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 .

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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 ) .

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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 ) .

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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).


Saline 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 ) .

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Saline 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 ) .

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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 .

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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 .

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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.


KIC 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 .

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KIC 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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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.


KIC 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 ) .

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KIC 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 .

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Tissues from (A) were stained for Ki67. Scale bars = 50 μm.


Overall survival of PDA patients from TCGA with and without TGFBR2 mutation .

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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 .

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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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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).


Control 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 .

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Control 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 .

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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 ) .

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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 ) .

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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 .

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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.


Control 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 .

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Control 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 ) .

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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 ) .

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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 .

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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 .

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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.


C , 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 .

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C, 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 ) .

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

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).


B , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

C, 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 .

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

The 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 .

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

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 .

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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 .

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

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 ) .

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

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

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.


, 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 ) .

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

, 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 .

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

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

E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants


A , Effects of FliMN - CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM ∆ N FliN ( A93D ) motors . Acetate concentration was 10 mM each ( pH 7 . 0 ) . FliMN - CheY and FliMN - CheY ~ P were produced by using ∆ cheA and ∆ cheZ backgrounds ( strains EW713 and EW714 ) , respectively . Each data point is the average of all measurements at a given FliMN - CheY concentration , weighted by the sample number of each experiment . The concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used . Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve . B , Distribution of clockwise interval lengths at different average clockwise levels in ∆ cheA cells containing FliM ∆ N FliN ( A93D ) motors and expressing FliMN - CheY ( strain EW713 ) , in the presence of acetate ( 10 mM , pH 7 . 0 ) . C , Average clockwise interval length , calculated as the inverse of the rate constants from mono - phasic fits of the distributions in ( B ) .

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

A, Effects of FliMN-CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM∆N FliN(A93D) motors. Acetate concentration was 10 mM each (pH 7.0). FliMN-CheY and FliMN-CheY~P were produced by using ∆cheA and ∆cheZ backgrounds (strains EW713 and EW714), respectively. Each data point is the average of all measurements at a given FliMN-CheY concentration, weighted by the sample number of each experiment. The concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used. Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve. B, Distribution of clockwise interval lengths at different average clockwise levels in ∆cheA cells containing FliM∆N FliN(A93D) motors and expressing FliMN-CheY (strain EW713), in the presence of acetate (10 mM, pH 7.0). C, Average clockwise interval length, calculated as the inverse of the rate constants from mono-phasic fits of the distributions in (B).


D , Response of tethered ∆ cheZ cells having FliM ∆ N FliN ( A93D ) motors to acetate addition and removal ( red ; strain EW714 ; FliMN - CheY concentration estimated at ~ 300 μM ) . Similar results were obtained with a ∆ cheA strain ( EW713 ) . Tethered cells having FliM ∆ N FliNwt motors and containing FliMN - CheY ( strain EW696 ) are shown Acetate concentration was 10 mM ( pH 7 . 0 ) . The data shown are the mean ± SEM . N is the number of cells . Black lines indicate the clockwise - decay rates of FliNwt motors ' slow phase and of FliN ( A93D ) motors following acetate removal .

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

D, Response of tethered ∆cheZ cells having FliM∆N FliN(A93D) motors to acetate addition and removal (red; strain EW714; FliMN-CheY concentration estimated at ~300 μM). Similar results were obtained with a ∆cheA strain (EW713). Tethered cells having FliM∆N FliNwt motors and containing FliMN-CheY (strain EW696) are shown Acetate concentration was 10 mM (pH 7.0). The data shown are the mean ± SEM. N is the number of cells. Black lines indicate the clockwise-decay rates of FliNwt motors' slow phase and of FliN(A93D) motors following acetate removal.


A , CheY crosslinks with FliM ∆ N - YPet in vivo . The cytoplasm of fliM ∆ N - YPet ΔcheZ cells overexpressing CheY ( strain EW694 ) or FliMN - CheY ( strain EW697 ) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS - PAGE . 1 , 2 , 3 stand for three different experiments that underwent this procedure . The plus sign before ~ 14 and ~ 15 stands for + 55 kDa of FliM ∆ N - YPet . Note that gel running suffered from parabolic distortion in band positions . The annotations relate to the lowest positions of the bands . The gel was imaged for FliM ∆ N - YPet fluorescence . To see the crosslinking products , the intense fluorescence of monomeric FliM ∆ N - YPet is shown at saturation .

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

A, CheY crosslinks with FliM∆N-YPet in vivo. The cytoplasm of fliM∆N-YPet ΔcheZ cells overexpressing CheY (strain EW694) or FliMN-CheY (strain EW697) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS-PAGE. 1, 2, 3 stand for three different experiments that underwent this procedure. The plus sign before ~14 and ~15 stands for +55 kDa of FliM∆N-YPet. Note that gel running suffered from parabolic distortion in band positions. The annotations relate to the lowest positions of the bands. The gel was imaged for FliM∆N-YPet fluorescence. To see the crosslinking products, the intense fluorescence of monomeric FliM∆N-YPet is shown at saturation.


C , Clockwise rotation of tethered fliM ( R94S ) ∆ N ∆ cheZ cells and fliM ( R94L ) ∆ N ∆ cheZ cells expressing FliMN - CheY ( strains EW731 and EW733 , respectively ) in the absence or presence of acetate ( 10 mM , pH 7 . 0 ) . FliMN - CheY concentration was estimated to be ~ 4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used . No clockwise rotation was observed when benzoate substituted for acetate . Each data point is the mean of a separate experiment . Black line is the mean of all experiments .

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

C, Clockwise rotation of tethered fliM(R94S)∆N ∆cheZ cells and fliM(R94L)∆N ∆cheZ cells expressing FliMN-CheY (strains EW731 and EW733, respectively) in the absence or presence of acetate (10 mM, pH 7.0). FliMN-CheY concentration was estimated to be ~4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used. No clockwise rotation was observed when benzoate substituted for acetate. Each data point is the mean of a separate experiment. Black line is the mean of all experiments.


D , Clockwise rotation of tethered fliM ( E214W ) ∆ N ΔcheZ cells ( strain EW718 ) at various concentrations of FliMN - CheY . The concentrations shown are estimates based on the calibration curve , for which a similar CheY expression system was used . Each data point is the average of all measurements at similar FliMN - CheY concentrations , weighted by the sample number of each experiment .

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

D, Clockwise rotation of tethered fliM(E214W)∆N ΔcheZ cells (strain EW718) at various concentrations of FliMN-CheY. The concentrations shown are estimates based on the calibration curve , for which a similar CheY expression system was used. Each data point is the average of all measurements at similar FliMN-CheY concentrations, weighted by the sample number of each experiment.


( D ) Absolute numbers of lymphocytes subpopulations in the bone marrow , thymus , and spleen . B cell development in the bone marrow was assessed by FACS analysis for surface expression of : B220 + CD43 + CD19 − IgM − IgD − ( pre - pro B ) ; B220 + CD43 + CD19 + IgM − IgD − ( pro B ) ; B220 + CD43 − CD19 + IgM − IgD − ( pre B ) ; CD19 + IgM + IgD − ( immature ) ; CD19 + IgM + IgD + ( mature ) . T cell development in the thymus was assessed by FACS analysis for surface expression of : CD4 − CD8 − ( double negative , DN ) ; CD4 + CD8 + ( double positive , DP ) ; CD4 + CD8 − ( CD4 single positive , SP ) ; CD4 − CD8 + ( CD8 SP ) . Splenic B cell populations were assessed by FACS analysis for surface expression of : B220 + CD21 + CD23 + ( follicular B cells , FOB ) ; B220 + CD21 + CD23low ( marginal zone B cells , MZB ) . Each symbol represents an individual mouse . Horizontal bars indicate mean values . Two - tailed Mann - Whitney test , ns = not significant , * P < 0 . 05 , and * * P < 0 . 01 .

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

(D) Absolute numbers of lymphocytes subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220+CD43+CD19−IgM−IgD− (pre-pro B); B220+CD43+CD19+IgM−IgD− (pro B); B220+CD43−CD19+IgM−IgD− (pre B); CD19+IgM+IgD− (immature); CD19+IgM+IgD+ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4−CD8− (double negative, DN); CD4+CD8+ (double positive, DP); CD4+CD8− (CD4 single positive, SP); CD4−CD8+ (CD8 SP). Splenic B cell populations were assessed by FACS analysis for surface expression of: B220+CD21+CD23+ (follicular B cells, FOB); B220+CD21+CD23low (marginal zone B cells, MZB). Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two-tailed Mann-Whitney test, ns = not significant, *P < 0.05, and **P < 0.01.


( B ) Maximum Ca2 + flux ( peak height ) at each anti - IgM concentration for FOB and MZB cells . Paired t test , ns = not significant , * P < 0 . 05 , and * * * P < 0 . 001 .

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(B) Maximum Ca2+ flux (peak height) at each anti-IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001.


( B ) Co - immunoprecipitation of HA - tagged Pacs1 by FLAG - Wdr37 and HA - Wdr37 by FLAG - Pacs1 in co - transfected 293T cells .

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(B) Co-immunoprecipitation of HA-tagged Pacs1 by FLAG-Wdr37 and HA-Wdr37 by FLAG-Pacs1 in co-transfected 293T cells.


( E ) Wdr37 + / + and Wdr37 − / − splenocytes were labeled with Indo - 1 , stained for cell surface markers to identify FOB cells , and stimulated with the indicated amounts of anti - IgM ( black arrowhead ) . Normalized traces from three ( 2 . 5 mcg / ml anti - IgM ) or four independent experiments ( 10 mcg / ml and 5 mcg / ml anti - IgM ) are shown ( Wdr37 + / + gray , Wdr37 − / − pink ) . Mean Ca2 + flux for each genotype is overlaid in bold ( Wdr37 + / + black , Wdr37 − / − red ) .

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(E) Wdr37+/+ and Wdr37−/− splenocytes were labeled with Indo-1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti-IgM (black arrowhead). Normalized traces from three (2.5 mcg/ml anti-IgM) or four independent experiments (10 mcg/ml and 5 mcg/ml anti-IgM) are shown (Wdr37+/+ gray, Wdr37−/− pink). Mean Ca2+ flux for each genotype is overlaid in bold (Wdr37+/+ black, Wdr37−/− red).


( A ) Immunoblot of ER mass , ER stress , and autophagy markers in Pacs1 + / + and Pacs1 - / - splenic B cells that were left unstimulated or stimulated overnight with 5 mcg / ml IgM .

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(A) Immunoblot of ER mass, ER stress, and autophagy markers in Pacs1+/+ and Pacs1-/- splenic B cells that were left unstimulated or stimulated overnight with 5 mcg/ml IgM.


( B ) Real - time quantitative PCR of IP3R and SERCA2 transcripts in B cells . Data are presented as mean ± SD from three independent Pacs1 + / + and Pacs1 - / - pairs of mice .

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(B) Real-time quantitative PCR of IP3R and SERCA2 transcripts in B cells. Data are presented as mean ± SD from three independent Pacs1+/+ and Pacs1-/- pairs of mice.


( D ) Peak cytosolic Ca2 + concentration based on aequorin measurements as performed in ( C ) . Symbols represent individual wells containing ~ 5 x 105 transfected 3T3 or Pacs1 - / - cells from one experiment . Horizontal bars indicate mean . Two - tailed unpaired t test , * * P < 0 . 01 .

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(D) Peak cytosolic Ca2+ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~5 x 105 transfected 3T3 or Pacs1-/- cells from one experiment. Horizontal bars indicate mean. Two-tailed unpaired t test, **P < 0.01.


( E ) Pacs1 + / + and Pacs1 - / - NIH - 3T3 cells ( C1 and C2 from ( A ) ) were transfected with ER - GCamP6 - 210 . ER Ca2 + was measured before and after treatment with 10 µM ATP . Kinetic traces show the mean 488 / 405 nm excitation ratio of each cell line with error bars indicating SEM . N = 30 cells ( 3T3 ) , 16 cells ( C1 ) , 25 cells ( C2 ) . Data are representative of 2 independent experiments .

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(E) Pacs1+/+ and Pacs1-/- NIH-3T3 cells (C1 and C2 from (A)) were transfected with ER-GCamP6-210. ER Ca2+ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N=30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments.


( B , C ) Pacs1 + / + and Pacs1 − / − mice were immunized with alum - ova and one week later with NP - Ficoll . Anti - ova IgG and anti - NP IgM titers were measured at 14 days and 7 days after immunization , respectively . Each symbol represents an individual mouse .

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(B, C) Pacs1+/+ and Pacs1−/− mice were immunized with alum-ova and one week later with NP-Ficoll. Anti-ova IgG and anti-NP IgM titers were measured at 14 days and 7 days after immunization, respectively. Each symbol represents an individual mouse.


( H , I ) Pacs1 + / + and Pacs1 - / - mice were injected with EdU and the fraction of EdU + FOB and MZB cells were measured in the spleen at 1 , 4 , and 7 days post - injection . Data are from one experiment .

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(H, I) Pacs1+/+ and Pacs1-/- mice were injected with EdU and the fraction of EdU+ FOB and MZB cells were measured in the spleen at 1, 4, and 7 days post-injection. Data are from one experiment.


( D , E ) The fraction of proliferating ( D ) and recovered ( E ) donor cells from the experiment in ( C ) . Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments . Two - tailed unpaired t test , * * P < 0 . 01 , * * * P < 0 . 001 .

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(D, E) The fraction of proliferating (D) and recovered (E) donor cells from the experiment in (C). Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments. Two-tailed unpaired t test, **P < 0.01, ***P < 0.001.


( I ) Enumeration of CD3 + B220 + cells in the peripheral blood and lymph nodes of Faslpr / lpr dependent on Pacs1 expression . Horizontal bars indicate median value . Two - tailed Mann - Whitney test , * * P < 0 . 01 .

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(I) Enumeration of CD3+B220+ cells in the peripheral blood and lymph nodes of Faslpr/lpr dependent on Pacs1 expression. Horizontal bars indicate median value. Two-tailed Mann-Whitney test, **P < 0.01.


( A ) Quantification of free cholesterol levels by lipidomics analysis of total homogenates ( TH ) , ER or MAM fractions isolated from WT and PS - DKO cells . Lipid units are represented as molar mass over total moles of lipids analyzed ( mol % ) . Graphs represent fold change over controls . Unpaired t - test ( n = 6 for TH , n = 3 for MAM or ER ; * p < 0 . 05 ) .

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(A) Quantification of free cholesterol levels by lipidomics analysis of total homogenates (TH), ER or MAM fractions isolated from WT and PS-DKO cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. Unpaired t-test (n= 6 for TH, n=3 for MAM or ER; * p<0.05).


( B ) Quantification of free cholesterol levels in total homogenates of APP - DKO cells transiently expressing C99 , C83 or AICD peptides , or treated with 5 µM Aβ42 oligomers for 16h . Cholesterol levels of WT cells are shown as a control . Lipid units are represented as molar mass over total moles of lipids analyzed ( mol % ) . Graphs represent fold change over controls . One - way ANOVA ( n = 4 ; * p < 0 . 05 ) .

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(B) Quantification of free cholesterol levels in total homogenates of APP-DKO cells transiently expressing C99, C83 or AICD peptides, or treated with 5 µM Aβ42 oligomers for 16h. Cholesterol levels of WT cells are shown as a control. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. One-way ANOVA (n = 4; * p<0.05).


( C ) Quantification of HMGCR enzymatic activity in PS - DKO and DAPT - treated WT cells . unpaired t - test ( n = 3 ; * p < 0 . 05 ) .

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(C) Quantification of HMGCR enzymatic activity in PS-DKO and DAPT-treated WT cells. unpaired t-test (n=3; * p< 0.05).


( D ) Quantification of cholesterol uptake by incubating WT and PS - DKO cells , treated or untreated with BACE inhibitor IV ( BI , 100 nM ) , with 2 . 5 µCi / mL 3H - cholesterol for the indicated times . Graph represents cholesterol radioactivity levels in total cell homogenates ( TH ) . Two - way repeated measures ANOVA ( Time , Group ) ( n = 3 independent experiments ; * p < 0 . 05 ) .

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(D) Quantification of cholesterol uptake by incubating WT and PS-DKO cells, treated or untreated with BACE inhibitor IV (BI, 100 nM), with 2.5 µCi/mL 3H-cholesterol for the indicated times. Graph represents cholesterol radioactivity levels in total cell homogenates (TH). Two-way repeated measures ANOVA (Time, Group)(n = 3 independent experiments; * p< 0.05).


( E - F ) Quantification of 3H - cholesterol delivery to MAM ( E ) or bulk ER ( F ) after the indicated times . Control and BI - treated or untreated PS - DKO cells incubated with 2 . 5 µCi / mL 3H - cholesterol for the indicated times , before subcellular fractionation ( Western blot of isolated MAM fractions shown in Fig . EV1F ) . Cholesterol delivery to MAM calculated by quantification of radioactivity levels in the isolated fractions . Two - way repeated measures ANOVA ( Time , Group ) ( n = 3 independent experiments ; * p < 0 . 05 )

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(E-F) Quantification of 3H-cholesterol delivery to MAM (E) or bulk ER (F) after the indicated times. Control and BI-treated or untreated PS-DKO cells incubated with 2.5 µCi/mL 3H-cholesterol for the indicated times, before subcellular fractionation (Western blot of isolated MAM fractions shown in Fig. EV1F). Cholesterol delivery to MAM calculated by quantification of radioactivity levels in the isolated fractions. Two-way repeated measures ANOVA (Time, Group)( n=3 independent experiments; * p< 0.05)


( G ) Measurement of cholesterol uptake in the indicated cells by internalization of a fluorescent cholesterol analog [ NBD - cholesterol ( 2 µM ; upper panel , nuclei in blue ) ] . Endogenous free cholesterol was stained with filipin ( lower panel ) . Graphs on the right represent fluorescence intensity ( using ImageJ ) and the percentage of cells with filipin punctae . Scale bar = 20 µm . One - way ANOVA ( 30 - 50 cells / condition from at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(G) Measurement of cholesterol uptake in the indicated cells by internalization of a fluorescent cholesterol analog [NBD-cholesterol (2 µM; upper panel, nuclei in blue)]. Endogenous free cholesterol was stained with filipin (lower panel). Graphs on the right represent fluorescence intensity (using ImageJ) and the percentage of cells with filipin punctae. Scale bar = 20 µm. One-way ANOVA (30-50 cells/condition from at least 3 independent experiments; * p<0.05, ** p<0.01, *** p<0.001).


Representative confocal images of WT and PS - DKO cells treated with the indicated SMase inhibitors ( 10 µM desipramine or 5 µM GW4869 ) or DMSO ( VEH ) for 12 - 16h . ( A ) Endogenous levels of lipid droplets ( LDs ) were visualized by incubation with LipidTox . Graph shows the Lipidtox fluorescence intensity / cell .

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Representative confocal images of WT and PS-DKO cells treated with the indicated SMase inhibitors (10 µM desipramine or 5 µM GW4869) or DMSO (VEH) for 12-16h. (A) Endogenous levels of lipid droplets (LDs) were visualized by incubation with LipidTox. Graph shows the Lipidtox fluorescence intensity/cell.


Endogenous levels of free cholesterol revealed by filipin staining of ( B ) WT cells under the indicated treatments . Graphs show the filipin fluorescence intensity as a percentage over VEH .

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Endogenous levels of free cholesterol revealed by filipin staining of (B) WT cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.


Endogenous levels of free cholesterol revealed by filipin staining of ( C ) PS - DKO cells under the indicated treatments . Graphs show the filipin fluorescence intensity as a percentage over VEH .

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Endogenous levels of free cholesterol revealed by filipin staining of (C) PS-DKO cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.


( A ) Crude membrane fractions from APP - DKO cells expressing C99WT or C99MUT were treated with 0 . 2 % Triton - X - 100 , loaded onto continuous density sucrose gradients and centrifuged for 16h . Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [ two parallel gels ( bold vertical line ) ] .

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(A) Crude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton-X-100, loaded onto continuous density sucrose gradients and centrifuged for 16h. Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)].


( B - G ) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient , as measured by densitometry analysis of western blot signals ( ImageJ ) . Note the reduced degree of co - migration of C99MUT with MAM markers ( Acsl4 ) , compared to C99WT .

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(B-G) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient, as measured by densitometry analysis of western blot signals (ImageJ). Note the reduced degree of co-migration of C99MUT with MAM markers (Acsl4), compared to C99WT.


( A ) Representative confocal images of APP - DKO cells expressing GFP - tagged C99WT or C99MUT ( in green ) and fluorescent markers of mitochondria ( MitoDsRed , in red ) and ER ( Sec61β - BFP , in blue ) , and treated with DAPT to prevent C99 cleavage . Note the different distribution of C99WT or C99MUT forms . Scale bar = 10 µm . Insets show 5X amplifications of individual ( C99 in green , and mitochondria in red ) and merged images . Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize . Upon thresholding each channel , a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask , so the positive pixels found in both channels are shown in black .

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(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black.


( B ) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ . Note the significantly decreased colocalization between C99MUT and mitochondria , when compared to that of C99WT . Unpaired t - test . ( 20 - 30 cells / condition from 3 independent experiments and 4 - 5 images / experiment ; * p < 0 . 05 ; n . s . , not significant ) .

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(B) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ. Note the significantly decreased colocalization between C99MUT and mitochondria, when compared to that of C99WT. Unpaired t-test. (20-30 cells/condition from 3 independent experiments and 4-5 images/experiment; * p<0.05; n.s., not significant).


( C ) Representative confocal images of APP - DKO cells expressing GFP - tagged C99WT or C99MUT and ER and mitochondria markers as in ( A ) ( upper panel ) . Individual zoomed single channels ( middle panel ) as well as areas of ER - mitochondria colocalization ( bottom panel ) are shown for each condition . Scale bar = 10 µm .

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(C) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT and ER and mitochondria markers as in (A) (upper panel). Individual zoomed single channels (middle panel) as well as areas of ER-mitochondria colocalization (bottom panel) are shown for each condition. Scale bar = 10 µm.


( D ) Quantification of ER - mitochondria colocalization per cell analyzed . Upon thresholding each channel , a mask for ER was generated and the mitochondria channel was superimposed on the ER mask , so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask . The number of cells analyzed is indicated in white text within each column . Levels of ER - mitochondria colocalization in APP - WT cells are shown for reference as a dashed line . EV , empty vector . One - way ANOVA ( n = 3 independent experiments and 5 - 8 images / experiment ; * p < 0 . 05 ) .

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(D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).


( B ) Representative immunoblot to reveal C99 levels in total homogenate ( TH ) and MAM fractions of APP - DKO cells expressing C99WT or C99MUT before ( input ) and after cholesterol pull - down . Acsl4 was used as a MAM marker . Streptavidin - HRP was used to detect total biotinylation ( biotin conjugated to PhotoClick cholesterol ) . ( C ) Quantification of pulled - down C99 levels versus input from the experiment in B . Two - way repeated measures ANOVA ( Fraction , mutation ) ( n = 5 ; * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol). (C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01).


( D ) Immunoblot showing the levels of pulled - down PhotoClick Cholesterol ( streptavidin - HRP ) in ER and MAM fractions . Note how the levels of pulled - down C99 in the ER are negligible when compared to those from MAM . Isolated MAM and ER fractions were assessed by western blot ( shown in AppendixFig S1E ) .

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(D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).


( E ) Quantification of free cholesterol levels ( FC ) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells . Lipid units are represented as molar mass over total moles of lipids analyzed ( mol % ) . Graphs represent percentage over controls . Dashed line represents control levels . One - way ANOVA . ( n = 4 ; * p < 0 . 05 )

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(E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05)


( F - G ) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H - cholesterol and subsequent analysis of radiolabel incorporation . The dashed line indicates control levels . Graphs represent fold change over controls . Treatment with Sandoz 58 - 035 , a specific ACAT1 inhibitor , caused a ~ 95 % reduction in cholesterol esterification . EV , empty vector . One - sample t - test . ( n = 3 - 4 ; * p < 0 . 05 ) .

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(F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05).


( H ) Ratio of cholesteryl esters : free cholesterol ( CE : FC ) in the indicated cells .

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(H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.


( A ) Fed rat liver lysosomes were extracted with 1 % of the indicated detergents and then subjected to sucrose gradient centrifugation . Aliquots collected from top to bottom were subjected to SDS - PAGE and immunoblotted for LAMP ‐ 2A and flotillin ‐ 1 . Stars indicate the aliquots of the gradient of translucent appearance ( highly enriched in lipids ) .

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(A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS-PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids).


( B ) The distribution of LAMP ‐ 2A through the sucrose gradient after extraction with 1 % Triton X ‐ 114 was calculated from the densitometric quantification of immunoblots as the one shown here . Values are expressed as percentage of the total lysosomal levels and are mean + s . e . of four experiments .

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(B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.


( A ) Lysosomes from livers of fed ( Fed ) or 24 h starved rats ( Stv ) were extracted with 1 % Triton X ‐ 114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent ‐ resistant ( DR ) fraction , and anything below this fraction was pooled , as the solubilized fraction ( Sol ) ( B ) . Lanes 5 - 6 are one ‐ tenth of the amount of lysosomes loaded in the gradient .

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(A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient.


( C ) Distribution of LAMP ‐ 2A ( L ‐ 2A ) and flotillin ‐ 1 ( Flot ‐ 1 ) in fed rats liver lysosomes extracted with 1 % Triton X ‐ 114 at 4 or 37 ° C . DR fractions were pooled as in ( B ) , and the aliquots below this region were divided in three fractions ( a soluble ( Sol ; aliquot 15 ) and two intermediate fractions ( light : aliquots 11 - 12 ; heavy : aliquots 13 - 14 ) .

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(C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).


( D ) Fed ratliverlysosomes were incubated with MBCD or filipin , as described under Material and methods , and then processed as in ( C ) . The mean value of the densitometric quantification of the LAMP ‐ from two experiments is shown at the bottom in ( C ) and ( D ) .

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(D) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).


( A ) Lysosomalmembranes from fed ratlivers and their corresponding 1 % Triton X ‐ 114 ‐ resistant regions , isolated as described under Material and methods , were subjected to one ( left ) ‐ or two ‐ dimensional ( right ) electrophoresis and stained with Sypro Ruby .

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(A) Lysosomalmembranes from fed ratlivers and their corresponding 1% Triton X‐114‐resistant regions, isolated as described under Material and methods, were subjected to one (left)‐ or two‐dimensional (right) electrophoresis and stained with Sypro Ruby.


( B ) Immunoblot for the indicated proteins of the detergent resistant ( DR ) , intermediate ( Int ) and solubilized ( Sol ) fractions of lysosomal membranes obtained as in Figure 2 .

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(B) Immunoblot for the indicated proteins of the detergent resistant (DR), intermediate (Int) and solubilized (Sol) fractions of lysosomal membranes obtained as in Figure 2.


LAMP ‐ 2A localizes in cholesterol ‐ and GM ‐ 1 ‐ enriched intracellular membrane domains . Mouse fibroblasts grown on coverslips in the presence ( + ) or absence ( − ) of serum were fixed and labeled with filipin ( red ) ( A ) or incubated with Texas ‐ Red ‐ cholera toxin B subunit to label GM ‐ 1 ‐ enriched membrane regions ( red ) and then fixed .

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LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed.


( B ) Both groups of cells were then subjected to indirect immunofluorescence for LAMP ‐ 2A ( green ) . Where indicated , cells were treated with MBCD ( 25 mM ) for 30 min before fixation . The merged images of both fluorophores are shown on the right . Insets show vesicles at higher magnification . Bar : 10 μm .

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(B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm.


The quantification of the colocalization of the two fluorophores is shown in ( C ) . Values are expressed as the mean + s . e . of percentage of colocalization in 20 - 40 cells .

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The quantification of the colocalization of the two fluorophores is shown in (C). Values are expressed as the mean+s.e. of percentage of colocalization in 20-40 cells.


( A , B ) Regions of the lysosomalmembrane resistant to 1 % Triton X ‐ 114 recovered from the sucrose gradients were fixed and processed for electron microscopy ( A ) or visualized without fixation by electron microscopy ( B ) . Arrows point to the membrane bilayer .

(A, B) Regions of the lysosomalmembrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer.


( C ) Intact ratliverlysosomes ( top ) and DR regions of the lysosomal membranes ( bottom ) were fixed and subjected to immunogold labeling for LAMP ‐ 2A and LAMP ‐ 2B , and a gold ‐ conjugated secondary antibody as described under Material and methods . Top right panels show ' clusters ' of gold particles ( black arrows ) in individual lysosomes at higher magnification . Graph shows the labeling density ( gold particles per μm ) in the indicated fractions for both proteins . Values are mean + s . e . from the quantification of 15 different micrographs .

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(C) Intact ratliverlysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show 'clusters' of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs.


( D ) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP ‐ 2A and flotillin ‐ 1 . Representative lysosomes are shown . Far right panel : zoomed ‐ in image of the distribution of LAMP ‐ 2A in regions containing ( black arrows ) and not containing ( white arrows ) flotillin ‐ 1 . Bars : 0 . 2 μm .

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(D) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP‐2A and flotillin‐1. Representative lysosomes are shown. Far right panel: zoomed‐in image of the distribution of LAMP‐2A in regions containing (black arrows) and not containing (white arrows) flotillin‐1. Bars: 0.2 μm.


( A ) Fed ratliverlysosomes were incubated in an isotonic buffer without additions , or in the presence of ratlivercytosol ( 25 μg ) , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ( GAPDH ) ( 25 μg ) , GST ‐ hsc70 ( 10 μg / ml ) , ATP ( 5 mM ) or the indicated combinations for 15 min at 37 ° C . Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel . At the end of the incubation , lysosomes were subjected to Triton X ‐ 114 extraction , apyrase density gradient centrifugation and immunoblot as in Figure 2C . Graph shows changes in LAMP ‐ 2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3 - 6 experiments as the one shown here . Because all the incubations were performed in the presence of protease inhibitors , total levels of LAMP ‐ 2A in lysosomes remained constant throughout the incubation .

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(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation.


( B ) Lysosomes from livers of fed rats , untreated or treated with paraquat ( + PQ ) , were processed as in ( A ) . Graph shows changes in LAMP ‐ 2A distribution induced by the PQ treatment , expressed as times the value in untreated animals and are the mean of three experiments .

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(B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments.


( C ) Lysosomes with high ( CMA + ) and low ( CMA − ) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in ( A ) . Top left : Levels of two CMA components ( LAMP ‐ 2A and hsc70 ) and of an unrelated lysosomalmembrane protein ( LAMP ‐ 1 ) in each of the four groups of lysosomes . Bottom left : Percentage of total lysosomal LAMP ‐ 2A present in each fraction , determined by densitometric quantification of the immunoblots .

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(C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.


( A ) Degradation of long ‐ lived proteins in mouse fibroblasts ( NIH3T3 cells ) treated or not with 25 mM MBCD . Where indicated , 15 mM NH4Cl / 100 μM leupeptin ( NH4Cl + Leup ) were added into the medium . Values are the mean + s . e . of three experiments with triplicate samples .

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(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.


( B ) Effect of MBCD on total rates of protein degradation , measured as in ( A ) , in wild ‐ type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP ‐ 2A ( LAMP ‐ 2A ( − ) ) . The ratio of degradation in MBCD ‐ treated and untreated cells is shown for cells maintained with or without 10 mM 3 ‐ methyladenine ( 3MA ) .

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(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).


( C ) Proteolysis of [ 14C ] GAPDH or a pool of [ 3H ] labeled cytosolic proteins ( pool proteins ) by isolated rat liver lysosomes , treated or not with MBCD . Broken : lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins . Values are expressed as percentage of the degradation by untreated lysosomes and are the mean + s . e . of three experiments with triplicate samples .

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(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.


( D ) Lysosomal - mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol ( 50 μM ) . Top : Distribution of LAMP ‐ 2A after Triton X ‐ 114 extraction and sucrose density gradient centrifugation . Bottom : Ability of intact or disrupted lysosomal - mitochondrial fractions to proteolize [ 14C ] GAPDH . Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean ± s . e . of triplicate samples .

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(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.


( A ) Lysosomes from mouse fibroblasts transfected with wild ‐ type hLAMP ‐ 2A and maintained in the presence ( Serum + ) or absence ( serum − ) of serum were collected by centrifugation and subjected to Triton X ‐ 114 extraction , sucrose density gradient centrifugation and immunobloting for human ( hL2A ) or mouse ( mL2A ) LAMP ‐ 2A . Lane 1 : homogenate .

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(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.


( B ) Amino - acid sequence of the transmembrane and cytosolic region of hLAMP - 2A and the mutations performed . Replaced amino acids are shadowed .

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(B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed.


( C ) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP ‐ 2A and maintained in the presence of serum were processed as in ( A ) .

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(C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A).


( D ) The percentage of total lysosomal hLAMP ‐ 2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here . Values , corrected for the amount of mLAMP ‐ 2A in each fraction , are mean + s . e . of 3 - 4 experiments .

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(D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3-4 experiments.


( E ) Changes in hLAMP ‐ 2A distribution induced by serum removal . Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments . Changes in the three nonDR regions are shown together ( no DR ) .

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(E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR).


( F ) Proteolysis of a pool of 3 [ H ] labeled cytosolic proteins by intact ( left ) or disrupted ( right ) lysosomes isolated from untransfected mouse fibroblasts ( none ) or fibroblasts transfected with wild ‐ type ( WT ) or the indicated mutant forms of hLAMP ‐ 2A . Values are the mean + s . e . of two experiments with triplicate samples .

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(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.


( G ) Part of the lysosomes from ( F ) was treated with MBCD before the incubation with the radiolabeled proteins . Values are expressed as percentage of degradation by untreated lysosomes and are the mean + s . e . of two experiments with triplicate samples .

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(G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.


( A ) The Triton X ‐ 114 ‐ resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous ( 10 - 80 % ) sucrose density gradient , SDS - PAGE and immunoblot for LAMP ‐ 2A . The molecular weight range of each fraction is as follows : fraction 1 ( 100 kDa ) ; fraction 2 ( 100 - 200 kDa ) ; fraction 3 ( 200 - 400 kDa ) ; fraction 4 ( 400 - 600 kDa ) ; fraction 5 ( > 600 kDa ) .

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(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).


( B ) Fed rat liver lysosomes were incubated with 1 mM CaCl2 ( + Ca2 + ) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors ( PI ) . Samples were subjected to Triton X ‐ 114 extraction , sucrose density gradients centrifugation and LAMP ‐ 2A immunoblot .

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(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.


( C ) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS - PAGE and immunobloted with antibodies against the cytosolic ( Top ; L ‐ 2A ) and luminal ( Bottom ; L ‐ 2 ) regions of LAMP ‐ 2A . Arrow : LAMP ‐ 2A proteolytic fragment ( Cuervo and Dice , 2000a ) .

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(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).