Journal: Nature Communications

Article Title: Systems analysis of intracellular pH vulnerabilities for cancer therapy

doi: 10.1038/s41467-018-05261-x

Figure Lengend Snippet: Validation of systems analyses in predicting pHi-sensitive metabolic vulnerabilities. a pHi measurements of naïve and acid-adapted (AA) breast cancer MCF7 cells, under normoxia and following the inhibition of NHE1 , by cariporide treatment. For pHi measurments at least 30 cells were analyzed. b Efficiency of knockdown of indicated targets at the mRNA and protein levels, following reverse transfection of MCF7 cells with the indicated siRNAs. qPCR was repeated at least three times with three replicates. c The effect of gene inhibition in normal and low extracellular pH (pHe) shown for naïve and AA MCF7 breast cancer cells. Similar color code to Fig. is applied. At low pHe where the lowest pHi was obtained there is a large reduction in the viability of cells. In AA cells, only the selective and pH-specific targets ( GAPDH , GPI , and ACAT2 ) achieve amplified anti-proliferative effects following NHE1 inhibition, despite the smaller reduction in the pHi of these cells. PFAS , a selective but not pH-specific target, is similar to control cells following NHE1 inhibition. Knockdown of RPIA had a weak effect in naïve cells and no/opposite effects in AA cells. The viability assay was done three times with four replicates each time. The bars depict the mean and the error bars depict the standard deviation of the mean

Article Snippet: Membranes were incubated with primary antibodies against GAPDH (Cat# 2118 Cell Signaling, 1:2000), GPI (ab68643, Abcam, 1:1000), ACAT2 (Cat# 13294s Cell Signaling 1:1000), RPIA (ab181235, abcam, 1:500), PFAS (PA554628, Thermofisher, 1:200), MCT1 (sc-365501, Santa Cruz Biotechnology, 1:500), MCT4 (sc-376140, Santa Cruz Biotechnology, 1:500), and β-Actin (A5441, Sigma, 1:6000).

Techniques: Inhibition, Transfection, Amplification, Viability Assay, Standard Deviation

Journal: Science signaling

Article Title: The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A 2 coordinates acute wound healing and repair

doi: 10.1126/scisignal.aav5918

Figure Lengend Snippet: (A) Migration velocity of pDFs from WT and cPLA2α mice treated with ethanol or the indicated concentrations of PGE2, 5-HETE, or 20-HETE. (B) Migration velocity of pDFs from KO mice treated with increasing concentrations of 5-HETE. (C–E) Migration velocity of pDFs from WT, KI, and KO mice in the presence of pyrrophenone (C), MK886 (D), or the indicated combinations of pyrrophenone, MK886, and 5-HETE (E). Samples were compared using ANOVA followed by Tukey’s post-hoc test. Data shown are means ± SD, n = 3–10 cell isolates per genotype (3–8 mice per genotype were used to generate the cell isolates) for each treatment group. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

Article Snippet: For exogenous addition of bioactive lipids and small molecule inhibitor studies, once a confluent monolayer was achieved, cells were placed in 2% FBS, High Glucose DMEM media containing either the addition of various eicosanoids at the following concentrations (20 ng/mL PGE 2 , 0.25ng/mL 5-HETE, 0.5ng/mL 5-HETE, 1.0 ng/mL 5-HETE, and 5.0 ng/mL 20-HETE) or MK886 (7.5 nM) (Cayman Chemical), pyrrophenone (50 nM) (Cayman Chemical), or a combination of MK886/pyrrophenone and 5-HETE (1.0 ng/ml) for two hours.

Techniques: Migration

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: Septins associate with lysosomes and promote lysosome positioning in the perinuclear cytoplasm. (A and B) Confocal images of COS-7 cells stained for SEPT9 (inverted magenta) and LAMTOR4 (inverted green; A) or EEA1 (inverted green; B). Scale bars, 20 µm. Insets show lysosomes from perinuclear regions in higher magnification (scale bars, 3 µm). Arrowheads point to SEPT9 puncta that localize on LAMTOR4-stained lysosomes. (C) Bar graph shows percentage (mean ± SEM; Mann–Whitney U test) of LAMTOR4 and EEA1 organelles with SEPT9 puncta per cell (n = 6 cells). (D) Super-resolution structured illumination microscopy images show SEPT9 puncta on the delimiting membrane of LAMP2-mCherry–labeled compartments in BSC-1 cells. (E and F) Spinning-disk confocal microscopy image (E) and still frames from time-lapse imaging (F) of COS-7 cells transfected with SEPT9-mCherry and LAMP1-mGFP. Insets show higher-magnification LAMP1-mGFP compartments with SEPT9-mCherry puncta (arrows; scale bars, 1 µm), and arrowheads point to retrograde cotraffic toward the nucleus. Scale bar, 5 µm. (G) Color-coded trajectory of the SEPT9-positive lysosome moving retrogradely in F. Arrowheads point to the early (blue), medial (green), and late (red) stages of movement. (H) COS-7 cells were transfected with mCherry or SEPT9-mCherry and stained with anti-LAMP1. Scale bars, 20 µm. (I) Plot shows the ratio (mean ± SEM) of perinuclear to peripheral LAMP1 fluorescence intensity per cell ( n = 70–79; Mann–Whitney U test). (J) Images of LAMP1-stained COS-7 cells after transfection with plasmids coexpressing scramble control or SEPT9-targeting shRNAs and GFP or SEPT9-GFP. Arrows point to lysosomes (LAMP1) with SEPT9-GFP. Scale bars, 20 µm. (K) Plot shows the ratio (mean ± SEM; Mann–Whitney U test) of perinuclear to peripheral LAMP1 fluorescence intensity per cell ( n = 45–47). (L) Images show lysosome (LAMP-1) distribution in COS-7 cells transfected with mCherry and GFP, SEPT9-mCherry and GFP-Rab7-DN, or SEPT9-mCherry and GFP. Scale bars, 20 µm. (M) Quantification shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral LAMP1 fluorescence intensity per cell ( n = 45–51). (N) Embryonic (E18) hippocampal neurons (DIV4) were transfected with LAMP1-mGFP and mCherry or rat SEPT9-mCherry, and axons were imaged live by TIRF microscopy. Kymographs show stationary or diffusive (blue), retrogradely (magenta), or anterogradely (green) moving particles with LAMP1-mGFP. Scale bar, 5 µm. (O) Bar graph shows the ratio (mean ± SEM; unpaired t test) of retrogradely to anterogradely moving LAMP-1-mGFP particles/2 min per axon ( n = 18–19). ns, nonsignificant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Staining, MANN-WHITNEY, Microscopy, Labeling, Confocal Microscopy, Imaging, Transfection, Fluorescence

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: Septin localization and effects on early endosomes and lysosomes. (A) Spinning-disk confocal image shows the distribution of SEPT9-mCherry, which was expressed under the PGK promoter, with respect to GFP-EEA1 in living COS-7 cells. Scale bar, 20 µm. Perinuclear area is shown in higher magnification. Scale bar, 5 µm. (B) Quantification shows the fraction (mean ± SEM; Mann–Whitney U test) of SEPT9-mCherry puncta colocalizing with LAMP1-mGFP and GFP-EEA1 in COS-7 cells ( n = 6). (C and D) PNSs of HEK-293 cell extracts were loaded on an iodixanol OPTIPREP density gradient (17%, 20%, 23%, 27%, and 30%), and fractions were blotted (C) for LAMP-1, Rab7, Rab5, GM130, BIP, SEPT9, SEPT2, SEPT6, and SEPT7. Plots (D) show quantification of indicated proteins in each fraction as percentage of total proteins across all fractions. Horizontal dashed lines provide a reference point for the percentage of septin protein in the top fraction. (E and F) Images (E) show the distribution of early endosomes (EEA1) in COS-7 cells that overexpress mCherry or SEPT9-mCherry (insets). Scale bars, 20 µm. Quantification (F) shows the ratio (mean ± SEM; unpaired t test) of perinuclear to peripheral fluorescence intensity of EEA1 per cell ( n = 20). (G and H) Images (G) of LAMP1-stained COS-7 cells expressing mCherry, SEPT2-mCherry, or mCherry-SEPT6 (insets). Scale bars, 20 µm. Plot (H) shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral fluorescence intensity of LAMP1 per cell ( n = 30–34). (I and J) Images (I) of COS-7 cells, which were transfected with plasmids that coexpress GFP and scramble or SEPT9 shRNA and stained with anti-SEPT9 antibody. Scale bars, 20 µm. Bar graph (J) shows fluorescence intensity (mean ± SEM; Mann–Whitney U test) of SEPT9 per shRNA-expressing cell ( n = 27–35). A.U., arbitrary units. (K and L) Images (K) of axon segments in hippocampal neurons (DIV4), which were cotransfected with rat SEPT9-mCherry and GFP-Rab5A or LAMP1-mGFP. Scale bars, 5 µm. Plot (L) shows percentage (mean ± SEM; Mann–Whitney U test) of GFP-Rab5A ( n = 13 axons) and LAMP1-mGFP ( n = 17 axons) puncta with SEPT9-mCherry per cell. ns, nonsignificant (P > 0.05); **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: MANN-WHITNEY, Fluorescence, Staining, Expressing, Transfection, shRNA

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: Membrane-associated SEPT9 recruits dynein and induces retrograde dynein-driven transport. (A) Images show COS-7 cells that were transfected with GFP-SEPT9-FRB and PEX-RFP-FKBP after 45 min of treatment with rapalog or control carrier. Bar graph shows percentage of cells ( n = 37–42; χ 2 test) with dispersed, partially clustered, and clustered peroxisomes. Scale bar, 20 µm. (B) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with DAPI. Bar graph shows perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell ( n = 20–44). Scale bar, 20 µm. (C) COS-7 cells were transfected with Mito-TagRFP or SEPT9-MitoTagRFP, treated with nocodazole (33 µM) or DMSO for 3 h, and stained with anti-GM130 and DAPI. Quantification shows the amount of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell ( n = 53–54). Scale bar, 10 µm. (D) Images show DAPI-stained COS-7 cells that were transfected with SEPT9-MitoTagRFP and GFP (control) or GFP-p50 (dynamitin). Bar graph shows quantification of perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total per cell ( n = 55–70). Scale bars, 20 µm. (E) COS-7 cells were transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with anti-DIC after 3-h treatment with DMSO or nocodazole (33 µM). Plot shows DIC fluorescence intensity (mean ± SEM) on MitoTagRFP-labeled mitochondria per cell ( n = 9–16; Kruskal–Wallis one-way ANOVA). Scale bars, 20 µm. (F) Lysates of HEK-293 cells expressing mCherry or mCherry-SEPT9 were incubated with mRFP trap beads, and immunoprecipitates were blotted with antibodies against mCherry (top), DIC, and p150 GLUED . A.U., arbitrary units. (G) Coomassie-stained gel (top) shows recombinant SEPT9 and SEPT2/6/7, which were used in pull-down assays with dynein–dynactin purified from HEK-293 cells. Western blots (bottom) were performed with antibodies against DHC and p150 GLUED . *, P < 0.05; *** P < 0.001.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Transfection, Staining, Fluorescence, Labeling, Expressing, Incubation, Recombinant, Purification, Western Blot

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: Targeting of septins to mitochondria induces perinuclear clustering in a SEPT9-dependent manner. (A and B) Images (A) show localization of MitoTagRFP- and SEPT9-MitoTagRFP-labeled mitochondria in living COS-7 cells, which were labeled with MitoSpy as an indicator of cell and mitochondria health. Plot (B) shows the fluorescence intensity (mean ± SEM; Mann–Whitney U test) of MitoSpy per mitochondria (MitoTagRFP) surface area per cell ( n = 13–19). Scale bars, 20 µm. (C and D) Images (C) show DAPI-stained COS-7 cells, which were transfected with MitoTagRFP (control) or MitoTagRFP-tagged SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. Bar graph (D) shows percentage (mean ± SEM; two-way ANOVA; n = 20–28 cells) of total mitochondria fluorescence intensity in the perinuclear and peripheral cytoplasm. (E) Images show COS-7 cells transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with antibodies against SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. (F) Images of DAPI-stained COS-7 cells after cotransfection with scramble or SEPT9 shRNAs (GFP) with MitoTagRFP-tagged SEPT2, SEPT6, or SEPT7. Scale bars, 20 µm. Bar graph shows the percentage of perinuclear and peripheral mitochondria (mean ± SEM; two-way ANOVA; n = 24–35 cells) in the indicated conditions. ns, nonsignificant (P > 0.05); ***, P < 0.001.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Labeling, Fluorescence, MANN-WHITNEY, Staining, Transfection, Cotransfection

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: SEPT9 provides a molecular link between dynein and dynactin through its GTP-binding and NTE domains, respectively. (A) Coomassie-stained gel shows the recombinant NTE and G domains of SEPT9, which were used in pull-down assays with purified dynein and dynactin. Pull-downs were Western blotted with antibodies against DHC and p150 GLUED . Bar graph shows the mean ratio of DHC to NTE or G domain and p150GLUED to NTE or G domain fragments of His-SEPT9 intensities from three independent experiments; error bars indicate SEM. Schematic depicts the domains of SEPT9 and the position of the catalytic T339 residue required for GTPase activity. (B) Coomassie-stained gel shows the results of protein binding assays between recombinant His-SEPT9 and GST (control) or the GST-tagged N-terminal (aa 1–389) and C-terminal (aa 389–523) halves of DLIC. Bar graph shows the mean ratio of SEPT9 to GST or GST-DLIC protein band intensities from three independent experiments; error bars indicate SEM. Schematic depicts the GTPase-like and adaptor-binding domains of DLIC. (C) Coomassie-stained gel shows results from pull-down assays of His-SEPT9 with GST (control) or GST-tagged DIC(1–108), DIC(108–268), and DIC(1–268). Schematic shows the major domains of DIC and their corresponding interactions with components of the dynein–dynactin complex. Bar graph shows quantification of the relative amount of SEPT9 pulled down as the ratio (mean ± SEM) of SEPT9 to GST protein band intensities from five independent experiments. (D) Coomassie-stained gel (top) and Western blot (bottom; anti-His) show results from pull-down assays of recombinant NTE and G domain fragments of His-SEPT9 with GST-DIC(108–268). (E) Bar graph shows the mean ratio of NTE or G domain fragments of SEPT9 to GST-DIC(108–268) protein band intensities from three independent experiments; error bars indicate SEM. (F) Gel shows the input and results from protein binding assays of equimolar (0.5 µM) recombinant p150 GLUED -CC1-Halo-His with GST-DIC(1–268) in the presence of increasing concentrations of His-mCherry-SEPT9 (0, 5, and 10 µM). Bar graph shows the relative increase in the amount of DIC(1–268)-bound p150 GLUED -CC1 with increasing concentrations of SEPT9 from three independent experiments; ratio was set to 100 for 0 µM of SEPT9. Error bars indicate SEM.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Binding Assay, Staining, Recombinant, Purification, Western Blot, Activity Assay, Protein Binding

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: The GDP-bound state of SEPT9 recruits dynein and induces retrograde transport. (A) Coomassie-stained gel (top) and Western blot (bottom; anti-His) show binding of the G domain of SEPT9 to GST-DIC(108–268) in the presence of GDP, GTP, or GTPγS (0.5 mM). Quantification shows the relative ratios of SEPT9 G domain to GST-DIC(108–268) protein band intensities from three independent experiments. (B) Gels show Western blots (anti-GST/anti-His) of pull-downs of recombinant His-tagged wild-type (wt) or GTPase-dead (T339G) SEPT9 with GST or GST-DIC(108–268). Bar graph shows the relative ratio of the wild-type or GTPase-dead (T339G) G domain of SEPT9 to GST-DIC(108–268) from four independent experiments. Error bars indicate SEM. (C) Images show DAPI-stained COS-7 cells transfected with mitochondria-targeted (Mito-TagRFP) wild-type and GTPase-dead (T339G) SEPT9. Scale bars, 20 µm. (D) Bar graph shows perinuclear and peripheral mitochondria as percentage (mean ± SEM; two-way ANOVA) of total mitochondria per cell ( n = 24–27). Error bars indicate SEM. (E) Images show COS-7 cells transfected with mitochondria-targeted (MitoTagRFP) wild-type or GTPase-dead (T339G) SEPT9 and stained with an antibody against DIC. Scale bars, 20 µm. (F) Plot shows DIC fluorescence intensity (mean ± SEM; Mann–Whitney U test) on mitochondria with SEPT9-Mito-TagRFP or SEPT9(T339G)-Mito-TagRFP per cell ( n = 40–41). (G) Images show the distribution of lysosomes (LAMP-1) in COS-7 cells transfected with wild-type SEPT9-mCherry or SEPT9(T339G)-mCherry (insets). Scale bars, 20 µm. (H) Plot shows the ratio (mean ± SEM; unpaired t test) of perinuclear to peripheral LAMP1 fluorescence intensity per cell ( n = 21–22). **, P < 0.01; ***, P < 0.001. A.U., arbitrary units.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Staining, Western Blot, Binding Assay, Recombinant, Transfection, Fluorescence, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: Oxidative stress increases lysosomal levels of SEPT9, which promotes perinuclear clustering. (A) Confocal microscopy images (inverted monochrome) of COS-7 cells treated with NaAsO 2 (300 µM) for 30 min or deprived of glucose for 1 h and stained for LAMTOR4 (inverted green) and SEPT9 (inverted magenta). Scale bars, 20 µm. Insets show the outlined perinuclear regions in higher magnification. Scale bars, 5 µm. (B) Bar graph shows the percentage (mean ± SEM; unpaired t test) of total lysosomal area (LAMTOR4) that overlaps with endogenous SEPT9 per cell ( n = 40–59) after NaAsO 2 treatment (300 µM, 30 min) or glucose deprivation (1 h). (C) COS-7 cells were transfected with plasmids that coexpress scramble control or SEPT9 shRNAs and GFP or shRNA-resistant SEPT9-GFP (wild type or T339G mutant). Images show lysosome (LAMP1) localization (green) and GFP fluorescence (magenta) after treatment with NaAsO 2 (300 µM) for 2 h. Arrows point to peripheral clusters of lysosomes. Scale bars, 20 µm. (D and E) Plots show the fraction (mean ± SEM; extra sum of squares F test) of total LAMP1 intensity across the distance of the cell radius, from the cell center to the periphery, after normalization for the length of each cell ( n = 31–37). (F and G) HeLa cells were transfected with plasmids that coexpress scramble control or SEPT9 shRNAs and GFP or shRNA-resistant SEPT9-GFP (wild type or T339G mutant). Cells were stained with anti-LAMP1 after treatment with NaAsO 2 (400 µM) for 1 h. Plots show the fraction (mean ± SEM; extra sum of squares F test) of total LAMP1 intensity across the distance of the cell radius after normalization for the length of each cell ( n = 55–64). ns, nonsignificant (P > 0.05); ***, P < 0.001.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Confocal Microscopy, Staining, Transfection, shRNA, Mutagenesis, Fluorescence

Journal: The Journal of Cell Biology

Article Title: A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

doi: 10.1083/jcb.202005219

Figure Lengend Snippet: SEPT9 promotes perinuclear clustering of lysosomes in HeLa cells under oxidative stress. (A) Images show SEPT9-stained HeLa cells after transfection with plasmids that coexpress GFP (magenta) and scramble or SEPT9 shRNAs. Scale bars, 20 µm. (B) Bar graph shows mean (± SEM; Mann–Whitney U test) fluorescence intensity of SEPT9 per shRNA-expressing cell ( n = 65–66). ***, P < 0.001. A.U., arbitrary units. (C) HeLa cells were transfected with plasmids that coexpress shRNAs (scramble control or SEPT9) and GFP or shRNA-resistant SEPT9-GFP (wild type or T339G mutant; magenta). After treatment with NaAsO 2 (400 µM) for 1 h, cells were stained with antibodies against LAMP1 (green). Representative images show LAMP1 (green) and GFP or SEPT9-GFP (magenta). Arrows point to lysosomes that localize to the periphery of SEPT9-depleted and SEPT9(T339G)-GFP–expressing cells. Quantifications of lysosome localization are shown in . Scale bars, 20 µm.

Article Snippet: Cells were immunostained with the following antibodies: mouse anti-Lamp1 (1:100; H4A3 clone; Iowa Developmental Studies Hybridoma Bank), rabbit anti-SEPT9 (1:100; 10769-1-AP; Proteintech Group), rabbit anti-SEPT9 (1:200; NBP2-13294; Novus Biologicals), mouse anti-SEPT9 clone 10C10 (1:100; MABE992; Millipore-Sigma), rabbit anti-SEPT2 (N5N; 1:300; gift from Makoto Kinoshita, Nagoya University, Nagoya, Japan), rabbit anti-SEPT7 (1:300; 18991; IBL America), rabbit anti-SEPT6 (1:300; gift from Makoto Kinoshita), mouse anti-GM130 (1:200; 610823; BD Biosciences), mouse anti-DIC antibody clone 74.1 (1:100; MAB1618; Millipore), rabbit anti-LAMTOR4 (1:200; 12284S; Cell Signaling), and mouse anti-EEA1 clone 14/EEA1 (1:100; 610456; BD Biosciences).

Techniques: Staining, Transfection, MANN-WHITNEY, Fluorescence, shRNA, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: SNX25 regulates proinflammatory cytokine expression via the NF-κB signal in macrophages

doi: 10.1371/journal.pone.0247840

Figure Lengend Snippet: SNX25 inhibits ubiquitination of IκBα and the dissociation of IκBα/NF-κB (p65 and p50) complex and prevents nuclear translocation of NF-κB and subsequent cytokine expression.

Article Snippet: After blocking with 5% non-fat milk in phosphate-buffered saline with 0.05% Tween 20 for 1.5 h, proteins of interest were allowed to bind, during an overnight incubation at 4°C, with anti-SNX25 (Proteintech, USA, 13294-1-AP, 1:1000), anti-IL-1β (abcam, UK, ab9722, 1:1000), anti-IL-6 (PeproTech, USA, 500-P56, 1:200), anti-TNF-α (Cell Signaling Technology, USA, #11948, 1:1000), anti-IκBα (Cell Signaling Technology, #9242, 1:1000), anti-phospho-IκBα (Cell Signaling Technology, #9246 1:1000), anti-p65 (Cell Signaling Technology, #8242, 1:1000), anti-phospho-p65 (Cell Signaling Technology, #3033, 1:1000), anti-JNK (Cell Signaling Technology, #9258, 1:1000), anti-phospho-JNK (Cell Signaling Technology, #4668, 1:1000), anti-p38 (Cell Signaling Technology, #9212, 1:1000), anti-phospho-p38 (Cell Signaling Technology, #4511, 1:1000), anti-ERK (Cell Signaling Technology, #4695, 1:1000), anti-phospho-ERK (Cell Signaling Technology, #4370, 1:1000), anti-phospho-IKKβ (Cell Signaling Technology, #2078, 1:1000), anti-α-Tubulin (Sigma-Aldrich, T5168, 1:4000), anti-LaminB1 (Proteintech, 12987-1-AP, 1:1000) or anti-GAPDH (Millipore, USA, ABS16, 1:2000) antibody.

Techniques: Translocation Assay, Expressing