of was assessed by confocal microscopy through of amoeba containing intact , P4C - mCherry - positive LCVs or ubiquitin - decorated , . ( A ) Representative of amoebae harboring L . pneumophila confined within LCVs or released from LCV to cytosol are shown ( scale bars : 5 μm ) .

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

D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).


( A ) Pancreatic cancer ( PANC - 1 ) normal human fibroblasts ( NHFs ) were treated with increasing concentrations of Green 1 NSC 51046 following treatment at indicated time points , were incubated with WST - 1 viability dye for 4 hours . Absorbance was read at 450 nm results were expressed as a of control . Values are expressed as mean ± SD from quadruplicates ; ( i ) PANC - 1 treated with NSC 51046 ( Interaction between each : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( ii ) NHF treated with NSC 51046 ( Interaction between each : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iii ) PANC - 1 treated with Green 1 ( Interaction between each : p = 0 . 9955 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iv ) NHF treated with Green 1 ( Interaction between each : p = 0 . 3642 ; Control versus column factor : * p = 0 . 0370 )

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O 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 O O O O O O O O O O O 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 O O O 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) Pancreatic cancer cells (PANC-1) and normal human fibroblasts (NHFs) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points, cells were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and results were expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates; (i) PANC-1 treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (ii) NHF treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (iii) PANC-1 treated with Green 1 (Interaction between each concentration: p = 0.9955; Control versus column factor: ****p<0.0001); (iv) NHF treated with Green 1 (Interaction between each concentration: p = 0.3642; Control versus column factor: *p = 0.0370)


( B ) Following treatment of human acute T leukemia ( Jurkat ) with Green 1 , a trypan blue excluding was carried out . were stained with trypan blue impermeable dye numbers of trypan blue positive were obtained using a hemocytometer . Results are expressed as of trypan blue positive , indicating dead with non - intact membranes . Values are expressed as mean ± SD from two independent experiments .

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(B) Following treatment of human acute T cell leukemia cells (Jurkat) with Green 1, a trypan blue excluding assay was carried out. Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer. Results are expressed as percent of trypan blue positive cells, indicating dead cells with non-intact cell membranes. Values are expressed as mean ± SD from two independent experiments.


Following treatment with either Green 1 ( A ) Pancreatic cancer ( BxPC - 3 PANC - 1 ) normal human fibroblasts ( NHFs NFFs ) were incubated with Hoechst 33342 dye to characterize detect induction of apoptosis . Brightly stained , condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis .

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Following treatment with either Green 1(A) Pancreatic cancer cells (BxPC-3 and PANC-1) and normal human and fetal fibroblasts (NHFs and NFFs) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis. Brightly stained, condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis.


( B ) PANC - 1 were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046 . were counterstained with Hoechst 33342 dye propidium iodide ( PI ) to visualize . shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 colchicine .

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(B) PANC-1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046. Cells were counterstained with Hoechst 33342 dye and propidium iodide (PI) to visualize nuclear morphology and cell death. Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine.


( A ) Pancreatic cancer , human leukemia ( Jurkat ) NHFs were incubated with Monodansylcadaverine ( MDC ) to stain autophagic vacuoles propidium iodide ( PI ) for to determine if autophagic induction led to . Bright , punctate is indicative of autophagy , as seen in our positive control ( Tamoxifen treated ) . were taken at 400X magnification on a microscope .

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(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope.


( B ) Western blot analyses of Beclin - 1 expression LC3 conversion were conducted on PANC - 1 treated with 5 . 0 µM Green 1 at various time points . β - actin was measured as an internal control . Samples treated for 3 6 hours were compared to early control , while sample treated for 48 hours was compared to 48 - hour control . Results are expressed as mean ± SD from two independent experiments for both Western blots .

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(B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.


( A ) Colchicine derivatives ( Green 1 NSC 51046 ) were incubated with porcine tubulin for an hour in a 96 - well plate . absorbance ( OD 340 nm ) was obtained every minute during one hour incubation . Colchicine was used for comparison to derivatives .

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(A) Colchicine derivatives (Green 1 and NSC 51046) were incubated with porcine tubulin for an hour in a 96-well plate. The absorbance (OD 340 nm) was obtained every minute during the one hour incubation. Colchicine was used for comparison to the derivatives.


( B ) Isolated mitochondria from PANC - 1 were treated with Green 1 ROS production was measured using Amplex Red substrate in presence of horseradish peroxidase ( HRP ) . Results were compared to control untreated mitochondria , colchicine treated mitochondria positive control , paraquat ( PQ ) . readings were taken in 5 min intervals for 4 hours at Ex . 560 nm Em . 590 nm expressed as relative units ( RFU ) . Results are a representative of three independent experiments .

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(B) Isolated mitochondria from PANC-1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria, colchicine treated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 hours at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Results are a representative of three independent experiments.


Following acclimatization , CD - 1 nu / nu mice were separated into two groups , one group was injected subcutaneously in right left hind flanks with Me2SO in PBS ( 10 µL in 200 µL PBS ) , while second group received subcutaneous injections of Green 1 ( 10 mg / kg / day for a total volume of 10 µL Green 1 / 200 µL PBS ) three times a week for a period of one month . ( A ) Mice weights were recorded twice a week for duration of study .

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Following acclimatization, CD-1 nu/nu mice were separated into two groups, one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS (10 µL in 200 µL PBS), while the second group received subcutaneous injections of Green 1 (10 mg/kg/day for a total volume of 10 µL Green 1/200 µL PBS) three times a week for a period of one month. (A) Mice weights were recorded twice a week for the duration of the study.


( B ) Following study , mice were sacrificed their organs were obtained for histopathological assessment . Hematoxylin Eosin was carried out on tissue samples .

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(B) Following the study, mice were sacrificed and their organs were obtained for histopathological assessment. Hematoxylin and Eosin staining was carried out on the tissue samples.


, Scaled reduced cost for uptake rates of at different growth rates . Colour represents scaled reduced cost value . A higher value indicates a greater impact of a given increase in acid uptake on growth rate .

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, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


, fold changes of CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated on 2 - sided two sample t - with a FDR threshold of 0 . 05 S 0 equal to 1 ( Tusher et al , 2001 ) . outside significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of are highlighted are labeled with their gene names .

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, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


( C ) Change in expression of 36 genes defining host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 are shown .

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(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.


( D ) Change in expression of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 are shown .

(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


( A ) Representative of Mock , SARS - CoV - 2 infected ( Ctrl ) , Salmeterol - treated wells analyzed with Multiwavelength Scoring application in MetaXpress . Grayscales of were adjusted to enable direct comparison of relative of among treatments : Segmentation show how were segmented identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm .

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(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.


( B ) Violin plots of Vero - E6 labeled for Spike . Multi - wavelength Scoring algorithm in MetaXpress was used to determine integrated signal in as a measure of amount of Spike within each . plots show population distribution of integrated signal for of treatments . Boxes in plot show interquartile range ( IQR ) with top bottom edges marking 75th 25th percentiles , respectively . horizontal in box is median value , whiskers are defined to be 1 . 5 IQR . ordinate is a log scale . effect of treatment is assessed quantitatively by changes in median signal level , qualitatively by observing changes in modes . dashed is 3 standard deviations above mean signal in Mock samples is used as a cut - off to quantify number of that are positive or negative for Spike signal . statistics table below plots shows number of counted in each treatment group , median of population .

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(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


HEK293 acceptor transfected with or without ACE2 TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of compounds for 1 - 2 h , co - cultured for 4 hours with HEK293 donor expressing SARS - CoV - 2 spike GFP , or donor expressing GFP only ( no spike ) . of GFP - positive objects were acquired on a confocal high - content imager analyzed for syncytia formation integrated GFP ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in Materials Methods . Data are aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

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HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


V . cholerae wild - type ΔrpoE strains carrying indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV VrrA was monitored on Northern blots . 5S rRNA served as loading control .

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V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.


V . cholerae wild - type , ΔvchM ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases analyzed for .

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V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.


V . cholerae wild - type ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) treated with ethanol ( 3 . 5 % final conc . ) , or water . was determined 180 min after ethanol treatment mKate2 of mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) treated with ethanol ( 3 . 5 % final conc . ) , or water . was determined 180 min after ethanol treatment mKate2 of mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates CFU / ml were determined .

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V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . were treated with L - arabinose ( 0 . 2 % final conc . ) samples were collected at indicated time points after induction . Northern blot was performed to determine VrrA , MicV ompT . 5S rRNA served as loading control . For comparison , samples of a wild - type carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold ~ 7 - fold higher of VrrA MicV expressed from pBAD plasmids , respectively ( see Source data for quantifications ) .

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V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).


V . cholerae ΔvrrA ΔmicV strains carrying indicated reporter plasmids ( x - axis ) either an empty vector control ( pCtr ) , pMicV , or pVrrA plasmid , were cultivated in M9 GFP was measured . of control strains was set to 1 . target genes were classified according to ( B ) : regulated by both

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V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAs


V . cholerae ΔvrrA ΔmicV strains carrying indicated reporter plasmids ( x - axis ) either an empty vector control ( pCtr ) , pMicV , or pVrrA plasmid , were cultivated in M9 GFP was measured . of control strains was set to 1 . target genes were classified according to regulated only by

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

V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicV


V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA