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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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


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

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


( E ) Wdr37 + / + 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 ) .

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


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

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


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

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


( D ) Peak 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 .

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


( E ) Pacs1 + / + 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 .

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


( B , C ) Pacs1 + / + 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 .

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


( H , I ) Pacs1 + / + 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 .

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


( D , E ) 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 .

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


( I ) Enumeration of CD3 + B220 + 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 O O O O O O O O O O O O O O O O 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) 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 ) .

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


( B ) 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 ) .

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


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

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


( D ) 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 ) .

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


( E - F ) 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 )

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


( G ) Measurement of cholesterol uptake in 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 ) .

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


Representative confocal 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 / .

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


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

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


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

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


( A ) Crude 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 ) ] .

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


( B - G ) Graphs represent 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 .

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


( A ) Representative confocal 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 .

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


( B ) 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 ) .

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


( C ) Representative confocal 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 .

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


( D ) 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 ) .

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


( B ) Representative immunoblot to reveal C99 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 ) .

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


( D ) Immunoblot showing 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 ) .

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


( E ) 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 )

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


( F - G ) 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 ) .

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


( H ) 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 ) .

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


( B ) 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 .

(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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( B ) - acid of transmembrane region of hLAMP - 2A mutations performed . Replaced are shadowed .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( D , E ) Rat liver lysosomes incubated or not with 1 mM CaCl2 ( + Ca2 + ) were subjected to Triton X ‐ 114 extraction sucrose density gradients centrifugation . of cathepsin A ( D ) carboxypeptidase A activity ( E ) were measured in each fraction by immunoblot or using a standardized colorimetric , respectively . Values in ( E ) are expressed as times activity in untreated membranes are mean + s . e . of two experiments .

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(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.


( B ) Dot plot indicating fraction of tf - Reporters delivered to lysosome by ATG7 - independent mechanisms . median red : green of each population was used to calculate fractional delivery according to following formula : ( ratiosgATG7 - ratiosgATG9A ) / ( ratioNegativeCtl1 - ratiosgATG9A ) , with assumption that sgATG9A yields a true autophagy - null phenotype . Mean values for each reporter are indicated by a black bar . n = 3 for each reporter .

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(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter.


( C ) Wild - type ( WT ) K562 indicated deletion isolates were treated with 100 nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h analyzed by flow cytometry for red : green of tf - NBR1 . Median values for each sample are identified by a black within each violin . red dotted across samples corresponds to red : green in wild - type . red solid across samples corresponds to observed under maximally inhibited conditions ( ATG9AKO ) . n = 10 , 000 .

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(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.


( D ) Representative confocal ( as maximum projections ) of wild - type K562 indicated deletion isolates expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from microscopy . Red ( RFP ) , magenta ; Green ( GFP ) , green ; Hoechst , blue . Scale bars : large panels , 5 µm ; small panels , 1 µm . scaled equally .

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(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.


( E ) Correlative electron microscopy ( CLEM ) of K562 ATG7KO expressing tf - NBR1 under basal conditions . workflow is indicated by green arrows . arrows indicate representative structures of interest . boxes demarcate zoomed areas in subsequent . NBR1 , green ; Hoechst , blue . Scale bar ( small ) , 2 . 5 µm . Scale bar ( large ) , 250 nm .

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(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.


( A ) Box plot of average beta scores ( similar to log - fold change ) for tf - NBR1 flux in indicated deletion lines . Median values ( central band ) , inner quartiles ( boxed regions ) , 1 . 5 interquartile ranges ( whiskers ) , outliers ( dots ) are indicated . Representative hits across three genetic backgrounds are indicated in blue .

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(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.


( B ) Gene correlation plot of average beta scores for lipidation dependent autophagy ( tf - NDP52 , from ( Shoemaker et al , 2019 ) ) lipidation independent autophagy ( average of tf - NBR1 from ATG7KO , ATG10 KO ATG3 KO Highlighted in red are genes with a beta score > 0 . 5 across three lipidation - deficient lines . In black are lipidation components . Dashed lines , top 1 % of beta scores .

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(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.


( C ) Wild - type K562 indicated deletion isolates expressing Cas9 tf - NDP52 or tf - NBR1 were transduced with sgRNAs for indicated genes . median red : green of each population was used to calculate fold - repression according to following formula : ( ratiosgGene - ratiosgATG9A ) / ( ratioNegativeCtl - ratiosgATG9A ) , with assumption that sgATG9A yields a true autophagy - null phenotype . Deeper shades of red indicate stronger suppressor . Genes were clustered on basis of their patterns of genetic interactions .

(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.


( A ) Wild - type ( WT ) K562 indicated deletion isolates were treated with 100nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h analyzed by flow cytometry for red : green of tf - NBR1 . Median values for each sample are identified by a black within each violin . red dotted across samples corresponds to red : green in wild - type . red solid across samples corresponds to observed under maximally inhibited conditions ( ATG9AKO ) . ( n > 10 , 000 ) .

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(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).


( B ) Representative confocal micrograph ( as maximum projections ) of ATG7KO / TAX1BP1KO K562 expressing tf - NBR1 . Selected region ( box ) of micrograph is shown as single merged channels from microscopy . RFP , magenta ; GFP , green ; merged , ; Hoechst , blue . Scale bars : large panel , 5 µm ; small panels , 1 µm .

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(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.


( C ) Correlative electron microscopy ( CLEM ) of K562 ATG7KO / TAX1BP1KO expressing tf - NBR1 under basal conditions . workflow is indicated by green arrows . arrow indicates a structure of interest . box demarcates zoomed in bottom . NBR1 , green ; Hoechst , blue .

(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.


( A ) Wild - type indicated K562 knock - out expressing tf - NBR1 were transfected with TagBFP or TagBFP - RavZ , BFP - positive were analyzed for red : green by flow cytometry . median red : green of each sample was used to calculate flux relative to WT + TagBFP . Bar graphs represent mean + / - SD from three independent experiments . n > 7 , 500 per sample .

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(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample.


( B ) K562 - derived extracts prepared from wild - type ( WT ) clonal deletion isolates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . samples were normalized by total using a BCA prior to loading . Bar graphs represent mean + / - SD from independent experiments ( n = 4 { NBR1 , NDP52 } ; n = 3 { SQSTM1 } ) . p values for WT vs ATG9AKO was determined using a one sample ( theoretical mean = 1 ) with Bonferroni correction .

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(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction.


( D ) Wild - type indicated K562 knock - out were treated corresponding extracts were resolved by SDS - PAGE analyzed by immunoblotting ( IB ) with indicated antibodies . Shown are representative from one experiment with mean from ( E ) indicated below each image ( NBR1 : n = 4 ; TAX1BP1 : n = 3 , NDP52 : n = 4 , tubulin : n = 4 ) . ( E ) Quantitation of protease - protection data from experiments in ( D ) . Bar graphs show mean + / - SD of each sample from independent experiments . Trypsin - treated samples were compared using a one - way ANOVA ( NBR1 : n = 4 , p = 0 . 0001 ; TAX1BP1 : n = 3 , p = 0 . 0098 ; NDP52 : n = 4 , p < 0 . 0001 ) with Tukey HSD post - . * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant


( A ) Extracts derived from wild - type deletion K562 expressing tf - NBR1 were normalized for total by BCA . NBR1 was immunoprecipitated using GFP - Trap dynabeads . Input eluate were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( B ) Representative confocal ( as maximum projections ) of wild - type K562 ATG9AKO expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . In conditions where were not observed , representative regions were selected to showcase diffuseness of signal . TAX1BP1 , SQSTM1 , or NDP52 , magenta ; NBR1 , green ; Hoechst , blue .

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(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.


( C ) Shown are representative confocal ( as maximum projections ) of K562 expressing tf - NDP52 ( or tf - TAX1BP1 ) transduced with sgRNAs targeting ATG9A ( sgATG9A ) or a negative control ( sgControl ) . Selected regions ( box ) of are shown as single merged channels from microscopy . RFP , magenta ; GFP , green ; Hoechst , blue .

(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.


( C ) HEK293T were transfected with either myc - TAX1BP1WT or myc - NBR1WT indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( D ) HEK293T were transfected with myc - NBR1WT indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( A ) Representative confocal ( as maximum projections ) from wild - type deletion K562 expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . P - TBK1 , magenta ; NBR1 , green ; Hoechst , blue . ( B ) Representative confocal ( as maximum projections ) from wild - type deletion K562 expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . FIP200 , magenta ; NBR1 , green ; Hoechst , blue .

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(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.


( C ) Representative confocal ( as maximum projections ) from wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO K562 expressing tf - NBR1 transduced with indicated sgRNA . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( D ) Quantitation of colocalization between NBR1 with FIP200 in wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO imaged in ( C ) Bar graphs represent mean + / - SD for three independently generated deletion lines ( dots ) . sgATG9A samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - . * * * , p < 0 . 001 . n > 200 NBR1 for each biological replicate . ( E ) Quantitation of FIP200 at NBR1 in wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO imaged in ( C ) . Bar graphs represent mean + / - SD for three independently generated deletion lines ( dots ) . n > 80 FIP200 - positive for each biological replicate . Samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - . * * * , p < 0 . 001 . a . u . , arbitrary units .

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(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.


( A ) Soft or stiff 4T1 or MCF - 7 were isolated by microfluidic chip , then seeded ( 500 ) in 90 Pa soft 3D fibrin gel for 3 days . of colonies formed was calculated colony was recorded . Scale bar , 100 μm . Data information : Two - Paired Student ' s t data represent mean ± SD . n = 3 independent experiments in ( A ) .

(A) Soft or stiff 4T1 or MCF-7 cells were isolated by the microfluidic chip, and then seeded (500 cells) in 90 Pa soft 3D fibrin gel for 3 days. The percentage of colonies formed was calculated and the colony size was recorded. Scale bar, 100 μm. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. n = 3 independent experiments in (A).


( F ) NSG mice were intravenously injected with 100 soft or stiff B16 or MP - 1 . Eight later , lung metastasis was analyzed by H & E . metastasis index was defined as of total metastatic nodule to total lung on calculation from 10 slides . Scale bar , 0 . 5 mm . n = 3 ( for B16 ) or 5 ( for MP - 1 ) mice with metastatic . Data information : Two - Paired Student ' s t data represent mean ± SD .

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(F) NSG mice were intravenously injected with 100 soft or stiff B16 or MP-1 cells. Eight weeks later, the lung metastasis was analyzed by H&E staining. The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides. Scale bar, 0.5 mm. n = 3 (for B16) or 5 (for MP-1) mice with metastatic tumor. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.


100 stiff ALDH + or soft ALDH - 4T1 were injected into mammary fat pads of NSG mice . Eight later , lung sections were H & E stained . metastasis index was calculated ( D ) . Scale bar , 0 . 5 mm . n = 5 mice with metastatic . Data information : two - Paired Student ' s t data represent mean ± SD .

The 100 stiff ALDH+ or soft ALDH- 4T1 cells were injected into the mammary fat pads of NSG mice. Eight weeks later, the lung sections were H&E stained. metastasis index was calculated (D). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor. Data information: two-tailed Paired Student's t test data represent mean ± SD.


( E ) of stemness association genes expression determined by mRNA sequencing comparing stiff soft MCF - 7 . n = 3 . unit for color scale was z - score of log2 expression data shown .

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(E) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF-7 cells. n = 3. The unit for the color scale was z-score of log2 expression data shown.


( A ) expression of BCL9L or β - catenin in stiff or soft 4T1 , MCF - 7 , B16 or MP - 1 was determined by western blot . ( B ) expression of β - catenin BCL9L from SGGFP , BCL9L - SGs - 4T1 , MCF - 7 , B16 or MP - 1 were determined by western blot . Data information n = 3 independent experiments in ( A B ) .

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(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot. (B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot. Data information n = 3 independent experiments in (A and B).


( E , F ) 100 soft SGGFP or BCL9L - SGs - 4T1 , B16 or MP - 1 were injected into mammary fat pads ( 4T1 ) of BALB / c mice or tail vein ( B16 MP - 1 ) of NSG mice . formation was recorded ( E ) . Six to eight later , lung sections were H & E stained . metastatic micronodules in lung were counted metastasis index was calculated ( F ) . Scale bar , 0 . 5 mm . n = 3 ( for B16 - F1 ) or 5 ( for MP - 1 ) mice with metastatic . Data information : Bonferroni data represent mean ± SD .

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(E, F) The 100 soft SGGFP or BCL9L-SGs- 4T1, B16 or MP-1 cells were injected into the mammary fat pads (4T1) of BALB/c mice or tail vein (B16 and MP-1) of NSG mice. The tumor formation was recorded (E). Six to eight weeks later, the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (F). Scale bar, 0.5 mm. n = 3 (for B16-F1) or 5 (for MP-1) mice with metastatic tumor. Data information: Bonferroni test The data represent mean ± SD.


( A ) stiffness of breast ( HBC , n = 3 ) colon ( HCC , n = 3 ) cancer screened from outlet or backflow inlet was measured by AFM . n = 100 from each . Data information : Mann - Whitney ( A ) data represent mean ± SD .

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(A) The stiffness of primary breast (HBC, n = 3) and colon (HCC, n = 3) cancer cells screened from the outlet or backflow inlet was measured by AFM. n = 100 from each patient. Data information: Mann-Whitney test (A) The data represent mean ± SD.


( C ) stiff soft were isolated from 3 with breast cancer stained with anti - BCL9L antibody . expression of BCL9L was observed under confocal microscope . Scale bar , 10 μ Data information : Paired Student ' s t ( C ) data represent mean ± SD .

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(C) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti-BCL9L antibody. The expression of BCL9L was observed under confocal microscope. Scale bar, 10 μ Data information: Paired Student's t test (C) The data represent mean ± SD.


( E ) correlation between expression of BCL9L from 18 melanoma grade of melanoma . Data information : Pearson ' s correlation E ) data represent mean ± SD .

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(E) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma. Data information: Pearson's correlation test E) The data represent mean ± SD.


( F ) Overall survival compared to BCL9L level in breast cancer ( BRCA , n = 536 ) or melanoma ( n = 376 ) . Data information : Log - rank survival ( F ) . data represent mean ± SD .

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(F) Overall survival compared to the BCL9L level in breast cancer (BRCA, n = 536) or melanoma (n = 376) patients. Data information: Log-rank survival analysis (F). The data represent mean ± SD.


Ribosome occupancy map at LINC00665 locus . MCF10A were either mock - treated or treated with TGF - β Ribosome profiling on three biological replicates were shown .

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Ribosome occupancy map at the LINC00665 locus. MCF10A were either mock-treated or treated with TGF-β and Ribosome profiling on three biological replicates were shown.


Relative of CIP2A - BP were determined by polysome profiling qRT - PCR in MCF - 10A TNBC either mock treated or treated with TGF - β . Relative of LINC00665 were determined by qRT - PCR in MCF - 10A TNBC either mock treated or treated with TGF - β .

Relative levels of CIP2A-BP were determined by polysome profiling and qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β. Relative levels of LINC00665 were determined by qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


Upper : putative ORFs in LINC00665 . Lower : ORFs were constructed into pcDNA3 . 1 vector transfected to MCF - 10A for 24h . ORFs - His fusion were determined by western blot with anti - His antibody .

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Upper: putative ORFs in LINC00665. Lower: The ORFs were constructed into pcDNA3.1 vector and transfected to MCF-10A cells for 24h. The ORFs-His fusion proteins were determined by western blot with anti-His antibody.


indicated constructs were transfected into MCF - 10A for 24h , then GFP was detected using microscope ( F ) fusion were determined by western blot with anti - GFP anti - CIP2A - BP antibodies

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The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectively


Diagram of His fusion constructs . start codon ATG of LINC00665 ORF is mutated to ATT . indicated constructs were stably expressed in MDA - MB - 231 , micropeptide CIP2A - BP was immuno - stained using anti - His ( I ) anti - CIP2A - BP antibodies ( K ) respectively ; CIP2A - BP - His fusion were determined by western blotting with anti - His CIP2A - BP

Diagram of the His fusion constructs. The start codon ATG of the LINC00665 ORF is mutated to ATT. The indicated constructs were stably expressed in MDA-MB-231 cells, micropeptide CIP2A-BP was immuno-stained using anti-His (I) and anti-CIP2A-BP antibodies (K) respectively; CIP2A-BP-His fusion protein levels were determined by western blotting with anti-His and CIP2A-BP antibodies


MCF - 10A incubated in absence or presence of puromycin ( 200μM ) were fractionated on sucrose gradients , distribution of CIP2A - BP transcripts was quantified by qPCR .

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MCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.


Micropeptide CIP2A - BP was immune - stained with anti - CIP2A - BP antibody in MCF - 10A MDA - MB - 231 either mock treated or treated with TGF - β .

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Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β.


Micropeptide CIP2A - BP were determined by western blot with anti - CIP2A - BP antibody in MCF - 10A TNBC either mock treated or treated with TGF - β .

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Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


MCF - 10A were first transfected with anti - LINC00665 translation - blocking antisense oligo , then micropeptide CIP2A - BP was detected .

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MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected.


Micropeptide CIP2A - BP were detected by western blot with anti - CIP2A - BP antibody in indicated non - metastatic TNBC tissues ( non - metastasis ) metastatic TNBC tissues ( metastasis ) . Micropeptide CIP2A - BP expression in MCF - 10A was used as positive control .

Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control.


Kaplan - Meier overall survival curves for TNBC with high or low micropeptide CIP2A - BP expression in Suzhou cohort ( discovery set , n = 112 ) Guangzhou cohort ( validation set , n = 105 ) .

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Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).


Migration invasion of TNBC transfected with LINC00665 ORF control lentiviruses for 48h . Right panel is of TNBC that migrated through without Matrigel ( upper ) invaded through Matrigel - coated ( lower ) .

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Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h. Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Migration invasion of wild - type CIP2A - BP knockout TNBC CIP2A - BP re - expressed Hs578TKO MDA - MB - 231KO . Right panel is of that migrated through without Matrigel ( upper ) invaded through Matrigel - coated ( lower ) .

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Migration and invasion of wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO cells. Right panel is the quantification of cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Wound - healing on Hs578T MDA - MB - 231 transfected with LINC00665 ORF expressing control lentiviruses . wound edges were photographed at indicated time points after wounding . Right panel is of relative wound healing .

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Wound-healing assay on Hs578T and MDA-MB-231 cells transfected with LINC00665 ORF expressing and control lentiviruses. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Wild - type CIP2A - BP knockout TNBC CIP2A - BP re - expressed Hs578TKO MDA - MB - 231KO were seeded on culture inserts for wound - healing . wound edges were photographed at indicated time points after wounding . Right panel is of relative wound healing .

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Wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound-healing assay. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Macroscopic histological of lungs of nude mice injected with indicated Hs578T ( G ) MDA - MB - 231 ( H ) ( 1 . 0 × 105 - 106 / mouse ) via tail vein ; lung metastatic nodules were visualized 8 post - transplantation ( 5 mice per group ) . Right panel is of pulmonary metastases .

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Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T (G) and MDA-MB-231 cells (H) (1.0×105-106 cells/mouse) via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Immunoblotting of expression of indicated in TNBC cultured with or without TGF - β .

Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF-β.


immunoprecipitation showing Smad4 occupancy at 4E - BP1 locus in TNBC . Co - precipitated was analyzed for amplicons A - E by qPCR . Values represent enrichment of bound fractions relative to input .

Chromatin immunoprecipitation showing Smad4 occupancy at the 4E-BP1 locus in TNBC cells. Co-precipitated DNA was analyzed for amplicons A-E by qPCR. Values represent the enrichment of bound protein fractions relative to input.


Luciferase reporter was performed in Hs578T ( F ) MDA - MB - 231 ( G ) following co - transfection with wildtype or mutant 4E - BP1 promoter fragment Smad4 over - expressed , or knocked down respective controls for 36h treatment with TGF - β for 24h . reporter constructs were expressing luciferase gene under 4E - BP1 promoter segment or 4E - BP1 promoter deleted 0 to + 200 region .

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Luciferase reporter assay was performed in Hs578T (F) and MDA-MB-231 (G) cells following co-transfection with wildtype or mutant 4E-BP1 promoter fragment and Smad4 over-expressed, or knocked down and respective controls for 36h and treatment with TGF-β for 24h. The reporter constructs were expressing the luciferase gene under 4E-BP1 promoter segment or 4E-BP1 promoter deleted 0 to +200 region.


Regulation of 4E - BP1 expression binding to eIF4E by TGF - β . TNBC were treated with TGF - β equal amounts of were subjected to immunoprecipitation / or examined by immunoblotting .

Regulation of 4E-BP1 expression and binding to eIF4E by TGF-β. TNBC cells were treated with TGF-β and equal amounts of proteins were subjected to immunoprecipitation and/or examined by immunoblotting analysis.


Immunoblotting of expression of micropeptide CIP2A - BP β - actin in TNBC cultured with or without TGF - β .

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Immunoblotting analysis of expression of micropeptide CIP2A-BP and β-actin in TNBC cells cultured with or without TGF-β.


Smad4 siRNA corresponding control transfected TNBC were cultured with or without TGF - β . indicated were determined by Immunoblotting .

Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by Immunoblotting analysis.


TNBC were pretreated with specific antagonist against Smad3 ( SIS3 , 3μM ) for 1h then cultured with or without TGF - β . indicated were determined by immunoblotting .

TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


4E - BP1 siRNA corresponding control transfected TNBC were cultured with or without TGF - β . indicated were determined by immunoblotting .

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4E-BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 . Whole - lysates were subjected to co - immunoprecipitation using anti - His antibody immunoblot with CIP2A antibody to verify that CIP2A bound to CIP2A - BP .

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LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 . Whole - lysates were subjected to co - immunoprecipitation by CIP2A antibody immunoblot with CIP2A - BP antibodies to verify that CIP2A - BP bound to CIP2A .

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.


Endogenous CIP2A - BP CIP2A were co - immunoprecipitated in MCF - 10A .

Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 . Whole - lysates were treated with 30 mg / mL RNase A for 30 min , indicated complexes were co - immunoprecipitated by anti - His or anti - CIP2A antibody , CIP2A or micropeptide CIP2A - BP were detected .

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LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.


Upper : A diagram of domain structure of full CIP2A . Red : RIP = Required for Interaction with PPP2R5C ; Orange : RH = Required for Homodimerization ; Blue , CC = Coiled coil . Lower : Co - immunoprecipitation coupled western blot assays revealed interaction of CIP2A - BP with CIP2A via N - terminus domain of CIP2A . Upper : A diagram of domain structure of CIP2A fragment ( 1 - 245 ) . Red : RIP = Required for Interaction with PPP2R5C . Lower : Co - immunoprecipitation coupled western blot assays revealed interaction of CIP2A - BP with CIP2A via N - terminus domain of CIP2A ( acid 159 - 245 ) .

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Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).


Co - immunoprecipitation assays showed that interaction between CIP2A B56γ in TNBC was affected by CIP2A - BP expression .

Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.


expression of indicated were detected by immunoblotting in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) respective controls of TNBC .

The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.


PP2A activity was measured using a phosphatase kit . PP2A activity was performed to evaluate PP2A activity in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) respective control TNBC .

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PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.


Immunohistochemistry ( ) of p - AKT from TNBC ( n = 20 ) with high or low micropeptide CIP2A - BP expression . Lower panels show higher - magnification of insets in upper panels . Right panel is of p - AKT .

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Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.


MDA - MB - 231 were transfected with anti - CIP2A siRNAs . Whole - lysates of these were subjected to immunoblot with CIP2A , CIP2A - BP , PP2Ac , p - AKT , AKT β - actin antibodies .

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MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.