( b , c ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were photoconverted maintained for 16 h in media supplemented ( + ) or not ( − ) with serum in presence of 3 - methyladenine ( 3 - MA ) to inhibit macroautophagy . ( b ) Representative of 3 - MA - treated . ( c ) of number of green per in 3 - MA treated or not maintained in presence ( bars ) or absence ( black bars ) of serum . Values are mean + s . e . of 3 different experiments with > 50 counted per experiment . No significant differences were detectable between samples supplemented or not with 3MA . Scale bars , 5 μm .

O O O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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.


( a ) Immunoblot with antibody that recognizes PS - CFP2 of homogenates ( Hom ) , cytosol ( Cyt ) lysosomal membranes ( MB ) matrices ( Mtx ) isolated from mouse fibroblasts stably expressing PS - CFP2 or KFERQ - PS - CFP2 16 h after serum removal .

O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(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 indicated of homogenates ( Hom ) , mitochondria ( Mit ) lysosomal membranes matrices isolated from stably expressing KFERQ - PS - CFP2 maintained for 16 h in media supplemented ( + ) or not ( − ) with serum . ( c ) of amount of KFERQ - PS - CFP2 associated to lysosomes ( matrix ) performed in blots as one shown in ( b ) . Values are expressed as of total KFERQ - PS - CFP2 in are mean + s . e . of three different experiments . Difference in lysosomal associated KFERQ - PS - CFP2 between serum + serum − condition ( * ) were significant for P = 0 . 033 ( t - ) .

O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 intracellular content of KFERQ - PS - CFP2 on serum removal . Values are expressed as of KFERQ - PS - CFP2 present in serum - supplemented mean + s . e . of three different experiments . Values are mean + s . e . of three different experiments differences are significant ( * ) for P = 0 . 007 ( t - ) .

O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) 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 lysosomal content of KFERQ - PS - CFP2 in maintained in serum - free media for 16 h . ( e ) Immunoblot for indicated of same fractions as described in ( b ) . amount of KFERQ - PS - CFP2 associated to lysosomes ( f ) changes in intracellular content of KFERQ - PS - CFP2 were calculated as in ( c , d ) . Values are mean + s . e . of three different experiments differences are significant ( * ) for P = 0 . 032 ( t - ) .

O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e-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).


( a , b ) Human neuroblastoma ( SH - SY5Y ) , hepatoma ( HuH - 7 ) breast adenocarcinoma ( MCF - 7 ) lines were transiently transfected with KFERQ - PS - CFP2 , photoconverted maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . show representative after immunostaining for LAMP - 2 ( red ) . ( b ) of number of green per under basal conditions ( black bars ) 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 counted per experiment . Differences with ( * ) NIH3T3 or ( § ) with supplemented with serum are significant for P0 . 05 ( range 0 . 002 - 0 . 03 , ANOVA - Bonferroni ) .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-CELL_LINE O O O O O O O O O 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-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a, b) 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 ) mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ - PS - CFP2 , photoconverted maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : representative image in green channel . Right : of number of green per in maintained in presence ( black bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of three different experiments with > 50 counted per experiment . ( P = 0 . 002 , ANOVA - Bonferroni ) .

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 presence ( bar ) or absence ( black bars ) of serum subjected to treatment with 3 - methyladenine ( 3 - MA ) , proteasome inhibitor MG - 132 or H2O2 . In both ( d , e ) , after fixation , were subjected to high content image of responders was calculated in > 200 per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - ) . Scale bars , 5 μm .

O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 presence of serum supplemented media leave untreated ( bar ) or subjected to indicated treatments ( black bars ) . In both ( d , e ) , after fixation , were subjected to high content image of responders was calculated in > 200 per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - ) . Scale bars , 5 μm .

O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) 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.


( b ) Mouse fibroblasts ( NIH3T3 ) stably expressing KFERQ - PA - mCherry1 were photoactivated maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : Representative . Nuclei are with DAPI . Right : of number of red per . Values are mean + s . e . of three different experiments with > 20 counted per experiment . * P = 0 . 001 , ANOVA - Bonferroni .

O O O B-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD 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-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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) 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 mCherry in stably expressing KFERQ - PA - mCherry1 at indicated times after photoactivation using FACS . were maintained in media supplemented ( squares ) or not ( black circles ) with serum , by MFI was calculated on events gated on living . Values are expressed as of at time 0 are mean + s . e . of 3 - 4 experiments .

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 effect of serum removal at 6 h ( top ) 42 h ( bottom ) . PS , photoswitching . Scale bars , 5 μm .

O O O O B-EXP_ASSAY O O O O 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) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.


Mouse qPCR array was performed with Colo357 MiaPaca - 2 orthotopic 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 ) .

B-ORGANISM B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Mouse 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 - Quansys mouse chemokine was performed with Capan - 1 ( Cap ) , MiaPaCa - 2 ( Mia ) , Colo357 ( Colo ) , C5LM2 ( C5 ) orthotopic samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect mouse chemokine changes after treatment . Change of mouse IL - 6 was shown .

B-EXP_ASSAY O O B-ORGANISM B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O

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 KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Tumors from were collected for mouse IL - 6 ELISA ( n = 6 / group ) .

O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O

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 KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . activation of SMAD2 ( P - Ser465 / 467 ) ( E H - I ) , STAT3 ( P - Tyr705 ) ( F J - K ) expression of IL - 6R ( G ) was detected by immunohistochemistry ( n = 4 / group ) .

O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O

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).


Single - sequencing was performed to profile populations in normal mouse pancreas ( n = 2 ) , early KIC ( 40 - day - old , n = 2 ) late KIC ( 60 - day - old , n = 3 ) pancreata . Samples from same stage were pooled . Violin plots of expression of Il6 , Tgfbr1 Tgfbr2 in distinct populations is shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O 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-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O

Single-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 TGFβR2 in lysates harvested from KIC ( mPLRB8 , mPLRB9 ) , KPC ( KPC - M01 , KPC - M09 ) KPfC ( BMFA3 , CT1BA5 ) mouse cancer , mouse macrophages ( RAW 264 . 7 ) , mouse fibroblasts ( NIH 3T3 pancreatic stellate ) . RAW 264 . 7 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 .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O 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-ORGANISM B-DISEASE O B-ORGANISM B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-ORGANISM B-CELL_TYPE O B-CELL_LINE I-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O

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 Spearman correlation of expression of IL6 TGFBR2 in PDA from TCGA ( n = 223 ) using R .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-DISEASE O O O O O O O O O O

Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.


Control , M1 ( LPS stimulated ) M2 ( IL - 4 stimulated ) RAW 264 . 7 , as well as NIH 3T3 were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . lysates were harvested blotted for P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , tubulin ( A ) .

O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O

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. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).


Control , M1 ( LPS stimulated ) M2 ( IL - 4 stimulated ) RAW 264 . 7 , as well as NIH 3T3 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 ) .

O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O

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 ( PSC ) ( E ) human CAF lines CAF - PC1 CAF - PC2 ( F ) were treated with TGFβ ( 30 ng / ml ) / or IL - 1α ( 1 ng / ml ) for 24 hours . CM was collected for mouse or human IL - 6 ELISA .

B-CELL_LINE I-CELL_LINE O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O

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 were treated with TGFβ ( 30 ng / ml ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . CM was collected subjected to human IL - 6 ELISA .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O

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 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 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 ) KPfC ( BMFA3 ) 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 ) . lysates were harvested blotted for P - STAT3 ( P - Tyr705 ) , STAT3 , P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , 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 : were seeded on poly - HEMA - coated 96 - well plates cultured for 4 days ( 5000 cancer for monoculture , 3000 cancer + 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 2G8 - treated MiaPaca - 2 Colo375 xenografts were subjected to - seq 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 2G8 - treated MiaPaca - 2 Colo375 xenografts were subjected to - seq for mouse gene expression changes . Molecules most significantly by 2G8 treatment . Y - axis values shown are log10 ( FPKM + 1 ) of transcript ( 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 cytotoxicity was performed . Human NKL were used as effector , mouse pancreatic cancer BMFA3 were used as target 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 were labeled with CFSE , dead were labeled with 7 - AAD . Samples were analyzed by flow cytometry . Cytotoxicity was calculated using formula ( 7 - AAD positive % ) / ( 7 - AAD positive % + CFSE positive % ) x 100 % . n = 4 / group , P values by t 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 Colo357 was orthotopically implanted into NOD SCID mice . After 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 . For NK 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 whole study . Tumors were harvested for metastatic burden was determined by histologic evaluation of H & E - stained liver tissue . Ten sections of anterior lobe of 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 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 , J - L ) T markers CD3 ( M - O ) , CD8 ( P - R ) FOXP3 ( regulatory T , S - U ) in KIC KPC mice . Scale bars = 50 μm . n = 4 / group , P values by t 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 of CD8 - positive T after 2G8 treatment . Details are described in Appendix Fig S7 . Sample of KPC tumors are shown ( V ) . Substantial cytotoxic T infiltration was observed after 2G8 treatment . Histological score was calculated using following formula ( W - X ) : low % + 2x ( medium % ) + 3x ( high % ) . Scale bars = 500 μm . n = 4 / group , P values vs . control by t 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 from TCGA with 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 mut2 ) in mouse PDA BMFA3 derived from KPfC model . Control ( Tgfbr2wt ) two Tgfbr2 - mutant lines ( Tgfbr2mut1 Tgfbr2mut2 ) were treated with TGFβ ( 30 ng / ml ) for 5 hours . lysates were harvested western blotting for P - SMAD2 , SMAD2 , SMAD4 , P - ERK1 / 2 , ERK1 / 2 , P - P38 , P38 , 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 expression localization of E - cadherin SMAD4 after TGFβ treatment ( 30 ng / ml for 24 hours ) in control two Tgfbr2 - mutant lines . Scale bars = 50 μm ( ) , 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 ( Tgfbr2wt ) ( A ) two Tgfbr2 - mutant lines ( Tgfbr2mut1 Tgfbr2mut2 ) ( B - C ) were treated with TGFβ at different time points , growth ( MTT ) assays were performed . P value by t 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 ( D ) two Tgfbr2 - mutant 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 injection ; mice were on therapy for 16 days . volume was measured twice per week . P values by t 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 ( Tgfbr2wt ; G & I ) Tgfbr2 - mutant ( 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 ( Tgfbr2wt ; G & I ) Tgfbr2 - mutant ( Tgfbr2mut1 ; H & J ) were orthotopically implanted in C57Bl / 6 mice . Mice were sacrificed when they became moribund . Tumors were harvested 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 samples from ( G - H ) for Ki67 ( M - O ) , P - STAT3 ( P - R ) , ZAP70 , CD3 ( S - X ) was performed . ZAP70 is a common marker for NK T was stained with a brown chromogen , CD3 is a specific T - marker was stained with a blue chromogen . Brown signal was converted to red signal blue signal was converted to green by ImageJ ( V ) ; therefore NK are highlighted as red ( ZAP70 + CD3 - ) T are highlighted as yellow ( ZAP70 + CD3 + ) . Scale bars outside magnification boxes = 50 μm , scale bars inside magnification boxes = 10 μm . n = 4 / group , P values vs . control by t 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 Tgfbr2 - mutant Tgfbr2mut1 . lysates were harvested western blotting for IL6RA 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 were seeded on poly - HEMA - coated 96 - well plates cultured for 4 days ( 5000 cancer for monoculture , 3000 cancer + 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 were seeded on poly - HEMA - coated 96 - well plates cultured for 4 days ( 5000 cancer for monoculture , 3000 cancer + 2000 NIH 3T3 for co - culture ) . Control IgG , 2G8 IL - 6 neutralizing antibody ( each 100 ng / ml , n = 5 / group ) . Scale bars = 50 μm . P values by t 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 IL6RA knockdown Tgfbr2 - mutant 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 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 whole study . Therapy started at day 12 post injection mice were on therapy for 16 days . volume was measured twice per week . P values by t 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 samples from ( D ) for Ki67 ( G K ) , P - STAT3 ( H L ) , ZAP70 , CD3 ( I - J M - N ) was performed . NK are highlighted as red ( ZAP70 + CD3 - ) T are highlighted as yellow ( ZAP70 + CD3 + ) by ImageJ ( N ) . Scale bars outside magnification boxes = 50 μm , scale bars inside magnification boxes = 10 μm . n = 5 / group , P values vs . control by t 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 . outline is shown as an ellipse . Left , Imaged FliM - YPet . dots indicate estimated motor locations . circles illustrate a 75 nm radius around these locations . Right , Contour map of normalized sum of probabilities of localization events of CheY ( I95V ) - Atto647 ( from blue to red is low to high ; black is zero probability ) . circles show motors ' locations from 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 this interaction is sensitive to chemotactic stimuli . Each curve is mean of two FRET measurements of in response to an attractant stimulus ( 0 . 1 mM serine ; in case of FliMwt , a single measurement was performed with serine ; another measurement of FliMwt with 1mM aspartate produced similar results ) . FRET is of mCherry to YPet . CheY - mCherry mCherry concentrations were ~ 170 µM in case of FliM ∆ N ~ 15 µM in case of FliMwt Strains used : EW677 ( FliMwt , red ) , EW659 ( negative control of FliM ∆ N without CheY , purple ) , EW637 ( FliM ∆ N , blue ) , EW636 ( FliM ∆ N ∆ cheA , yellow ) .

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 ; negative positive values ( below above blue bar ) are counterclockwise clockwise , respectively . switch from negative to positive rotation rate at 3 s is shown in montage to right .

O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY I-EXP_ASSAY 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 , response of tethered fliM ∆ N ΔcheZ ( EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) , to positive stimuli . Lines shaded regions are mean time spent in clockwise rotation ± . arrow marks estimated time at which stimulant arrived to , or left ( as indicated ) , flow chamber . N is number of . α - Methyl - DL - aspartate ( MeAsp ) leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM .

O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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.


response of tethered fliM ∆ N ΔcheZ ( EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) Lines shaded regions are mean time spent in clockwise rotation ± . arrow marks estimated time at which stimulant arrived to , or left ( as indicated ) , flow chamber . N is number of . α - Methyl - DL - aspartate ( MeAsp ) leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM . D , negative stimuli .

O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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 ( EW634 ) to acetate ( 50 mM , pH 7 . 0 ) at different CheY concentrations . Lines shaded regions are mean ± .

O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD 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 ( EW635 ) to acetate .

O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O

F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate.


G , Contribution of acetylation phosphorylation of CheY as well as its D13K mutation to clockwise rotation . points are mean clockwise rotation at time segments 0 - 20 s 160 - 180 s . variants shown are fliM ∆ N ∆ cheZ expressing CheY in absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( i . e . , CheY ~ P CheY ~ P ~ Ac ; blue green curves ; EW635 ) , fliM ∆ N ∆ cheA expressing CheY , in absence of presence of acetate ( CheY CheY ~ Ac ; purple red , respectively ; EW634 ) , fliM ∆ N ∆ cheA expressing CheY ( D13K ) ( burgundy ; EW737 ) . Each data point is average of experiments at a given CheY , weighted by sample number of each experiment . CheY concentrations shown are estimates on calibration curves for which a similar CheY expression system was used .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O 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-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD 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 . variants shown are fliM ∆ N ∆ cheZ expressing FliMN - CheY ( i . e . , FliMN - CheY ~ P ; blue curve ; EW697 ) , fliM ∆ N ∆ cheA expressing FliMN - CheY , in absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( FliMN - CheY FliMN - CheY ~ Ac ; purple red , respectively ; EW696 ) , fliM ∆ N ∆ cheA expressing FliMN - CheY ( D13K ) ( burgundy ; EW739 ) .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 expressing FliMN - CheY ( EW696 ) to acetate benzoate ( 10 mM each ; pH 7 . 0 ) . FliMN - CheY expression was induced with 800 μM IPTG . Lines shaded regions are mean time spent in clockwise rotation ± . black arrow indicates estimated time point at which stimulus entered flow chamber .

O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O 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 level of FliMN - CheY ~ Ac . red purple curves are two experimental days in which clockwise production by fliM ∆ N ∆ cheA expressing FliMN - CheY was measured as a function of FliMN - CheY ( EW696 ; Concentrations are estimates on calibration curves or which a similar CheY expression system was used . ach data point is a mean ± of data or second third experimental days of this ( in presence of acetate ) .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O 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-SMALL_MOLECULE 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 to addition removal of varying acetate concentrations ( pH 7 . 0 ) . FliMN - CheY was estimated to be ~ 300 μM . Lines shaded regions are mean time spent in clockwise rotation ± . arrows indicate estimated time points at which acetate entered or left flow chamber . Black lines mark linear part of biphasic response to acetate removal . N is number of .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 , calculated as inverse of fast ( blue ) slow ( red ) rate

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


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

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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 having FliM ∆ N FliN ( A93D ) motors to acetate addition removal ( red ; EW714 ; FliMN - CheY estimated at ~ 300 μM ) . Similar results were obtained with a ∆ cheA ( EW713 ) . Tethered having FliM ∆ N FliNwt motors containing FliMN - CheY ( EW696 ) are shown Acetate was 10 mM ( pH 7 . 0 ) . data shown are mean ± . N is number of . Black lines indicate clockwise - decay rates of FliNwt motors ' slow phase of FliN ( A93D ) motors following acetate removal .

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

O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O 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-GENEPROD O O O B-GENEPROD O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 fliM ( R94L ) ∆ N ∆ cheZ expressing FliMN - CheY ( strains EW731 EW733 , respectively ) in absence or presence of acetate ( 10 mM , pH 7 . 0 ) . FliMN - CheY was estimated to be ~ 4 mM by extrapolation of 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 mean of a separate experiment . Black is mean of experiments .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 ( EW718 ) at various concentrations of FliMN - CheY . concentrations shown are estimates on calibration curve , for which a similar CheY expression system was used . Each data point is average of measurements at similar FliMN - CheY concentrations , weighted by sample number of each experiment .

O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 bone marrow , thymus , spleen . B development in bone marrow was assessed by FACS 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 development in thymus was assessed by FACS 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 populations were assessed by FACS for surface expression of : B220 + CD21 + CD23 + ( follicular B , FOB ) ; B220 + CD21 + CD23low ( marginal zone B , MZB ) . Each symbol represents an mouse . Horizontal bars indicate mean values . Two - Mann - Whitney , ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-CELL_TYPE O O B-TISSUE O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O O O O 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 for FOB MZB . Paired t , ns = not significant , * P < 0 . 05 , * * * P < 0 . 001 .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O 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) 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 HA - Wdr37 by FLAG - Pacs1 in co - transfected 293T .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O

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


( E ) Wdr37 + / + Wdr37 − / − splenocytes were labeled with Indo - 1 , stained for surface markers to identify FOB , stimulated with indicated amounts of anti - IgM ( black arrowhead ) . Normalized traces from three ( 2 . 5 mcg / ml anti - IgM ) or four independent experiments ( 10 mcg / ml 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 ) .

O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

(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 , autophagy markers in Pacs1 + / + Pacs1 - / - splenic B that were left unstimulated or stimulated overnight with 5 mcg / ml IgM .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O

(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 SERCA2 transcripts in B . Data are presented as mean ± SD from three independent Pacs1 + / + Pacs1 - / - pairs of mice .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM O

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

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O 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) 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 + / + Pacs1 - / - NIH - 3T3 ( C1 C2 from ( A ) ) were transfected with ER - GCamP6 - 210 . ER Ca2 + was measured before after treatment with 10 µM ATP . Kinetic traces show mean 488 / 405 nm excitation of each with error bars indicating . N = 30 ( 3T3 ) , 16 ( C1 ) , 25 ( C2 ) . Data are representative of 2 independent experiments .

O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O

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

O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) 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 + in peripheral blood lymph nodes of Faslpr / lpr dependent on Pacs1 expression . Horizontal bars indicate median value . Two - Mann - Whitney , * * P < 0 . 01 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O

(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 ) of free cholesterol in total homogenates of APP - DKO transiently expressing C99 , C83 or AICD peptides , or treated with 5 µM Aβ42 oligomers for 16h . Cholesterol of WT 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 ) .

O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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) 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 ) of HMGCR enzymatic activity in PS - DKO DAPT - treated WT . unpaired t - ( n = 3 ; * p < 0 . 05 ) .

O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

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


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

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

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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 of WT PS - DKO treated with indicated SMase inhibitors ( 10 µM desipramine or 5 µM GW4869 ) or DMSO ( VEH ) for 12 - 16h . ( A ) Endogenous of lipid droplets ( LDs ) were visualized by incubation with LipidTox . Graph shows Lipidtox / .

O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

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 of free cholesterol revealed by filipin of ( B ) WT under indicated treatments . Graphs show filipin as a over VEH .

B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

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 of free cholesterol revealed by filipin of ( C ) PS - DKO under indicated treatments . Graphs show filipin as a over VEH .

B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

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

O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O

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

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

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

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) 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 of APP - DKO expressing GFP - tagged C99WT or C99MUT ER mitochondria markers as in ( A ) ( upper panel ) . zoomed single channels ( middle panel ) as well as areas of ER - mitochondria colocalization ( bottom panel ) are shown for each condition . Scale bar = 10 µm .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O

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

O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O 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) 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 in total homogenate ( TH ) MAM fractions of APP - DKO expressing C99WT or C99MUT before ( input ) 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 ) of pulled - down C99 versus input from experiment in B . Two - way repeated measures ANOVA ( Fraction , mutation ) ( n = 5 ; * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O 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) 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 of pulled - down PhotoClick Cholesterol ( streptavidin - HRP ) in ER MAM fractions . Note how of pulled - down C99 in ER are negligible when compared to those from MAM . Isolated MAM ER fractions were assessed by western blot ( shown in AppendixFig S1E ) .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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

O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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 ) of cholesterol uptake esterification in indicated was measured by 4h incubation with 3H - cholesterol subsequent of radiolabel incorporation . dashed indicates control . 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 - . ( n = 3 - 4 ; * p < 0 . 05 ) .

O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-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 ) of cholesteryl esters : free cholesterol ( CE : FC ) in indicated .

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


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

O O O O B-ORGANISM B-TISSUE B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(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 processed as in Figure 1 or aliquots in translucent region were pooled together as detergent ‐ resistant ( DR ) fraction , anything below this fraction was pooled , as solubilized fraction ( Sol ) ( B ) . Lanes 5 - 6 are one ‐ tenth of amount of lysosomes loaded in gradient .

O O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O

(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.