Figure 1Human red blood cells ( RBCs ) were incubated with 400 nM pigment or control ΔcylE extract , and kinetics of K + and Hb release was monitored . Data shown are the average and SEM of six independent experiments . The time to 50 % K + and 50 % Hb release with pigment was 4 . 8 min and 8 . 4 min , respectively ; n = 6 , P < 0 . 0001 , extra sum - of - squares F test . Role of osmoprotectants in pigment - treated RBC . HumanRBCs were pre - incubated with GBSpigment for 2 min at RT , centrifuged , and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0 . 40 nm ( PEG200 ) , 0 . 56 nm ( PEG400 ) , 0 . 89 nm ( PEG1000 ) , 1 . 1 nm ( PEG1500 ) , or 1 . 6 nm ( PEG3000 ) , respectively . Release of Hb was measured after 1 h of incubation at 37 ° C . Data shown are the average and SEM of three independent experiments . Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers . Lipid bilayers were generated using diphytanoylphosphatidylcholine ( DPhPC ) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract . In the pigment - treated sample , channel conductance indicating disruption of the membrane is seen within 45 s . Erratic and non - discrete fluctuations in current are observed , suggesting the formation of multiple , small membrane defects . The bilayer eventually breaks at 120 s . In lipid bilayers treated with the control ΔcylE extract , the mean ionic current trace remains constant at 0 pA , showing no membrane disruption . Data shown are representative of three independent experiments .

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

Figure 1Human red blood cells (RBCs) were incubated with 400 nM pigment or control ΔcylE extract, and kinetics of K+ and Hb release was monitored. Data shown are the average and SEM of six independent experiments. The time to 50% K+ and 50% Hb release with pigment was 4.8 min and 8.4 min, respectively; n = 6, P < 0.0001, extra sum-of-squares F test.Role of osmoprotectants in pigment-treated RBC. HumanRBCs were pre-incubated with GBSpigment for 2 min at RT, centrifuged, and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0.40 nm (PEG200), 0.56 nm (PEG400), 0.89 nm (PEG1000), 1.1 nm (PEG1500), or 1.6 nm (PEG3000), respectively. Release of Hb was measured after 1 h of incubation at 37°C. Data shown are the average and SEM of three independent experiments.Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers. Lipid bilayers were generated using diphytanoylphosphatidylcholine (DPhPC) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract. In the pigment-treated sample, channel conductance indicating disruption of the membrane is seen within 45 s. Erratic and non-discrete fluctuations in current are observed, suggesting the formation of multiple, small membrane defects. The bilayer eventually breaks at 120 s. In lipid bilayers treated with the control ΔcylE extract, the mean ionic current trace remains constant at 0 pA, showing no membrane disruption. Data shown are representative of three independent experiments.


Figure 2A , B PBMC - derived macrophages were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by ELISA ( B ) . Data information : Data shown are the average of four independent experiments performed in triplicate , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA . ( A ) n = 4 , * P = 0 . 021 for WT versus ΔcovR ; * P = 0 . 01 for ΔcovR versus ΔcovRΔcylE . ( B ) n = 4 , * P = 0 . 031 for WT versus ΔcovR ; * P = 0 . 036 for ΔcovR versus ΔcovRΔcylE . ( C ) n = 4 , * * * * P < 0 . 0001 , * * P = 0 . 002 . ( D ) n = 4 , * * P = 0 . 005 . C , D PBMC - derived macrophages primed with 100 ng / ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 4 , * * * * P < 0 . 0001 , * * P = 0 . 002 . ( D ) n = 4 , * * P = 0 . 005 .

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

Figure 2A, B PBMC-derived macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by ELISA (B).Data information: Data shown are the average of four independent experiments performed in triplicate, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 4, *P = 0.021 for WT versus ΔcovR; *P = 0.01 for ΔcovR versus ΔcovRΔcylE. (B) n = 4, *P = 0.031 for WT versus ΔcovR; *P = 0.036 for ΔcovR versus ΔcovRΔcylE. (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.


Figure 3A , B WT THP - 1macrophages were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data information : Data from three independent experiments performed in triplicate are shown , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA . ( A ) n = 3 , * * P = 0 . 0080 for WT versus ΔcovR ; * * P = 0 . 0016 for ΔcovR versus ΔcovRΔcylE . ( B ) n = 3 , * P = 0 . 03 for WT versus ΔcovR ; * P = 0 . 02 for ΔcovR versus ΔcovRΔcylE . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * P = 0 . 01 . C , D WT THP - 1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * P = 0 . 01 .

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

Figure 3A, B WT THP-1macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.


Figure 4A , B THP - 1macrophages transfected with empty vector , scrambled control , shASC , or shNLRP3 were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data from three independent experiments performed in triplicate are shown . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( A ) n = 3 , for WT : * * P = 0 . 001 ( vector versus shNLRP3 ) , * * P = 0 . 0016 ( vector versus shASC ) ; for ΔcovR : * * * P = 0 . 0008 ( vector versus shNLRP3 ) , * * * P = 0 . 0001 ( vector versus shASC ) , * P = 0 . 047 ( scrambled versus shNLRP3 ) , * * P = 0 . 0054 ( scrambled versus shASC ) . Data obtained from THP - 1 / vector was not significantly different from THP - 1 / scrambled , P = 0 . 18 . ( B ) n = 3 , * P = 0 . 025 . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 89 . C , D The shRNA THP - 1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release in supernatants was measured by Luminex assay ( D ) . Pigment - mediated cytotoxicity is dependent on the NLRP3 inflammasome components , suggesting that pigment is inducing a programmed cell death . Data shown are the average of at least three independent experiments performed in triplicate . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( C ) n = 3 , for 4 μM pigment : * * P = 0 . 0063 ( vector versus shNLRP3 ) , * * P = 0 . 0035 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 ) , * * P = 0 . 009 ( scrambled versus shASC ) ; for 2 μM pigment : * * P = 0 . 0088 ( vector versus shNLRP3 ) , * * P = 0 . 0066 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 or shASC ) ; for 1 μM pigment : * P = 0 . 02 ( vector versus shNLRP3 ) , * P = 0 . 01 ( vector versus shASC ) . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 99 . ( D ) n = 3 , * * * * P < 0 . 0001 , * P = 0 . 015 .

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

Figure 4A, B THP-1macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89.C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.


Figure 5A , B THP - 1macrophages proficient for NLRP3 ( transfected with scrambled control , A ) or deficient for NLRP3 ( shNLRP3 , B ) were treated with 1 μM pigment or ΔcylE extract for 20 min , and propidium iodide ( PI ) was added during the final 10 min . PI uptake was measured by flow cytometry , and data shown are representative of two independent experiments . C , D Intracellular potassium concentration was measured by ICP - AES . THP - 1macrophages transfected with the scrambled control ( C ) or shNLRP3 ( D ) were treated with GBSpigment ( 1 μM ) or an equivalent amount of the ΔcylE extract . At various time points , cells were lysed and intracellular [ K + ] was measured relative to untreated cells ( see bars and left y - axis ) , and percent cell death was quantified by alamar blue ( see squares , dotted connecting lines and right y - axis ) . Both NLRP3 - proficient and NLRP3 - deficient macrophages initially lose intracellular K + due to GBSpigment ( compare t = 0 min to t = 30 min ) , but the NLRP3 - deficient cells ( shNLRP3 ) are able to recover , while the scrambled control do not , demonstrating that initial K + loss occurs independently of NLRP3 . Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett ' s multiple comparison test following ANOVA ; all data were compared to control at t = 0 , error bars ± SEM . ( C ) n = 3 , * * * P = 0 . 0002 , * P = 0 . 019 , * * P = 0 . 0032 for 120 min , * * P = 0 . 0072 for 180 min . ( D ) n = 3 , * * P = 0 . 0043 , * P = 0 . 031 . ( E ) WT THP - 1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride , and cytotoxicity was measured by alamar blue assay . The addition of potassium chloride is able to protect the macrophages from cytolysis , demonstrating that K + efflux is essential for this process . Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM ( n = 3 , * * * * P < 0 . 0001 , * * P = 0 . 0036 ) .

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

Figure 5A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031.(E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).


Figure 6THP - 1macrophages were treated with GBSpigment , and caspase 1 activation was measured by flow cytometry using a FLICA reagent . Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line , demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome . Results are representative of three independent experiments . WT THP - 1macrophages were treated with the caspase 1 inhibitor Z - YVAD - FMK , the caspase 3 / 7 inhibitor Z - DEVD - FMK , or DMSO only prior to treatment with the GBSpigment . YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment , demonstrating that caspase 1 is required for GBSpigment - mediated cell death , characteristic of pyroptosis , while DEVD had no effect . Data are average of three independent experiments , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA ( n = 3 , * * * * P < 0 . 0001 , * * * P = 0 . 0002 , * P = 0 . 014 ) .

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

Figure 6THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).


Figure 7In utero fetal death in wild - type CD - 1mice due to infection with GBS WT , hyperhemolytic ΔcovR , and non - hemolytic ΔcovRΔcylE . Fetal death is represented by the number of dead fetuses / total number of fetuses obtained from six pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * * * * P < 0 . 0001 , Fisher ' s exact test . H & amp ; amp ; E staining of uterine tissue . Open arrow indicates the presence of few mononuclear cells , whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris . Fetal death due to infection with hyperhemolytic GBS ΔcovR and non - hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome - deficient mice ; fetal death is represented by the number of dead fetuses / total number of fetuses obtained from 6 pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * P = 0 . 011 , * * P = 0 . 0015 , * * * * P < 0 . 0001 , Fisher ' s exact test . Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS ; P = 0 . 31 , Fisher ' s exact test . Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains ( n = 6 / pup ; of note , pups that were delivered preterm were excluded from CFU enumeration . Scheme of pup numbering is shown in ( A ) . RUH and LUH indicate right uterine horn and left uterine horn , respectively . CFUs are not significantly different between any of the groups ( ANOVA , P = 0 . 6 , error bars ± SEM ) . Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . IL - 1β levels in GBS - infected tissues ( placenta and fetus , n = 6 / group ) was measured by Luminex assay ( * P = 0 . 025 , * * * P = 0 . 0002 , * * * * P < 0 . 0001 , Bonferroni ' s multiple comparison test following ANOVA ) . IL - 1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS ; P = 0 . 99 .

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

Figure 7In utero fetal death in wild-type CD-1mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. 'n' indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&amp;E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. 'n' indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS; P = 0.99.


f1 ( d ) NIH3T3 cells stably expressing PS - CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels . ( e , f ) Mouse fibroblasts ( NIH3T3 ) stably expressing PS - CFP2 or KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . ( e ) Representative images in both channels . ( f ) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( white bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of four different experiments with > 50 cells counted per experiment . ( * ) Differences with PS - CFP2 in the green channel ( P = 0 . 005 , ANOVA - Bonferroni ) . ( § ) Differences with samples in the presence of serum ( P = 0 . 03 , ANOVA - Bonferroni ) . ( g ) Green fluorescence intensity at excitation 450 nm and emission 510 - 550 nm . Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence ( white bars ) or absence ( gray bars ) of serum was calculated for each condition . Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means + s . e . of 3 independent experiments . Differences with serum ( * ) were significant for P = 0 . 04 ( t - test ) . Scale bars , 5 μm .

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

f1(d) NIH3T3 cells stably expressing PS-CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels.(e, f) Mouse fibroblasts (NIH3T3) stably expressing PS-CFP2 or KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. (e) Representative images in both channels. (f) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (white bars) or absence (gray bars) of serum. Values are mean+s.e. of four different experiments with >50 cells counted per experiment. (*) Differences with PS-CFP2 in the green channel (P=0.005, ANOVA-Bonferroni). (§) Differences with samples in the presence of serum (P=0.03, ANOVA-Bonferroni). (g) Green fluorescence intensity at excitation 450 nm and emission 510-550 nm. Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence (white bars) or absence (gray bars) of serum was calculated for each condition. Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means+s.e. of 3 independent experiments. Differences with serum (*) were significant for P=0.04 (t-test). Scale bars, 5 μm.


f2 ( a - c ) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ - PS - CFP2 maintained in the absence ( black circles ) or presence ( white squares ) of serum for the indicated times . ( a ) Representative images of cells maintained in serum - free media . Quantification of the number of puncta per cell ( b ) and green fluorescence intensity ( c ) . Values are expressed as percentage of fluorescence at time 0 and half - lives ( t1 / 2 ) are indicated . Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum ( * ) were significant for P = 0 . 048 ( 16 h ) and P = 0 . 04 ( 24 h ) ( t - test ) . ( d ) The same cells imaged in the cyan channel , before and at the indicated times after photoconversion ( arrow ) by exposure to 405 nm light . Top : Representative images of the cyan fluorescence . Bottom : Quantification of the intensity of the cyan fluorescence . Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1 . Values are mean + s . e . of 3 different experiments with > 10 cells counted per experiment . Scale bars , 5 μm .

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

f2(a-c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta per cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for P=0.048 (16 h) and P=0.04 (24 h) (t-test).(d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405 nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean+s.e. of 3 different experiments with >10 cells counted per experiment. Scale bars, 5 μm.


f3 ( a ) Localization of photoconverted KFERQ - PS - CFP2 ( green ) in LysoTracker - stained compartments ( red ) in mouse fibroblasts maintained in the presence or absence of serum . ( b ) Percentage of co - localization of KFERQ - PS - CFP2 with the indicated proteins . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment . ( c ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LAMP - 1 , LAMP - 2 or LAMP - 2A . ( d ) Immunofluorescence with antibodies that recognize PS - CFP2 ( green ) and hsc70 ( red ) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction . ( e , f ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LC3 , CD - M6PR , Rab5 ( e ) and ubiquitin ( f ) in mouse fibroblasts maintained in serum - free media . Scale bars , 5 μm .

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f3(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum.(b) Percentage of co-localization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment.(c) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A.(d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction.(e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.


f4 ( a ) Mouse fibroblasts knockeddown for LAMP - 2A and transfected with KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Representative images of cells also immunostained for LAMP - 1 are shown . ( b , c ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were photoconverted and maintained for 16 h in media supplemented ( + ) or not ( − ) with serum in the presence of 3 - methyladenine ( 3 - MA ) to inhibit macroautophagy . ( b ) Representative images of the 3 - MA - treated cells . ( c ) Quantification of the number of green fluorescence puncta per cell in 3 - MA treated or not cells maintained in the presence ( white bars ) or absence ( black bars ) of serum . Values are mean + s . e . of 3 different experiments with > 50 cells counted per experiment . No significant differences were detectable between samples supplemented or not with 3MA . Scale bars , 5 μm .

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

f4(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown.(b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.


f5 ( a ) Immunoblot with the antibody that recognizes PS - CFP2 of homogenates ( Hom ) , cytosol ( Cyt ) and lysosomal membranes ( MB ) and matrices ( Mtx ) isolated from mouse fibroblasts stably expressing PS - CFP2 or KFERQ - PS - CFP2 proteins 16 h after serum removal . ( b ) Immunoblot for the indicated proteins of homogenates ( Hom ) , mitochondria ( Mit ) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ - PS - CFP2 maintained for 16 h in media supplemented ( + ) or not ( − ) with serum . ( c ) Quantification of the amount of KFERQ - PS - CFP2 associated to lysosomes ( matrix and membrane ) performed in blots as the one shown in ( b ) . Values are expressed as percentage of the total KFERQ - PS - CFP2 in the cell and are mean + s . e . of three different experiments . Difference in the lysosomal associated KFERQ - PS - CFP2 between serum + and serum − condition ( * ) were significant for P = 0 . 033 ( t - test ) . ( d ) Changes in the intracellular content of KFERQ - PS - CFP2 on serum removal . Values are expressed as percentage of KFERQ - PS - CFP2 present in serum - supplemented cells and area mean + s . e . of three different experiments . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 007 ( t - test ) . ( e - g ) Effect of inhibition of macroautophagy by treatment with 3 - methyladenine ( 3 - MA ) on the lysosomal content of KFERQ - PS - CFP2 in cells maintained in serum - free media for 16 h . ( e ) Immunoblot for the indicated proteins of the same fractions as described in ( b ) . The amount of KFERQ - PS - CFP2 associated to lysosomes ( f ) and the changes in the intracellular content of KFERQ - PS - CFP2 were calculated as in ( c , d ) . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 032 ( t - test ) .

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

f5(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16 h after serum removal.(b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16 h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in (b). Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean+s.e. of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for P=0.033 (t-test).(d) Changes in the intracellular content of KFERQ-PS-CFP2 on serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean+s.e. of three different experiments. Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.007 (t-test).(e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).


f6 ( a , b ) Human neuroblastoma ( SH - SY5Y ) , hepatoma ( HuH - 7 ) and breast adenocarcinoma ( MCF - 7 ) cell lines were transiently transfected with KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Images show representative cells after immunostaining for LAMP - 2 ( red ) . ( b ) Quantification of the number of green fluorescent puncta per cell under basal conditions ( black bars ) and after serum removal ( gray bars ) . Values of NIH3T3 are included as reference . Values are mean + s . e . of three - to - five different experiments with > 50 cells counted per experiment . Differences with ( * ) NIH3T3 or ( § ) with cell supplemented with serum are significant for P0 . 05 ( range 0 . 002 - 0 . 03 , ANOVA - Bonferroni ) . ( c ) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : representative image in the green channel . Right : Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( black bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment . ( P = 0 . 002 , ANOVA - Bonferroni ) . ( d ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence ( white bar ) or absence ( black bars ) of serum and subjected to treatment with 3 - methyladenine ( 3 - MA ) , the proteasome inhibitor MG - 132 or H2O2 . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . Scale bars , 5 μm . ( e ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence of serum supplemented media and leave untreated ( white bar ) or subjected to the indicated treatments ( black bars ) . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . Scale bars , 5 μm .

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

f6(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002-0.03, ANOVA-Bonferroni).(c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni).(d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.(e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.


f7 ( b ) Mouse fibroblasts ( NIH3T3 ) stably expressing KFERQ - PA - mCherry1 were photoactivated and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : Representative images . Nuclei are staining with DAPI . Right : Quantification of the number of red fluorescent puncta per cell . Values are mean + s . e . of three different experiments with > 20 cells counted per experiment . * P = 0 . 001 , ANOVA - Bonferroni . ( c ) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ - PA - mCherry1 at the indicated times after photoactivation using FACS analysis . Cells were maintained in media supplemented ( white squares ) or not ( black circles ) with serum , and fluorescence by the MFI was calculated on events gated on living cells . Values are expressed as percentage of the intensity at time 0 and are mean + s . e . of 3 - 4 individual experiments . ( d ) Representative FACS experiment showing the effect of serum removal at 6 h ( top ) and 42 h ( bottom ) . PS , photoswitching . Scale bars , 5 μm .

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

f7(b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: Representative images. Nuclei are staining with DAPI. Right: Quantification of the number of red fluorescent puncta per cell. Values are mean+s.e. of three different experiments with >20 cells counted per experiment. *P=0.001, ANOVA-Bonferroni.(c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black circles) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean+s.e. of 3-4 individual experiments.(d) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.


Figure 1Mouse qPCR array analysis was performed with Colo357 and MiaPaca - 2 orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect changes of mouse stromal gene expression after treatment . A heat map was generated with ratios of gene expression ( 2G8 vs . control ) . ELISA - based Quansys mouse chemokine assay was performed with Capan - 1 ( Cap ) , MiaPaCa - 2 ( Mia ) , Colo357 ( Colo ) , and C5LM2 ( C5 ) orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect mouse chemokine changes after treatment . Change of mouse IL - 6 was shown . KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Tumors from were collected for mouse IL - 6 ELISA ( n = 6 / group ) . KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . The activation of SMAD2 ( P - Ser465 / 467 ) ( E and H - I ) , STAT3 ( P - Tyr705 ) ( F and J - K ) and expression of IL - 6R ( G ) was detected by immunohistochemistry ( n = 4 / group ) .

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

Figure 1Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control).ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown.KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group).KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).


Figure 2Single - cell RNA sequencing was performed to profile cell populations in normal mouse pancreas ( n = 2 ) , early KIC ( 40 - day - old , n = 2 ) and late KIC ( 60 - day - old , n = 3 ) pancreata . Samples from the same stage were pooled . Violin plots of expression of Il6 , Tgfbr1 and Tgfbr2 in distinct cell populations is shown . Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC ( mPLRB8 , mPLRB9 ) , KPC ( KPC - M01 , KPC - M09 ) and KPfC ( BMFA3 , CT1BA5 ) mouse cancer cells , mouse macrophages ( RAW 264 . 7 ) , and mouse fibroblasts ( NIH 3T3 and pancreatic stellate cells ) . RAW 264 . 7 cells were induced into M1 ( 30 ng / ml LPS for 18 hours ) or M2 ( 20 ng / ml IL - 4 for 18 hours ) macrophages . Tubulin was used as a loading control . Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA ( n = 223 ) using R .

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Figure 2Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown.Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.


Figure 3Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Cell lysates were harvested and blotted for P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( A ) . Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Conditioned media ( CM ) was collected mouse IL - 6 ELISA ( B - C ) . NIH 3T3 ( D ) , pancreatic stellate cells ( PSC ) ( E ) and human CAF cell lines CAF - PC1 and CAF - PC2 ( F ) were treated with TGFβ ( 30 ng / ml ) and / or IL - 1α ( 1 ng / ml ) for 24 hours . CM was collected for mouse or human IL - 6 ELISA . CAF - PC2 cells were treated with TGFβ ( 30 ng / ml ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . CM was collected and subjected to human IL - 6 ELISA . CAF - PC2 cells were treated with IL - 1α ( 1 ng / ml ) ( H ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . M was collected and subjected to human IL - 6 ELISA . KIC ( mPLRB9 ) , KPC ( KPC - M09 ) and KPfC ( BMFA3 ) cell lines were treated with normal DMEM ( CTRL ) , CM from NIH 3T3 ( CM ) , CM from TGFβ - treated NIH 3T3 ( TGFβ - CM ) , CM from TGFβ - treated NIH 3T3 + 2G8 ( TGFβ - CM + 2G8 ) normal DMEM + TGFβ ( TGFβ ) , CM from TGFβ - treated NIH 3T3 + IL - 6 neutralizing antibody ( TGFβ - CM + IL - 6 Ab ) . Cell lysates were harvested and blotted for P - STAT3 ( P - Tyr705 ) , STAT3 , P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( J ) . 3D culture : cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . IL - 6 neutralizing antibody ( 100 ng / ml ) . 3D culture : cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . IL - 6 neutralizing antibody ( 100 ng / ml ) .

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

Figure 3Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA.CAF-PC2 cells were treated with TGFβ (30 ng/ml) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA.CAF-PC2 cells were treated with IL-1α (1 ng/ml) (H) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. M was collected and subjected to human IL-6 ELISA.KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).


Figure 4Saline and 2G8 - treated MiaPaca - 2 and Colo375 xenografts were subjected to RNA - seq analysis for mouse gene expression changes . A heat map was generated with gene clustering ( A ) . Saline and 2G8 - treated MiaPaca - 2 and Colo375 xenografts were subjected to RNA - seq analysis for mouse gene expression changes . Molecules most significantly by 2G8 treatment . Y - axis values shown are log10 ( FPKM + 1 ) of the transcript levels ( D - F ) . An in vitro NK cell cytotoxicity assay was performed . Human NKL cells were used as effector cells , and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM ( control ) , conditioned medium collected from TGFβ ( 30 ng / ml ) - treated human pancreatic CAFs ( CAF - PC2 ; CAF TGFβ - CM ) , CAF TGFβ - CM + IL - 6 antibody ( 100 ng / ml ) or normal DMEM + TGFβ ( TGFβ ) . Living cells were labeled with CFSE , and dead cells were labeled with 7 - AAD . Samples were analyzed by flow cytometry . Cytotoxicity percentage was calculated using the formula ( 7 - AAD positive cells % ) / ( 7 - AAD positive cells % + CFSE positive cells % ) x 100 % . n = 4 / group , P values by t test are shown . Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice . After tumor establishment , mice were randomized to receive rat IgG Mac84 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 6 / group ) for 3 weeks . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H & E - stained liver tissue . Ten sections of the anterior lobe of the liver ( n = 6 per group ) were scored for lesions .

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

Figure 4Saline and 2G8-treated MiaPaca-2 and Colo375 xenografts were subjected to RNA-seq analysis for mouse gene expression changes. A heat map was generated with gene clustering (A).Saline and 2G8-treated MiaPaca-2 and Colo375 xenografts were subjected to RNA-seq analysis for mouse gene expression changes. Molecules most significantly by 2G8 treatment. Y-axis values shown are log10(FPKM+1) of the transcript levels (D-F).An in vitro NK cell cytotoxicity assay was performed. Human NKL cells were used as effector cells, and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM (control), conditioned medium collected from TGFβ (30 ng/ml)-treated human pancreatic CAFs (CAF-PC2; CAF TGFβ-CM), CAF TGFβ-CM + IL-6 antibody (100 ng/ml) or normal DMEM + TGFβ (TGFβ). Living cells were labeled with CFSE, and dead cells were labeled with 7-AAD. Samples were analyzed by flow cytometry. Cytotoxicity percentage was calculated using the formula (7-AAD positive cells %) / (7-AAD positive cells % + CFSE positive cells %) x 100%. n = 4/group, P values by t test are shown.Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H&E-stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions.


Figure 5KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Immune landscape changes were detected by immunohistochemistry for macrophage makers F4 / 80 ( total , A - C ) , iNOS ( M1 , D - F ) , ARG1 ( M2 , G - I ) , CD11b ( myeloid - derived suppressor cells , J - L ) and T cell markers CD3 ( M - O ) , CD8 ( P - R ) and FOXP3 ( regulatory T cells , S - U ) in KIC and KPC mice . Scale bars = 50 μm . n = 4 / group , P values by t test are shown . Spatial distribution analysis of CD8 - positive T cells after 2G8 treatment . Details are described in Appendix Fig S7 . Sample images of KPC tumors are shown ( V ) . Substantial cytotoxic T cell infiltration was observed after 2G8 treatment . Histological score was calculated using the following formula ( W - X ) : low IHC cells % + 2x ( medium IHC cells % ) + 3x ( high IHC cells % ) . Scale bars = 500 μm . n = 4 / group , P values vs . control by t test are shown .

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Figure 5KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Immune landscape changes were detected by immunohistochemistry for macrophage makers F4/80 (total, A-C), iNOS (M1, D-F), ARG1 (M2, G-I), CD11b (myeloid-derived suppressor cells, J-L) and T cell markers CD3 (M-O), CD8 (P-R) and FOXP3 (regulatory T cells, S-U) in KIC and KPC mice. Scale bars = 50 μm. n = 4/group, P values by t test are shown.Spatial distribution analysis of CD8-positive T cells after 2G8 treatment. Details are described in Appendix Fig S7. Sample images of KPC tumors are shown (V). Substantial cytotoxic T cell infiltration was observed after 2G8 treatment. Histological score was calculated using the following formula (W-X): low IHC cells % + 2x (medium IHC cells %) + 3x (high IHC cells %). Scale bars = 500 μm. n = 4/group, P values vs. control by t test are shown.


Figure 6KIC mice were treated in a survival study with rat IgG Mac48 ( control ) or 2G8 . Median overall survival of rat IgG treatment was 72 days , while median overall survival of 2G8 treatment was 63 days ( B ) . Tissues from ( A ) were stained for Ki67 . Scale bars = 50 μm . Overall survival of PDA patients from TCGA with and without TGFBR2 mutation . Loss - of - function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs ( mut1 and mut2 ) in the mouse PDA cell line BMFA3 derived from KPfC model . Control cell line ( Tgfbr2wt ) and two Tgfbr2 - mutant cell lines ( Tgfbr2mut1 and Tgfbr2mut2 ) were treated with TGFβ ( 30 ng / ml ) for 5 hours . Cell lysates were harvested and western blotting for P - SMAD2 , SMAD2 , SMAD4 , P - ERK1 / 2 , ERK1 / 2 , P - P38 , P38 , and actin was performed . Immunofluorescence was performed to study the expression and localization of E - cadherin and SMAD4 after TGFβ treatment ( 30 ng / ml for 24 hours ) in control cell line and two Tgfbr2 - mutant cell lines . Scale bars = 50 μm ( fluorescent ) , scale bars = 100 μm ( brightfield ) .

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Figure 6KIC mice were treated in a survival study with rat IgG Mac48 (control) or 2G8. Median overall survival of rat IgG treatment was 72 days, while median overall survival of 2G8 treatment was 63 days (B).Tissues from (A) were stained for Ki67. Scale bars = 50 μm.Overall survival of PDA patients from TCGA with and without TGFBR2 mutation.Loss-of-function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs (mut1 and mut2) in the mouse PDA cell line BMFA3 derived from KPfC model. Control cell line (Tgfbr2wt) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) were treated with TGFβ (30 ng/ml) for 5 hours. Cell lysates were harvested and western blotting for P-SMAD2, SMAD2, SMAD4, P-ERK1/2, ERK1/2, P-P38, P38, and actin was performed.Immunofluorescence was performed to study the expression and localization of E-cadherin and SMAD4 after TGFβ treatment (30 ng/ml for 24 hours) in control cell line and two Tgfbr2-mutant cell lines. Scale bars = 50 μm (fluorescent), scale bars = 100 μm (brightfield).


Figure 7Control cell line ( Tgfbr2wt ) ( A ) and two Tgfbr2 - mutant cell lines ( Tgfbr2mut1 and Tgfbr2mut2 ) ( B - C ) were treated with TGFβ at different time points , and cell growth ( MTT ) assays were performed . P value by t test is shown . NS , not significant . Subcutaneous tumors established from control cell line ( D ) and two Tgfbr2 - mutant cell lines ( E - F ) in C57Bl / 6 mice received rat IgG Mac48 ( control ) or 2G8 ( each 30 mg / kg 2x / week , n = 4 / group ) . Therapy started at day 12 post tumor cell injection ; mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are shown . Control cell line ( Tgfbr2wt ; G & I ) and the Tgfbr2 - mutant cell line ( Tgfbr2mut1 ; H & J ) were orthotopically implanted in C57Bl / 6 mice . Mice received rat IgG Mac48 or 2G8 , n = 9 - 10 / group for survival study ( each 30 mg / kg 2x / week ) . Control cell line ( Tgfbr2wt ; G & I ) and the Tgfbr2 - mutant cell line ( Tgfbr2mut1 ; H & J ) were orthotopically implanted in C57Bl / 6 mice . Mice were sacrificed when they became moribund . Tumors were harvested and weighed ( K - L ) . Immunohistochemistry with tumor samples from ( G - H ) for Ki67 ( M - O ) , P - STAT3 ( P - R ) , ZAP70 , and CD3 ( S - X ) was performed . ZAP70 is a common marker for NK cells and T cells and was stained with a brown chromogen , and CD3 is a specific T - cell marker and was stained with a blue chromogen . Brown signal was converted to red signal and blue signal was converted to green by ImageJ ( V ) ; therefore NK cells are highlighted as red ( ZAP70 + CD3 - ) and T cells are highlighted as yellow ( ZAP70 + CD3 + ) . Scale bars outside the magnification boxes = 50 μm , scale bars inside the magnification boxes = 10 μm . n = 4 / group , P values vs . control by t test are shown .

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

Figure 7Control cell line (Tgfbr2wt) (A) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) (B-C) were treated with TGFβ at different time points, and cell growth (MTT) assays were performed. P value by t test is shown. NS, not significant.Subcutaneous tumors established from control cell line (D) and two Tgfbr2-mutant cell lines (E-F) in C57Bl/6 mice received rat IgG Mac48 (control) or 2G8 (each 30 mg/kg 2x/week, n = 4/group). Therapy started at day 12 post tumor cell injection; mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are shown.Control cell line (Tgfbr2wt; G&I) and the Tgfbr2-mutant cell line (Tgfbr2mut1; H&J) were orthotopically implanted in C57Bl/6 mice. Mice received rat IgG Mac48 or 2G8, n = 9-10/group for survival study (each 30 mg/kg 2x/week).Control cell line (Tgfbr2wt; G&I) and the Tgfbr2-mutant cell line (Tgfbr2mut1; H&J) were orthotopically implanted in C57Bl/6 mice. Mice were sacrificed when they became moribund. Tumors were harvested and weighed (K-L).Immunohistochemistry with tumor samples from (G-H) for Ki67 (M-O), P-STAT3 (P-R), ZAP70, and CD3 (S-X) was performed. ZAP70 is a common marker for NK cells and T cells and was stained with a brown chromogen, and CD3 is a specific T-cell marker and was stained with a blue chromogen. Brown signal was converted to red signal and blue signal was converted to green by ImageJ (V); therefore NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 4/group, P values vs. control by t test are shown.


Figure 8Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2 - mutant cell line Tgfbr2mut1 . Cell lysates were harvested and western blotting for IL6RA and tubulin . 3D culture : control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . 3D culture : control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . Control IgG , 2G8 and IL - 6 neutralizing antibody ( each 100 ng / ml , n = 5 / group ) . Scale bars = 50 μm . P values by t test are shown ( C ) . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Immunohistochemistry with tumor samples from ( D ) for Ki67 ( G and K ) , P - STAT3 ( H and L ) , ZAP70 , and CD3 ( I - J and M - N ) was performed . NK cells are highlighted as red ( ZAP70 + CD3 - ) and T cells are highlighted as yellow ( ZAP70 + CD3 + ) by ImageJ ( N ) . Scale bars outside the magnification boxes = 50 μm , scale bars inside the magnification boxes = 10 μm . n = 5 / group , P values vs . control by t test are shown .

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

Figure 8Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2-mutant cell line Tgfbr2mut1. Cell lysates were harvested and western blotting for IL6RA and tubulin.3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture).3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). Control IgG, 2G8 and IL-6 neutralizing antibody (each 100 ng/ml, n = 5/group). Scale bars = 50 μm. P values by t test are shown (C).Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Immunohistochemistry with tumor samples from (D) for Ki67 (G and K), P-STAT3 (H and L), ZAP70, and CD3 (I-J and M-N) was performed. NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+) by ImageJ (N). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 5/group, P values vs. control by t test are shown.


Figure 1C , Demonstration of single - molecule tracking in a single cell . Cell outline is shown as an ellipse . Left , Imaged FliM - YPet fluorescence . White dots indicate the estimated motor locations . White circles illustrate a 75 nm radius around these locations . Right , Contour map of the normalized sum of probabilities of the localization events of CheY ( I95V ) - Atto647 ( from blue to red is low to high ; black is zero probability ) . White circles show the motors ' locations from the left panel . I , CheY interacts with FliM ∆ N and this interaction is sensitive to chemotactic stimuli . Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus ( 0 . 1 mM serine ; in the case of FliMwt , a single measurement was performed with serine ; another measurement of FliMwt with 1mM aspartate produced similar results ) . FRET ratio is the ratio of mCherry to YPet fluorescence . CheY - mCherry and mCherry concentrations were ~ 170 µM in the case of FliM ∆ N and ~ 15 µM in the case of FliMwt Strains used : EW677 ( FliMwt , red ) , EW659 ( negative control of FliM ∆ N without CheY , purple ) , EW637 ( FliM ∆ N , blue ) , and EW636 ( FliM ∆ N ∆ cheA , yellow ) .

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Figure 1C, Demonstration of single-molecule tracking in a single cell. Cell outline is shown as an ellipse. Left, Imaged FliM-YPet fluorescence. White dots indicate the estimated motor locations. White circles illustrate a 75 nm radius around these locations. Right, Contour map of the normalized sum of probabilities of the localization events of CheY(I95V)-Atto647 (from blue to red is low to high; black is zero probability). White circles show the motors' locations from the left panel.I, CheY interacts with FliM∆N and this interaction is sensitive to chemotactic stimuli. Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus (0.1 mM serine; in the case of FliMwt, a single measurement was performed with serine; another measurement of FliMwt with 1mM aspartate produced similar results). FRET ratio is the ratio of mCherry to YPet fluorescence. CheY-mCherry and mCherry concentrations were ~170 µM in the case of FliM∆N and ~15 µM in the case of FliMwt Strains used: EW677 (FliMwt, red), EW659 (negative control of FliM∆N without CheY, purple), EW637 (FliM∆N, blue), and EW636 (FliM∆N ∆cheA, yellow).


Figure 2B , Demonstration of rotation rate measured from a single cell ; negative and positive values ( below and above the blue bar ) are counterclockwise and clockwise , respectively . The switch from negative to positive rotation rate at 3 s is shown in the montage to the right . C , The response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) , to positive stimuli . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrow marks the estimated time at which the stimulant arrived to , or left ( as indicated ) , the flow chamber . N is the number of cells . α - Methyl - DL - aspartate ( MeAsp ) and leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM . The response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrow marks the estimated time at which the stimulant arrived to , or left ( as indicated ) , the flow chamber . N is the number of cells . α - Methyl - DL - aspartate ( MeAsp ) and leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM . D , negative stimuli . E , Response of tethered fliM ∆ N ΔcheA cells ( strain EW634 ) to acetate ( 50 mM , pH 7 . 0 ) at different CheY concentrations . Lines and shaded regions are mean ± SEM . F , Response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) to acetate . G , Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation . The points are the mean clockwise rotation at time segments 0 - 20 s and 160 - 180 s . The variants shown are fliM ∆ N ∆ cheZ cells expressing CheY in the absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( i . e . , CheY ~ P and CheY ~ P ~ Ac ; blue and green curves ; strain EW635 ) , fliM ∆ N ∆ cheA cells expressing CheY , in the absence of presence of acetate ( CheY and CheY ~ Ac ; purple and red , respectively ; EW634 ) , and fliM ∆ N ∆ cheA cells expressing CheY ( D13K ) ( burgundy ; EW737 ) . Each data point is the average of all experiments at a given CheY concentration , weighted by the sample number of each experiment . The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used . H , FliMN further activates CheY variants to produce clockwise rotation . The variants shown are fliM ∆ N ∆ cheZ cells expressing FliMN - CheY ( i . e . , FliMN - CheY ~ P ; blue curve ; strain EW697 ) , fliM ∆ N ∆ cheA cells expressing FliMN - CheY , in the absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( FliMN - CheY and FliMN - CheY ~ Ac ; purple and red , respectively ; EW696 ) , and fliM ∆ N ∆ cheA cells expressing FliMN - CheY ( D13K ) ( burgundy ; EW739 ) . I , Response of fliM ∆ N ∆ cheA cells expressing FliMN - CheY ( strain EW696 ) to acetate and benzoate ( 10 mM each ; pH 7 . 0 ) . FliMN - CheY expression was induced with 800 μM IPTG . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The black arrow indicates the estimated time point at which the stimulus entered the flow chamber .

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

Figure 2B, Demonstration of rotation rate measured from a single cell; negative and positive values (below and above the blue bar) are counterclockwise and clockwise, respectively. The switch from negative to positive rotation rate at 3 s is shown in the montage to the right.C, The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM.The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. D, negative stimuli.E, Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM.F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate.G, Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation at time segments 0-20 s and 160-180 s. The variants shown are fliM∆N ∆cheZ cells expressing CheY in the absence of presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ∆cheA cells expressing CheY, in the absence of presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ∆cheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used.H, FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ∆cheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ∆cheA cells expressing FliMN-CheY, in the absence of presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ∆cheA cells expressing FliMN-CheY(D13K) (burgundy; EW739). I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber.


Figure 3 , Dependence of clockwise rotation on the level of FliMN - CheY ~ Ac . The red and purple curves are two experimental days in which the clockwise production by fliM ∆ N ∆ cheA cells expressing FliMN - CheY was measured as a function of FliMN - CheY concentration ( strain EW696 ; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used . ach data point is a mean ± SEM of the data or the second and third experimental days of this strain ( in the presence of acetate ) . B , Response of the strain to addition and removal of varying acetate concentrations ( pH 7 . 0 ) . FliMN - CheY concentration was estimated to be ~ 300 μM . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrows indicate the estimated time points at which acetate entered or left the flow chamber . Black lines mark the linear part of the biphasic response to acetate removal . N is the number of cells . E , Average clockwise interval length , calculated as the inverse of the fast ( blue ) and slow ( red ) rate

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

Figure 3, Dependence of clockwise rotation on the level of FliMN-CheY~Ac. The red and purple curves are two experimental days in which the clockwise production by fliM∆N ∆cheA cells expressing FliMN-CheY was measured as a function of FliMN-CheY concentration (strain EW696; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used. ach data point is a mean ± SEM of the data or the second and third experimental days of this strain (in the presence of acetate).B, Response of the strain to addition and removal of varying acetate concentrations (pH 7.0). FliMN-CheY concentration was estimated to be ~300 μM. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrows indicate the estimated time points at which acetate entered or left the flow chamber. Black lines mark the linear part of the biphasic response to acetate removal. N is the number of cells.E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants