Figure 6 ( B ) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database . TPM : transcripts per million of reads . The central band shows the median value and the box indicates the first and third quartiles . The sample size of human samples used for quantification in GTEx Portal database ( from testis to pancreas ) : n = 361 / 429 / 432 / 555 / 803 / 240 / 432 / 226 / 406 / 578 / 241 / 202 / 85 / 328 / 755 . ( C ) Cellular localization of FAM210A in primary mouse ACMs detected by IF . Scale bar : 30 μm . ( D ) Cellular localization of recombinant FAM210A in human AC16 CM cells . FAM210A - 3xFlag expression plasmid was transfected into AC16 cells for 48 hrs . Scale bar : 40 μm . ( E ) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts . VDAC1 and tubulin are used as fraction controls . Cyto : cytoplasm ; Mito : mitochondria . A representative image is shown from three biological replicates . ( F ) Sub - organelle localization of FAM210A in isolated mouse cardiac mitochondria . TOM20 and ATP5B are used as the outer membrane ( OM ) and mitoplast ( MP ) markers , respectively .

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

Figure 6(B) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database. TPM: transcripts per million of reads. The central band shows the median value and the box indicates the first and third quartiles. The sample size of human samples used for quantification in GTEx Portal database (from testis to pancreas): n=361/429/432/555/803/240/432/226/406/578/241/202/85/328/755.(C) Cellular localization of FAM210A in primary mouse ACMs detected by IF. Scale bar: 30 μm.(D) Cellular localization of recombinant FAM210A in human AC16 CM cells. FAM210A-3xFlag expression plasmid was transfected into AC16 cells for 48 hrs. Scale bar: 40 μm.(E) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts. VDAC1 and tubulin are used as fraction controls. Cyto: cytoplasm; Mito: mitochondria. A representative image is shown from three biological replicates.(F) Sub-organelle localization of FAM210A in isolated mouse cardiac mitochondria. TOM20 and ATP5B are used as the outer membrane (OM) and mitoplast (MP) markers, respectively.


Figure 7 ( B ) Top list of FAM210A - interacting proteins in IP - MS ( immunoprecipitation - mass spectrometry ) analysis . PSM : peptide spectrum match . C ) Confocal imaging of co - localization of FAM210A - 3xFLAG and endogenous EF - Tu by immunostaining . Scale bar : 40 μm . ( D ) Left panel : IP - IB validates FAM210A - interacting proteins using pre - immune IgG and FAM210A antibodies . Right panel : IP - IB validates FAM210A - interacting proteins in HEK293T cell lysates subjected to IP using pre - immune IgG and anti - FLAG antibodies . A representative image is shown in three biological replicates . ( E ) Mapping of interacting domains in FAM210A for binding EF - Tu . ( F , G ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( F ) siRNA knockdown of FAM210A . ( G ) FAM210A overexpression .

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

Figure 7(B) Top list of FAM210A-interacting proteins in IP-MS (immunoprecipitation-mass spectrometry) analysis. PSM: peptide spectrum match.C) Confocal imaging of co-localization of FAM210A-3xFLAG and endogenous EF-Tu by immunostaining. Scale bar: 40 μm.(D) Left panel: IP-IB validates FAM210A-interacting proteins using pre-immune IgG and FAM210A antibodies. Right panel: IP-IB validates FAM210A-interacting proteins in HEK293T cell lysates subjected to IP using pre-immune IgG and anti-FLAG antibodies. A representative image is shown in three biological replicates.(E) Mapping of interacting domains in FAM210A for binding EF-Tu.(F,G) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (F) siRNA knockdown of FAM210A. (G) FAM210A overexpression.


Figure 8 ( A ) miR - 574 - 5p and miR - 574 - 3p overexpression reduced FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) . ( B ) miR - 574 - 5p and miR - 574 - 3p inhibition increased FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) . ( C , D ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( C ) miR - 574 - 5p or miR - 574 - 3p overexpression . ( D ) Transfection of anti - miR - 574 - 5p or anti - miR - 574 - 3p inhibitor . ( E , F ) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment ( 10 μM for 24 hr ) with overexpression of miR - 574 - 5p or miR - 574 - 3p . > 120 cells / group were quantified in ( E ) and n = 4 biological replicates in ( F ) . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group . ( G , H ) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR - 574 - / - mice under ISO treatment . Scale bar : 10 μm . > 110 cells / group were quantified in bottom panel of ( G ) and n = 5 in ( H ) . ( I ) transfection of miR - 574 - 5p / 3p in the absence or presence of FAM210A overexpression . All the Western blot images were provided as representative data from 3 - 4 biologically replicated experiments . ( J ) Increased protein expression of FAM210A and ND1 in miR - 574 KO hearts compared to WT hearts upon TAC surgery . N = 5 hearts ( ~ 110 - 150 CM cells ) were measured per group . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group .

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

Figure 8(A) miR-574-5p and miR-574-3p overexpression reduced FAM210A mRNA expression in AC16 cells (n=3 in each group). (B) miR-574-5p and miR-574-3p inhibition increased FAM210A mRNA expression in AC16 cells (n=3 in each group). (C,D) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (C) miR-574-5p or miR-574-3p overexpression. (D) Transfection of anti-miR-574-5p or anti-miR-574-3p inhibitor.(E,F) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment (10 μM for 24 hr) with overexpression of miR-574-5p or miR-574-3p. >120 cells/group were quantified in (E) and n=4 biological replicates in (F). The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.(G,H) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR-574-/- mice under ISO treatment. Scale bar: 10 μm. >110 cells/group were quantified in bottom panel of (G) and n=5 in (H).(I) transfection of miR-574-5p/3p in the absence or presence of FAM210A overexpression. All the Western blot images were provided as representative data from 3-4 biologically replicated experiments.(J) Increased protein expression of FAM210A and ND1 in miR-574 KO hearts compared to WT hearts upon TAC surgery. N=5 hearts (~110-150 CM cells) were measured per group. The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.


Figure 1A . Raw 264 . 7 macrophage cells were treated with DMSO ( 0 . 05 % ) , 1 μM control antisense oligonucleotide ( ASO ) , or Rab10 - directed ASO ( 1 μM ) , for four days . Immunoblots are representative of three independent experiments , with similar results obtained . B . Calculated reduction of Rab10 ( n = 3 biologically independent experiments ) . Each dot in the plot presents one independent experiment . Group are shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 . C . Raw 264 . 7 cells were treated with the indicated ASO for four days , or 50 μM EIPA ( 5 - [ N - ethyl - N - isopropyl ] amiloride ) for 20 min , prior to incubation for 60 min with fluorescent ( tetramethylrhodamine , TRITC ) - labeled dextran ( 70 kDa , shown as red color ) , transferrin , or IgG - conjugated agarose beads . Representative photomicrographs ( from 30 images analyzed for each condition , from three biologically independent experiments ) are shown . Cells were washed , fixed , and stained with DAPI ( shown as blue color ) . Scale bars show 200 μm . D - F . Relative fluorescent signals were calculated as a percent of control - ASO treated cells to indicate ( D ) dextran or ( E ) transferrin uptake level . ( F ) For quantification of phagocytosis , the average number of IgG - agarose beads internalized were calculated as a percent of control - ASO treated cells . 30 images across three independent experiments were quantified for each condition , with each dot in the plot representing results from one image . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * representing P < 0 . 0001 , N . S P > 0 . 05 . G . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male C57BL / 6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage ( mBMDM ) , dendritic lineage ( mBMDC ) , or microglia - like lineage ( mMicroglia , see Methods section ) . Cells were incubated with TRITC - dextran for 30 min prior to washing , fixing , and staining with DAPI . Representative photomicrographs from 30 images analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . H - J . Relative fluorescent signals were calculated as a percent of control - ASO treated cells . 30 images across 3 independent experiments were quantified for each condition , with each dot in the dotted plot representing 1 image quantified . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 , N . S P > 0 . 05 . K . Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage ( hMDM ) , dendritic lineage ( hMDC ) , or microglia - like lineage ( hMicroglia ) . Cells were incubated with TRITC - dextran for 30 min prior to washing , fixing , and staining with DAPI . Representative photomicrographs from 30 images analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . L - N . Relative fluorescent signals were calculated as a percent of control - ASO treated cells . 30 images across 3 independent experiments were quantified for each condition , with each dot in the plot representing one image quantified . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 .

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

Figure 1A. Raw 264.7 macrophage cells were treated with DMSO (0.05%), 1 μM control antisense oligonucleotide (ASO), or Rab10-directed ASO (1 μM), for four days. Immunoblots are representative of three independent experiments, with similar results obtained. B. Calculated reduction of Rab10 (n=3 biologically independent experiments). Each dot in the plot presents one independent experiment. Group are shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.C. Raw 264.7 cells were treated with the indicated ASO for four days, or 50 μM EIPA (5-[N-ethyl-N-isopropyl]amiloride) for 20 min, prior to incubation for 60 min with fluorescent (tetramethylrhodamine, TRITC)-labeled dextran (70 kDa, shown as red color), transferrin, or IgG-conjugated agarose beads. Representative photomicrographs (from 30 images analyzed for each condition, from three biologically independent experiments) are shown. Cells were washed, fixed, and stained with DAPI (shown as blue color). Scale bars show 200 μm. D-F. Relative fluorescent signals were calculated as a percent of control-ASO treated cells to indicate (D) dextran or (E) transferrin uptake level. (F) For quantification of phagocytosis, the average number of IgG-agarose beads internalized were calculated as a percent of control-ASO treated cells. 30 images across three independent experiments were quantified for each condition, with each dot in the plot representing results from one image. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P<0.0001, N.S P>0.05. G. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male C57BL/6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage (mBMDM), dendritic lineage (mBMDC), or microglia-like lineage (mMicroglia, see Methods section). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. H-J. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the dotted plot representing 1 image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with ****representing P <0.0001, N.S P>0.05. K. Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage (hMDM), dendritic lineage (hMDC), or microglia-like lineage (hMicroglia). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. L-N. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the plot representing one image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.


Figure 2A . Raw 264 . 7 macrophage cells were transfected with FLAG ( N - term ) - Rab10 and eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker ) , or mkate2 ( N - term ) - 2xTAPP1 - PH domain ( PI ( 3 , 4 ) P2 marker ) . Representative photomicrographs ( from 25 images analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed cells immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . White bounding boxes show " High Mag " panels that magnify representative individual vesicles . The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the " CoLoc " panels . The colocalized pixels are shown in white with the same intensity . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker . B . At least 25 cells across three independent experiments were analyzed for each condition ( see Materials and Methods section ) . % of Rab10 vesicles positive with the indicated marker within each cell was quantified . Each dot represents the mean value from one cell analyzed . Group means are given , with error bars representing ± SEM . C . Raw 264 . 7 macrophage cells were transfected with FLAG ( N - term ) - Rab10 and eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) and treated with wortmannin ( 1 μM ) for one hour . Representative photomicrographs ( from 25 cells analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed cells immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . Scale bars are 10 μm , or 1 μm in " Hig Mag " panel . D . Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from > 30 cells across five independent experiments . Each dot on the plot represents mean values from one experiment . Error bars represent ± SEM and significance was assessed by Mann - Whitney test ( owing to non - normal distributions ) with * * indicating P < 0 . 01 . E . Raw 264 . 7 cells were co - transfected with eGFP ( N - term ) - Rab10 and mRuby ( N - term ) - Rab5 for 48 hours , with live - cell recordings for ~ 10 minutes . A representative vesicle is shown over the presented time - lapse . Scale bar represents 1 μm . F . Fluorescence intensities of individual vesicles are calculated over time . Dots show mean values calculated from n = 8 recorded vesicles from cells from n = 3 biologically independent experiments . Error bars show ± SEM .

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

Figure 2A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM. C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01. E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm. F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.


Figure 3Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , cells were incubated with ( A ) TRITC - dextran ( 70 kDa , shown as red signal ) Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker . Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , cells were incubated with ( B ) DQ - ovalbumin , for 30 min before washing and fixation . Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker . Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . ( C , D ) Cells were further stained for ( C ) EEA1 , or ( D ) Lamp1 , which was detected with Cy5 dye ( show as red signal ) . Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker . E ) 25 cells across three independent experiments were analyzed for each condition . % of Rab10 vesicles positive with the indicated marker in each cell were quantified . Each dot represents the mean results from vesicles analyze in one cell . The group means of % Rab10 vesicles positive with the marker are shown , with error bars representing ± SEM . Significance was assessed by Mann - Whitney test for each marker , where * * representing p < 0 . 01 , * * * * p < 0 . 0001 and N . S p > 0 . 05 .

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

Figure 3Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.E) 25 cells across three independent experiments were analyzed for each condition. % of Rab10 vesicles positive with the indicated marker in each cell were quantified. Each dot represents the mean results from vesicles analyze in one cell. The group means of % Rab10 vesicles positive with the marker are shown, with error bars representing ± SEM. Significance was assessed by Mann-Whitney test for each marker, where ** representing p<0.01, **** p<0.0001 and N.S p>0.05.


Figure 4A . Raw 264 . 7 macrophage cells were transfected with Flag ( N - term ) - Rab10 and eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker , epifluorescence , shown in blue ) followed by anti - Flag immunofluorescence ( shown in green ) and pT73 - Rab10 antibody ( shown in red ) . To evaluate colocalization of phospho - Rab10 with early endosomes or late endosome / lysosome markers , immunofluorescence detection was performed for endogenous EEA1 or Lamp1 ( shown in blue ) . Raw 264 . 7 macrophage cells were further fed 0 . 05 mg mL - 1 of 70 kDa TRITC - conjugated dextran ( epifluorescence , shown in blue ) or 0 . 05 mg mL - 1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis . Representative images were selected from 25 cells analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are " High Mag " panels that show individual vesicles analyzed . Scale bars represent 10 μm and 1 μm for " High Mag " . Positive colocalization signal ( depicted as white ) in the " CoLoc ( pRab10 / Rab10 ) ' and ' CoLoc ( pRab10 / Marker ) ' columns are indicated . White arrow heads highlight vesicles scored as co - positive between pT73 - Rab10 and Rab10 or the indicated marker . B . 25 cells across three independent experiments were analyzed for each condition , with each dot representing the mean of the vesicle analysis across individual cells . The group means of % pT73 - Rab10 vesicles positive for the indicated marker are given , with error bars showing ± SEM .

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

Figure 4A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in the "CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker. B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM.


Figure 5A . Raw264 . 7 cells were transfected with Flag ( N - term ) - LRRK2 and eGFP ( N - term ) - Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from > 20 images analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed cells with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) , and immuno - stained for pT73 - Rab10 ( shown as red signal ) and LRRK2 ( shown as blue signal ) . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Scale bars represent 10 μm or 1 μm in " Hig Mag " panels . B . Raw 264 . 7 macrophage cells transfected with eGFP ( N - term ) - Rab10 and Flag ( N - term ) - LRRK2 were sequentially lysed mechanically into buffer to create a ' soluble ' protein fraction , and then insoluble material lysed into triton X - 100 buffer to create a " triton x - 100 " fraction . The " soluble " and triton X - 100 buffer solubilized fractions were analyzed using SDS - PAGE ( TGX gels ) , and representative immunoblots from n = 3 independent experiments are shown . C . The " triton X - 100 " fraction was used as input for LRRK2 immunoprecipitation , with subsequent detection of total Rab10 and pT73 - Rab10 proteins in LRRK2 : Rab10 immunocomplexes using SDS - PAGE ( TGX gels ) . To measure the stoichiometry of pT73 - Rab10 to total Rab10 , the same immunocomplexes were analyzed using phos - tag SDS - PAGE method , with unphosphorylated eGFP ( N - term ) - Rab10 migrating below the phospho - Rab10 protein . Immunoblots are representative of three independent experiments , with similar results obtained . D . Calculated levels of total LRRK2 : Rab10 protein complex with and without LRRK2 kinase inhibition . Data are from n = 3 biologically independent experiments with error bars showing ± SEM . Significance was assessed by a two - tailed t - test with N . S representing P > 0 . 05 . E . The signal intensities from pT73 - Rab10 and unphosphorylated Rab10 in the input lysate co - immunoprecipitated with LRRK2 antibodies . The stoichiometry of pT73 - Rab10 was calculated by dividing the pT73 - Rab10 signal by total Rab10 signal ( total Rab10 signal equivalent to pT73 - Rab10 signal + unphosphorylated Rab10 signal ) . Data are from n = 3 biologically independent experiments with error bars showing ± SEM ; Significance was assessed by two - tailed t - test with * * * representing P < 0 . 0005 .

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

Figure 5A. Raw264.7 cells were transfected with Flag(N-term)-LRRK2 and eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells with eGFP(N-term)-Rab10 epifluorescence (shown as green signal), and immuno-stained for pT73-Rab10 (shown as red signal) and LRRK2 (shown as blue signal). White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm or 1 μm in "Hig Mag" panels.B. Raw 264.7 macrophage cells transfected with eGFP(N-term)-Rab10 and Flag(N-term)-LRRK2 were sequentially lysed mechanically into buffer to create a 'soluble' protein fraction, and then insoluble material lysed into triton X-100 buffer to create a "triton x-100" fraction. The "soluble" and triton X-100 buffer solubilized fractions were analyzed using SDS-PAGE (TGX gels), and representative immunoblots from n=3 independent experiments are shown.C. The "triton X-100" fraction was used as input for LRRK2 immunoprecipitation, with subsequent detection of total Rab10 and pT73-Rab10 proteins in LRRK2:Rab10 immunocomplexes using SDS-PAGE (TGX gels). To measure the stoichiometry of pT73-Rab10 to total Rab10, the same immunocomplexes were analyzed using phos-tag SDS-PAGE method, with unphosphorylated eGFP(N-term)-Rab10 migrating below the phospho-Rab10 protein. Immunoblots are representative of three independent experiments, with similar results obtained.D. Calculated levels of total LRRK2:Rab10 protein complex with and without LRRK2 kinase inhibition. Data are from n=3 biologically independent experiments with error bars showing ± SEM. Significance was assessed by a two-tailed t-test with N.S representing P>0.05.E. The signal intensities from pT73-Rab10 and unphosphorylated Rab10 in the input lysate co-immunoprecipitated with LRRK2 antibodies. The stoichiometry of pT73-Rab10 was calculated by dividing the pT73-Rab10 signal by total Rab10 signal (total Rab10 signal equivalent to pT73-Rab10 signal+ unphosphorylated Rab10 signal). Data are from n=3 biologically independent experiments with error bars showing ± SEM; Significance was assessed by two-tailed t-test with *** representing P<0.0005.


Figure 6A , B . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male WT - LRRK2 mBAC transgenic mice were pre - incubated with FITC - conjugated dextran ( 70 kDa , green signal ) prior to washing , fixing , and immunostaining with total - Rab10 ( shown as red signal ) , or pT73 - Rab10 antibody ( shown as red signal ) . Alternatively , cells were co - stained with antibodies to Rab5 , Rab7 or Lamp1 ( shown as green signal in separate images ) . Representative photomicrographs ( from > 30 images analyzed for each condition from n = 3 biologically independent experiments ) are shown . White bounding boxes are " High Mag " panels that show individual vesicles analyzed . Positive colocalization signal ( depicted as white ) in " CoLoc " panels are given with white arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 and the indicated marker . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . C . Analysis of 20 cells across three independent experiments are given for each indicated co - localization , where % of Rab10 or pT73 - Rab10 vesicles positive with indicated markers was calculated for each cell ( with signal inside of vesicles analyzed for TRITC - dextran and DQ - ovalbumin ) . Each dot represents the mean value of vesicles analyzed inside one cell . The group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated . Error bars represent ± SEM . Significance was assessed by Mann - Whitney tests for each marker with * * * * representing p < 0 . 0001 and N . S representing p > 0 . 05 . D . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male WT - LRRK2 mBAC transgenic mice were treated with 0 . 001 % DMSO or 100 nM MLi2 ( i . e . , IC90 concentration ) for two hours prior to immunostaining with total Rab10 or pT73 - Rab10 antibody ( shown as red signal ) , and anti - Rab5 or anti - Rab7antibody ( shown as green signal ) . Representative photomicrographs ( from 20 cells analyzed for each condition from n = 3 biologically independent experiments ) are shown . White bounding boxes are " High Mag " panels that show individual vesicles analyzed . Scale bars represent 10 μm and 1 μm for " High Mag " . Positive colocalization signal ( depicted as white ) in " CoLoc " panels are given with white arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 and the indicated marker . E . Calculated percentage of double - positive vesicles ( with > 50 % of the vesicle boundary demarcated by white signal in the colocalization analysis ) from 20 cells counted from n = 3 biologically independent experiments are shown . Each dot represents 1 cell in the dotted plot . The group means of % Rab10 or pT73 - Rab10 vesicles positive with indicated markers were calculated and shown . Error bars represent ± SEM . Significance was assessed by Mann - Whitney test for each marker with * representing p < 0 . 05 , * * P < 0 . 01 . F . Raw 264 . 7 macrophage cells were transfected with eGFP ( N - term ) - Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 ( 100 nM ) for two hours . Representative photomicrographs ( from 25 images analyzed for each condition across n = 3 biologically independent experiments ) are shown with eGFP ( N - term ) - Rab10 epifluorescence ( shown as green signal ) and pT73 - Rab10 ( shown as red signal ) , with EEA1 ( shown as blue signal ) . White bounding boxes are " High Mag " panels that show individual vesicles analyzed . Scale bars represent 10 μm and 1 μm for " High Mag " . Positive colocalization signal ( depicted as white ) in " CoLoc " panels are given with white arrow heads highlighting vesicles scored as co - positive between pT73 - Rab10 Rab10 and the indicated marker . G . Calculated percentage of colocalized vesicles , with each dot representing mean vesicle frequency in each cell analyzed . The group mean of % Rab10 vesicles positive with EEA1 were calculated , and error bars represent ± SEM . Significance was assessed by Mann - Whitney test for each marker with * * * representing P < 0 . 005 .

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

Figure 6A,B. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were pre-incubated with FITC-conjugated dextran (70 kDa, green signal) prior to washing, fixing, and immunostaining with total-Rab10 (shown as red signal), or pT73-Rab10 antibody (shown as red signal). Alternatively, cells were co-stained with antibodies to Rab5, Rab7 or Lamp1 (shown as green signal in separate images). Representative photomicrographs (from >30 images analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels.C. Analysis of 20 cells across three independent experiments are given for each indicated co-localization, where % of Rab10 or pT73-Rab10 vesicles positive with indicated markers was calculated for each cell (with signal inside of vesicles analyzed for TRITC-dextran and DQ-ovalbumin). Each dot represents the mean value of vesicles analyzed inside one cell. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated. Error bars represent ± SEM. Significance was assessed by Mann-Whitney tests for each marker with **** representing p<0.0001 and N.S representing p>0.05.D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were treated with 0.001% DMSO or 100 nM MLi2 (i.e., IC90 concentration) for two hours prior to immunostaining with total Rab10 or pT73-Rab10 antibody (shown as red signal), and anti-Rab5 or anti-Rab7antibody (shown as green signal). Representative photomicrographs (from 20 cells analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker.E. Calculated percentage of double-positive vesicles (with >50% of the vesicle boundary demarcated by white signal in the colocalization analysis) from 20 cells counted from n=3 biologically independent experiments are shown. Each dot represents 1 cell in the dotted plot. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated and shown. Error bars represent± SEM. Significance was assessed by Mann-Whitney test for each marker with * representing p<0.05, ** P<0.01.F. Raw 264.7 macrophage cells were transfected with eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from 25 images analyzed for each condition across n=3 biologically independent experiments) are shown with eGFP(N-term)-Rab10 epifluorescence (shown as green signal) and pT73-Rab10 (shown as red signal), with EEA1 (shown as blue signal). White bounding boxes are "High Mag" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for "High Mag". Positive colocalization signal (depicted as white) in "CoLoc" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 Rab10 and the indicated marker. G. Calculated percentage of colocalized vesicles, with each dot representing mean vesicle frequency in each cell analyzed. The group mean of % Rab10 vesicles positive with EEA1 were calculated, and error bars represent ± SEM. Significance was assessed by Mann-Whitney test for each marker with *** representing P<0.005.


Figure 7A . Raw264 . 7 cells were transfected with Flag - Rab10 or control empty vector . The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP . The Flag - Rab10 complex was immunoprecipitated with Flag - resin followed by extensive washing and elution of complexes with 200 μg mL - 1 3xFlag - tide . Eluates were analyzed by SDS - PAGE followed by immuno - blotting with anti - Flag - HRP antibody . C . Flag ( N - term ) - Rab10 protein was purified from HEK293 - FT cells transfected with Flag ( N - term ) - Rab10 plasmids and immobilized on Flag - resin in lysis buffer containing 2 mM GTPγS , or 2 mM GDP , as indicated . Beads were mixed with lysates from cells transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to generate Rab10 : EHBP1L1 complexes . Total Rab10 on the beads was detected using anti - Flag antibody , and EHBP1L1 immobilized to total Rab10 detected with an anti - Myc antibody . Representative immunoblots from n = 3 biologically independent experiments are shown . D . Quantification of relative EHBP1L1 pulled down . Data are from n = 3 biologically independent experiments . Error bars represent ± SEM . . . E . HEK293 - FT cells were transfected with Flag ( N - term ) - Rab10 and Myc ( N - term ) - LRRK2R1441C to induce Rab10 phosphorylation . Membrane bound Flag ( N - term ) - Rab10 protein was extracted with Flag - resin to generate ' pRab10 beads , ' or extracted from cells treated with MLi2 ( 100 nM ) to generate ' Rab10 beads . ' Beads were mixed with lysates from HEK293 - FT cells transfected with Myc - mkate2 ( N - term ) - EHBP1L1 to form on - bead Rab10 : EHBP1L1 complexes . The total Rab10 and pT73 - Rab10 on the beads was detected using anti - Flag antibody and pT73 - Rab10 antibody . The EHBP1L1 in the lysates and immobilized on the beads was detected using anti - Myc tag antibody . Representative immunoblots are shown . F . The relative EHBP1L1 pulled down , normalized to total Rab10 protein on - bead , was determined from n = 3 biologically independent experiments . Error bars represent ± SEM . Each dot represents one experiment , with significance assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc test P < 0 . 0001 . G . Raw 264 . 7 cells were co - transfected with eGFP ( N - term ) - Rab10 ( epifluorescence shown in green ) and Myc - mkate2 ( N - term ) - EHBP1L1 ( epifluorescence shown in blue ) , or Flag ( C - term ) - RILPL2 ( anti - Flag signal shown in blue ) . Cells were stained for pT73 - Rab10 ( shown in red ) . Representative photomicrographs of individual vesicles ( from > 10 cells analyzed for each condition from n = 3 biologically independent experiments ) are shown . Scale bar represents 1 μm . H . Pearson ' s colocalization coefficient for Rab10 and pT73 - Rab10 , EHBP1L1 and RILPL2 on vesicle bodies and tubular vesicles . Data are from n = 20 vesicles with tubular structures positive for pT73 - Rab10 , and n = 12 vesicles with tubular structures positive for EHBP1L1 , or RILPL2 , calculated from cells from three independent experiments . Each dot represents one vesicle body or tubular structure , with significance assessed by Kruskal - Wallis test followed by Dunn ' s multiple comparison test with * * * representing P < 0 . 0005 and * * * * P < 0 . 0001 . Error bars represent ± SEM . . I . Raw264 . 7 cells were co - transfected with Flag ( N - term ) - Rab10 , Flag ( N - term ) - LRRK2R1441C , and Myc ( N - term ) - EHBP1L1 , with representative immunoblots shown . J . Relative pT73 - Rab10 levels were calculated as fold of cells transfected with Flag ( N - term ) - Rab10 and Flag ( N - term ) - LRRK2 only . Data are from n = 3 biologically independent experiments . Each dot represents one independent experiment . Significance was assessed by one - way ANOVA with * * * * representing Tukey ' s post hoc test P < 0 . 0001 . K . Raw264 . 7 cells were co - transfected with eGFP ( N - term ) - Rab10 ( epifluorescence , shown in green ) and Myc - mkate2 ( N - term ) - EHBP1L1 or Myc - mkate2 control plasmid ( epifluorescence , shown in red ) . Representative photomicrographs are shown . Bounding boxes highlight vesicles in " High Mag " panels . Scale bars represent 10 μm , and 1 μm for " High Mag " images . L . % of eGFP ( N - term ) - Rab10 vesicles positive with pT73 - Rab10 were calculated ( from 25 cells analyzed for each condition from n = 3 biologically independent experiments ) . Each dot represents the mean frequency observed in one cell . Error bars show ± SEM with significance was assessed by Mann - Whitney test . * * * represents P < 0 . 0005 .

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

Figure 7A. Raw264.7 cells were transfected with Flag-Rab10 or control empty vector. The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP. The Flag-Rab10 complex was immunoprecipitated with Flag-resin followed by extensive washing and elution of complexes with 200 μg mL-1 3xFlag-tide. Eluates were analyzed by SDS-PAGE followed by immuno-blotting with anti-Flag-HRP antibody.C. Flag(N-term)-Rab10 protein was purified from HEK293-FT cells transfected with Flag(N-term)-Rab10 plasmids and immobilized on Flag-resin in lysis buffer containing 2 mM GTPγS, or 2 mM GDP, as indicated. Beads were mixed with lysates from cells transfected with Myc-mkate2(N-term)-EHBP1L1 to generate Rab10:EHBP1L1 complexes. Total Rab10 on the beads was detected using anti-Flag antibody, and EHBP1L1 immobilized to total Rab10 detected with an anti-Myc antibody. Representative immunoblots from n=3 biologically independent experiments are shown. D. Quantification of relative EHBP1L1 pulled down. Data are from n=3 biologically independent experiments. Error bars represent ± SEM... E. HEK293-FT cells were transfected with Flag(N-term)-Rab10 and Myc(N-term)-LRRK2R1441C to induce Rab10 phosphorylation. Membrane bound Flag(N-term)-Rab10 protein was extracted with Flag-resin to generate 'pRab10 beads,' or extracted from cells treated with MLi2 (100 nM) to generate 'Rab10 beads.' Beads were mixed with lysates from HEK293-FT cells transfected with Myc-mkate2(N-term)-EHBP1L1 to form on-bead Rab10:EHBP1L1 complexes. The total Rab10 and pT73-Rab10 on the beads was detected using anti-Flag antibody and pT73-Rab10 antibody. The EHBP1L1 in the lysates and immobilized on the beads was detected using anti-Myc tag antibody. Representative immunoblots are shown. F. The relative EHBP1L1 pulled down, normalized to total Rab10 protein on-bead, was determined from n=3 biologically independent experiments. Error bars represent ± SEM. Each dot represents one experiment, with significance assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001. G. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence shown in green) and Myc-mkate2(N-term)-EHBP1L1 (epifluorescence shown in blue), or Flag(C-term)-RILPL2 (anti-Flag signal shown in blue). Cells were stained for pT73-Rab10 (shown in red). Representative photomicrographs of individual vesicles (from >10 cells analyzed for each condition from n=3 biologically independent experiments) are shown. Scale bar represents 1 μm. H. Pearson's colocalization coefficient for Rab10 and pT73-Rab10, EHBP1L1 and RILPL2 on vesicle bodies and tubular vesicles. Data are from n=20 vesicles with tubular structures positive for pT73-Rab10, and n=12 vesicles with tubular structures positive for EHBP1L1, or RILPL2, calculated from cells from three independent experiments. Each dot represents one vesicle body or tubular structure, with significance assessed by Kruskal-Wallis test followed by Dunn's multiple comparison test with *** representing P<0.0005 and **** P<0.0001. Error bars represent ± SEM.. I. Raw264.7 cells were co-transfected with Flag(N-term)-Rab10, Flag(N-term)-LRRK2R1441C, and Myc(N-term)-EHBP1L1, with representative immunoblots shown. J. Relative pT73-Rab10 levels were calculated as fold of cells transfected with Flag(N-term)-Rab10 and Flag(N-term)-LRRK2 only. Data are from n=3 biologically independent experiments. Each dot represents one independent experiment. Significance was assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001.K. Raw264.7 cells were co-transfected with eGFP(N-term)-Rab10 (epifluorescence, shown in green) and Myc-mkate2(N-term)-EHBP1L1 or Myc-mkate2 control plasmid (epifluorescence, shown in red). Representative photomicrographs are shown. Bounding boxes highlight vesicles in "High Mag" panels. Scale bars represent 10 μm, and 1 μm for "High Mag" images. L. % of eGFP(N-term)-Rab10 vesicles positive with pT73-Rab10 were calculated (from 25 cells analyzed for each condition from n=3 biologically independent experiments). Each dot represents the mean frequency observed in one cell. Error bars show ± SEM with significance was assessed by Mann-Whitney test. *** represents P<0.0005.


Figure 8A - D . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male WT - mLRRK2 BAC transgenic mice were immunostained with anti - mouse CCR5 , or CD11b , or MHC II antibody ( shown as green signal ) and ( A ) total Rab10 antibody ( red signal ) , or ( B ) pT73 - Rab10 antibody ( red signal ) as indicated . Representative photomicrographs ( from > 20 images analyzed for each condition from n = 3 biologically independent experiments ) are shown . ( C ) Photomicrographs showing the different cargo with respect to Rab10 tubular structures . ( D ) EEA1 ( shown as green signal ) with total Rab10 or pT73 - Rab10 ( shown as red signals ) as indicated . Bounding boxes in panels A - D are magnified in " High Mag " panels that show individual vesicles . Scale bars represent 10 μm and 2 μm for " High Mag " images . E . Calculated percentage of vesicles positive with tubular structures ( n > 30 vesicles analyzed from cells from n = 3 biologically independent experiments ) are shown . Each dot represents one vesicle , with significance assessed by Mann - Whitney test . * * represents P < 0 . 01 . Error bars represent ± SEM . F . BMDM cells were treated with 0 . 004 % DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa - 594 conjugated rat anti - mouse CD11b antibody on ice for ten minutes . Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding ( shown in red as indicated ) . Six wells of cells from each condition were further incubated at 37 ° C to allow rapid internalization of CD11b - antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0 . 5 % acetic acid on ice for one min . Three of the six wells were fixed and surface - stained with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes remaining on the cell surface ( shown in green as indicated ) . Internalized CD11b - antibody complexes are shown in red . The other three wells of cells were further incubated at 37 ° C again for ten min to allow recycling of internalized CD11b - antibody complex followed by surface staining with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes recycled back to the surface . Representative images from n = 3 biologically independent experiments are shown . Scale bars represent 30 μm . G - I . ( G ) The initial surface CD11b , or ( H ) internalized CD11b , or ( I ) recycled CD11b were calculated from > 20 images analyzed for each condition from n = 3 biologically independent experiments . Each dot represents the mean value from one experiment . Error bars show ± SEM with significance assessed by Mann - Whitney test . * represents P < 0 . 05 .

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

Figure 8A-D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were immunostained with anti-mouse CCR5, or CD11b, or MHC II antibody (shown as green signal) and (A) total Rab10 antibody (red signal), or (B) pT73-Rab10 antibody (red signal) as indicated. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown. (C) Photomicrographs showing the different cargo with respect to Rab10 tubular structures. (D) EEA1 (shown as green signal) with total Rab10 or pT73-Rab10 (shown as red signals) as indicated. Bounding boxes in panels A-D are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm and 2 μm for "High Mag" images.E. Calculated percentage of vesicles positive with tubular structures (n>30 vesicles analyzed from cells from n=3 biologically independent experiments) are shown. Each dot represents one vesicle, with significance assessed by Mann-Whitney test. ** represents P<0.01. Error bars represent ± SEM.F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05.


Figure 9A . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male WT - mLRRK2 BAC transgenic mice were treated with DMSO , 1 μM Rab10 anti - sense oligonucleotide for 4 days , 100 nM MLi2 for 12 hours , as indicated , before stimulation with 100 nM CCL5 for the indicated time ( hours ) . Representative immunoblots from n = 3 biologically independent experiments are shown . B . Relative pSer473 - Akt levels were normalized to total Akt and calculated as fold of naïve cells ( without CCL5 stimulation ) . Data shown are group means ± SEM from n = 3 biologically independent replicates , with significance assessed by one - way ANOVA with Tukey ' s post hoc test with treated groups compared to matched control groups , with * * representing P < 0 . 01 . C . BMDM cells , as described in panel ( A ) , were additionally treated with DMSO ( control ) , EIPA , or Akti - 1 / 2 ( Akt1 / 2 inhibitor ) as indicated , for 1 hour after CCL5 stimulation . Representative immunoblots from n = 3 biologically independent experiments are shown . D . Relative pSer473 - Akt levels were normalized to total Akt and calculated as fold of naïve cells ( without CCL5 stimulation ) . Each dot represents one experiment , with error bars showing ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * representing P < 0 . 0005 , * * * * P < 0 . 0001 . E . BMDM cells , as described in panels ( A and C ) , were seeded in Transwell ( Sigma ) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers . After two hours , the cells failing to migrate through the matrices were removed with a cotton swab . Migrated cells were counted with DAPI images , with representative images shown at low magnification . Representative images from n = 3 biologically independent experiments are shown . F . Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5 . Each dot represents the mean of images collected from one experiment . Data are group means ± SEM , with significance assessed by multiple t test . , with * representing P < 0 . 05 .

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Figure 9A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown. B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01. C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001. E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown. F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05.


Figure 1 ( E ) Representative images of the pilot validation screen for the negative control ( no siRNA ) and for five sets of siRNA - treated cells with reduced TGN retrieval ratio . Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text . Scale bar , 100 μm ( top two rows ) and 38 μm ( bottom row ) .

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Figure 1(E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).


Figure 3 ( A ) Primary screen anti - CD8 antibody - uptake images for control cells ( top ) and SFT2D2 KD cells ( bottom ) . ( B ) Cells stably expressing SFT2D2 - Myc show localization of SFT2D2 to perinuclear membranes and endosomes , including VPS35 - positive endosomes ( arrowheads in inset ) . Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2 - Myc and VPS35 ( arrowheads in inset ) . ( C ) Cells stably expressing SFT2D2 - Myc were costained with antibodies against various post - Golgi SNARE proteins . Colocalization was quantified by Pearson ' s correlation coefficient ( right - hand graph ) and indicates very extensive colocalization with STX6 , STX7 , and VAMP8 . The image panels illustrate colocalization of SFT2D2 - Myc and STX5 at the Golgi ( top , arrowhead ) but much more extensive colocalization of SFT2D2 - Myc and STX6 ( bottom , arrowheads ) . In ( B ) and ( C ) , the white dashed box delineates the area magnified in the insets . ( D ) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified . The graph shows the change in cellular intensity measured for each post - Golgi SNARE investigated . Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells . ( E ) Control and SFT2D2 KD HeLa cell lysates were separated by LDS - PAGE and blotted for the indicated SNARE proteins or actin .

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Figure 3(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.


Figure 4 ( A ) Primary screen anti - CD8 antibody - uptake images for control cells ( top ) and ZDHHC5 KD cells ( bottom ) . ( B ) Cells stably expressing ZDHHC5 - Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles . Some colocalization between ZDHHC5 and retromer VPS35 is observed ( arrowheads in inset ) . Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 ( arrowheads in inset ) . ( C ) Control and ZDHHC5 siRNA - treated SFT2D2 - Myc cells were mixed and stained for ZDHHC5 , Myc , and VPS35 . KD cells are marked by an asterisk . ( D ) Control and ZDHHC5 siRNA - treated HeLa cells were mixed and stained for ZDHHC5 , α5 - integrin , and VPS35 . KD cells are indicated with an asterisk . ( E ) Control ( top ) and ZDHHC5 KD ( bottom ) HeLa cells were fixed and stained for TGN46 and β1 - integrin .

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Figure 4(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.


Figure 5 ( A ) Primary screen anti - CD8 antibody uptake images for control cells ( top ) and GRINA KD cells ( bottom ) . ( B ) Control ( top ) and GRINA KD ( bottom ) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein - 1 ( GLG1 ) . ( C ) Cells stably expressing GFP - Rab6 were treated with GRINA siRNA ( bottom ) and compared to control cells ( top ) upon staining for TGN46 and VPS35 . ( D ) Quantitation by western blotting of lysates from control and GRINA - silenced HeLa cells and GFP - Rab6 cells . ( E - G ) HeLa cells were transiently transfected with GRINA - Myc for 24 hr before fixing and staining . ( E and F ) GRINA - Myc colocalizes with both TGN46 and VPS35 . In this example , GRINA - Myc expression ( in the cell marked by an asterisk in E ) reduced the cell ' s TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35 - positive endosomes . The area in ( E ) magnified in ( F ) is indicated by a dashed line . ( G ) GRINA - Myc expression also perturbs CIMPR localization . In this example , GRINA - Myc transfection ( in the cell marked by an asterisk ) caused CIMPR to localize to round vesicular structures positive for GRINA - Myc , some of which appeared larger than regular endosomes , whereas TGN46 staining was almost absent in the transfected cell .

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Figure 5(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.(E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.


Figure 6 ( A ) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide , lysed , and incubated with agarose - bound wheat germ agglutinin to capture glycosylated membrane proteins . Total cell lysates ( left ) and lectin pull - down samples ( right ) were assayed by western blotting . The experiment was repeated three times , and representative data are shown . ( B - E ) Quantitative analysis of SFT2D2 , ZDHHC5 , and GRINA KD cells immunofluorescence using automated microscopy ( see Experimental Procedures ) . ( B ) Representative images showing VPS35 , TGN46 , and CIMPR staining . Scale bar , 50 μm . ( C ) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2 , ZDHHC5 , or GRINA KD cells . ( D ) SFT2D2 , ZDHHC5 , and GRINA KD increase the Pearson ' s correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130 . In some cases , identical correlation coefficients were measured in the replicate experiments . ( E ) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells .

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Figure 6(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.(B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.


Figure 5 ( C ) Benign breast epithelial MCF10A and breast carcinoma BT - 474 cells were embedded in 3D collagen as single cells or as spheroids , respectively , and the growth was followed for 5 days . Light micrographs show filamentous actin ( phalloidin ) and nuclei ( Hoechst ) in representative cell colonies . Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout . At 72 hours , MCF10A mock versus ECHDC1 _ sgRNA _ 1 and ECHDC1 _ sgRNA _ 2 P < 0 . 05 ; at 96 hours mock versus ECHDC1 _ sgRNA _ 1 , ECHDC1 _ sgRNA _ 2 and ECHDC1 _ sgRNA _ 3 P < 0 . 001 ; at 120 hours mock versus ECHDC1 _ sgRNA _ 1 , ECHDC1 _ sgRNA _ 2 and ECHDC1 _ sgRNA _ 3 P < 0 . 0001 . Nuclei count relative to mock 0 hours . Error bars indicate mean ± SEM ; n ≥ 10 colonies . Statistical significance was assessed with one - way ANOVA with Tukey ' s multiple comparison test . Scale bar 50 µm . ( E ) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status ( n = 7 in both groups ) . Boxes represent the interquartile range , whiskers represent the range of the values and solid line within the box correspond to the median value . Outlier points indicates values not included between the whiskers . Statistical significance was assessed with Wilcoxon test .

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Figure 5(C) Benign breast epithelial MCF10A and breast carcinoma BT-474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 hours, MCF10A mock versus ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 hours. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one-way ANOVA with Tukey's multiple comparison test. Scale bar 50 µm.(E) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status (n=7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.


Figure 6 ( H ) Survival analysis based on mRNA expression levels of DDX27 in patients with endometrial cancer in the TCGA dataset . Expression levels were divided into 2 classes , high ( n = 203 ) and low ( n = 322 ) , based on mean expression level of DDX27 ( logFPKM = 18 . 27 ) . Patients in the high class showed lower survival probability than those in the low class ( P = . 4 . 2 × 10 - 4 ; log - rank test ) . ( I ) mRNA expression levels of DDX27 in PTEN mutated ( Mut , n = 302 ) and PTEN wild type ( WT , n = 224 ) endometrial patient tumors in the TCGA dataset . Triangles correspond to the median value . P - value was calculated with Wilcoxon test .

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Figure 6(H) Survival analysis based on mRNA expression levels of DDX27 in patients with endometrial cancer in the TCGA dataset. Expression levels were divided into 2 classes, high (n=203) and low (n=322), based on mean expression level of DDX27 (logFPKM=18.27). Patients in the high class showed lower survival probability than those in the low class (P=.4.2×10-4; log-rank test).(I) mRNA expression levels of DDX27 in PTEN mutated (Mut, n=302) and PTEN wild type (WT, n=224) endometrial patient tumors in the TCGA dataset. Triangles correspond to the median value. P-value was calculated with Wilcoxon test.


Figure 1 ( A ) Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01 . ( B ) Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01 . ( C ) Livedo reticularis in the left hand of Pt02 after the first infusion . ( D ) Brain MRI acquired one day after the MAB infusion showing acute small thalamic stroke in Pt03 . Axial diffusion - weighted imaging ( left ) and fluid - attenuated inversion recovery ( FLAIR ; right ) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke . ( E ) FLAIR MRI axial image obtained in Pt 03 one month after the acute stroke , showing the expected evolution of the right thalamic lesion .

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Figure 1(A) Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01.(B) Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01.(C) Livedo reticularis in the left hand of Pt02 after the first infusion.(D) Brain MRI acquired one day after the MAB infusion showing acute small thalamic stroke in Pt03. Axial diffusion-weighted imaging (left) and fluid-attenuated inversion recovery (FLAIR; right) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke.(E) FLAIR MRI axial image obtained in Pt 03 one month after the acute stroke, showing the expected evolution of the right thalamic lesion.


Figure 2 ( A ) Haematoxilin and Eosin staining of muscle biopsies from Pt 01 , Pt 02 and Pt 03 performed 2 months after the last MAB infusion . Images show diffuse increase of connective tissue , atrophic and hypertrophic fibers , degenerating fibers , and diffuse centralization of nuclei . ( B ) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion . Images show diffuse increase of connective tissue , atrophic and hypertrophic fibers , degenerating fibers , and diffuse centralization of nuclei . No evident differences were observed between biopsy performed after and before treatment . ( C ) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion . The staining was performed in tibialis anterior muscle for Pt 01 , Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06 . A higher number of positive ( brownish staining ) fibers ( representing regenerating fibers ) were observed in Pt 02 , Pt 05 and Pt 06 .

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Figure 2(A) Haematoxilin and Eosin staining of muscle biopsies from Pt 01, Pt 02 and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei.(B) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. No evident differences were observed between biopsy performed after and before treatment.(C) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion. The staining was performed in tibialis anterior muscle for Pt 01, Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06. A higher number of positive (brownish staining) fibers (representing regenerating fibers) were observed in Pt 02, Pt 05 and Pt 06.