Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet: ( A,B ) qPCR quantification (2 -ΔCt ) of RNA levels in mCherry+ macrophages FACS sorted from srpHemo-3xmCherry wild type embryos reveals no significant change in the expression of ( A ) the C1GalTA galactose transferase or ( B ) the Ugalt Gal transporter during Stage 9–17 (n = 7 biological replicates, three independent FACS sorts). ( C ) Schematic made with Protter showing the predicted 12 transmembrane domains of CG8602. Blue lines indicate regions displaying higher than 20% identity to the correspondingly numbered Drosophila protein indicated below, along with the homologous protein’s predicted or determined function. ( D ) Quantification by qPCR of CG8602 RNA levels in FACS sorted mCherry+ macrophages compared to other mCherry- cells obtained from srpHemo-3xmCherry wild type embryos at Stage 9–10, Stage 12 and Stage 13–17. CG8602 macrophage expression peaks at Stage 12, during macrophage germband entry (n = 3–7 biological replicates, four independent FACS sorts, p=0.036). ( E ) qPCR quantification in FACS sorted srpHemo-3xmCherry labeled macrophages from control and CG8602 EP3102 mutant Stage 12 embryos shows an extremely strong decrease in CG8602 RNA expression in the P element insertion mutant used in this study (n = 7 biological replicates, three independent FACS sorts, p=0.0024). ( F ) Confocal images of Stage 12 control and CG8602 EP3102 mutant embryos with macrophages (red) visualized by srpHemo-mCherry expression and T antigen by antibody staining (green). Schematic at left depicts macrophages (red) entering the germband. Black box indicates the region next to the germband imaged at right. We observe decreased T antigen staining on macrophages in the CG8602 EP3102 mutant compared to the control. ( G ) qPCR quantification (2 -ΔCt ) of C1GalTA and Ugalt RNA levels in FACS sorted macrophages from Stage 12 embryos from control and CG8602 EP3102 mutant embryos shows no significant change in expression of the Gal transferase, or the Gal and GalNAc transporter in the mutant compared to the control (n = 7 biological replicates, three independent FACS sorts). ( H ) Quantitation using Fiji of the colocalization of transfected MT-CG8602::FLAG::HA in fixed S2R+ cells with markers for the ER (Cnx99a), the Golgi (Golgin 84, Golgin 245, and GMAP), the early endosome (Hrs), the late endosome (Rab7), and live S2R+ cells transfected with srp-CG8602::3xmCherry with dyes that mark the lysosome (Lysotracker) and the nucleus (DAPI). Representative images are shown in . n = 24, 23, 23, 17, 6, 22, 6 and 13 cells analyzed per respective marker. ( I ) Macrophages near the germband extracted from srpHemo >CG8602 HA Stage 11/12 embryos show partial colocalization of the HA antibody labeling CG8602 (red) and a Golgin 84 or Hrs antibody marking the Golgi or endosome respectively (green). Nucleus is stained by DAPI (blue). For all qPCR experiments values are normalized to expression of a housekeeping gene RpL32. Scale bars are 5 μm in F, 3 μm in I . Significance was assessed by Kruskal-Wallis test in A, B , One way Anova in D and Student’s t-test in E, G . ns = p > 0.05, *p<0.05, ***p<0.001. See also . 10.7554/eLife.41801.008 Figure 2—source data 1. Source data on the quantification of C1GalTA, Ugalt, and CG8602 expression in macrophages by qPCR (shown in ) and the Pearson’s coefficient for CG8602 colocalization with different markers .

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Expressing, Labeling, Control, Mutagenesis, RNA Expression, Staining, Quantitation Assay, Transfection, Marker, Antibody Labeling

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet: ( A ) Schematic depicting the CG8602 gene and the insertion site of the EP3102 P element and the Δ33 excision mutant induced by P element mobilization which removes 914 bp of the ORF. ( B–J ) Confocal images of S2R+ cells transfected with ( B–G ) MT-CG8602::FLAG::HA and then fixed and visualized by HA antibody staining (red) or ( H–J ) srpHemo-CG8602::3xmCherry ( red) with different parts of the endomembrane system visualized by live markers as indicated (green). DAPI (blue) marks the nucleus. CG8602 showed ( B ) no colocalization with the ER marker Calnexin, partial colocalization with the ( C,D ) Golgi markers GMAP and Golgin 245, ( E ) late endosomal marker Rab7, ( F ) recycling endosome marker Rab11-YFP, and ( G ) endosomal marker Hrs8.2, no colocalization with the ( H ) lysosome marker lysotracker, ( I ) mitochondrial marker mitotracker and ( J ) peroxisomal marker PTS1-GFP in fixed ( B–G ) or live ( H–J ) S2R+ cells. Scale bar is 3 μm in B-J.

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Mutagenesis, Transfection, Staining, Marker

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet: ( A ) Representative confocal images of early Stage 12 embryos from control and P{SUPor-P}Qsox1KG04615 = qsox1 KG04615 . ( B–C ) Quantification in early Stage 12 embryos showing a significant reduction in germband macrophages ( B ) in the P-element mutant qsox1 KG04615 located in the Qsox1 5’UTR (n = 18, p=0.0012) and ( C ) upon the expression in macrophages under srpHemo-GAL4 control of an RNAi line ( v108288 ) against Qsox1 (n = 24, 23 embryos, p=0.001). ( D ) Images from two-photon movies from control and qsox1 KG04615 . Macrophage nuclei (red) are labeled with srpHemo-H2A::3xmCherry. Stills at 0, 60, 90, 120, 150 and 180 min. ( E ) Quantification of macrophage speed reveals 18% slower macrophage migration in the head towards the yolk neighboring the germband in the qsox1 KG04615 mutant compared to the control (n = 3 movies for each, #tracks: control = 329, mutant = 396, p=0.0056). ( F ) Quantification of the time required for macrophage entry into the germband in qsox1 KG04615 compared to the control. n = 3 movies for each, p=0.043. ( G ) Quantification of macrophage speed in the germband in the qsox1 KG04615 mutant compared to the control (n = 3 movies for each, #tracks: control = 21, mutant = 19, p=0.300). ( H ) Pearson’s Coefficient analysis indicating the level of colocalisation of a MT-Qsox1::FLAG::HA construct transfected into S2R+ cells visualized with an HA antibody and antibodies against markers for the ER (Cnx99a), the Golgi (Golgin 84, Golgin 245, and GMAP), the early endosome (Hrs), the late endosome (Rab7) and the nucleus (DAPI) (n = 11–15) as well as with a srpHemo-mrva::3xmCherry construct (n = 18). ( I ) Western blot of concentrated supernatant collected from S2R+ cells transfected with srpGal4 UAS-qsox1::FLAG::HA (first three lines) and S2R+ cells that are untransfected. ( J ) Quantification of intracellular LanA intensity along a 4 μm line in macrophages (as indicated in schematic) from the control (black), minerva 3102 (blue) and the qsox1 KG04615 mutants (orange) (n = 4–5 embryos, 80–100 cells, 240–300 lines). For the whole graph see . Scale bars 50 μm for A, 30 μm in D. B-C, E-G and J were analyzed with Student’s test. ns = p > 0.05, *p<0.05, **p<0.01, ***p<0.001. See also . 10.7554/eLife.41801.020 Figure 5—source data 1. Source data on the quantification of macrophages in the germband shown in , on the yolk shown in , on the vnc shown in , and in the whole embryo shown in . Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in . Source data on the quantification of the Pearson's coefficient for Qsox1 colocalization with different markers shown in and the quantification of LanA intensity shown in and . Source data on the xyz position of macrophages in the movies of the qsox1 KG04615 mutant underlying the analysis shown in and .

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Control, Mutagenesis, Expressing, Labeling, Migration, Construct, Transfection, Western Blot

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet: ( A ) Topology prediction of mouse MFSD1 (NP_080089.1) using the online tools TMPred and Protter . 50% of amino acids are identical between the M. musculus MFSD1 and D. melanogaster sequence of mrva (CG8602) (NP_648103.1) and are highlighted in dark blue, similar amino acids are in light blue. ( B ) Confocal images of MC-38 colon carcinoma cells showing colocalization of MFSD1-eGFP (green) with the Golgi marker GRASP65 (red). DAPI labels the nucleus (blue). ( C ) Quantitation using Fiji of the colocalization of MFSD1-eGFP with the Golgi marker (GRASP65), early endosome marker (Rab5), late endosome marker (Rab7), and lysosome marker (LAMP1) in MC-38 colon carcinoma, B16-BL6 melanoma, LLC1 Lewis lung carcinoma, and 4T1 breast carcinoma cells. Representative images are shown in (n = 8–15, 5–9, 4–9, 5–10 cells per condition within the respective cancer types). ( D ) Confocal image of a Stage 12 fixed embryo showing that expression of mmMFSD1 in macrophages under the direct control of the srpHemo(macro) promoter in the mrva 3102 mutant can rescue the defect in macrophage migration into the germband. Compare to . Macrophages visualized with srpHemo-H2A::3xmCherry for D-E. ( E ) Quantitation of the number of macrophages in the germband of early Stage 12 embryos from the control (n = 25), mrva 3102 mutants (n = 29), and mrva 3102 srpHemo(macro)-mmMFSD1 (n = 13, p=0.0005 for mutant vs control, p<0.0001 for mutant vs rescue). ( F ) Quantification of T antigen levels on macrophages in late Stage 11 embryos from control, mrva 3102 mutant and mrva 3102 srpHemo(macro)-mmMFSD1 embryos. T antigen levels normalized to those observed in the control (n = 8–9 embryos, 280, 333, and 289 cells quantified respectively, p<0.0001 for both). ( G ) Confocal images of macrophages (red) on the germband border stained with T antigen antibody (green) in the control, the mrva 3102 mutant, and mrva 3102 srpHemo(macro)-mmMFSD1 shows that mmMFSD1 expression in macrophages can rescue the decrease of macrophage T antigen observed in the mrva 3102 mutant. Macrophages visualized with srpHemo-3xmCherry for F-G. ( H ) Model for Minerva’s function during macrophage invasion based on our findings and the literature: Minerva in the Golgi (grey) leads to increases in T antigen levels on a subset of proteins that aid invasion, including Qsox1 which regulates protein folding through disulfide bond formation and isomerization. We propose that increased T antigen on Qsox1 facilitates its sulfhydryl oxidase activity that aids the formation of a robust crosslinked ECM which macrophages utilize during tissue entry. Significance was assessed by Kruskal-Wallis test with Conover post test analysis in E,F ). ***p<0.001, ****p<0.0001. Scale bars are 10 μm in B, 50 μm in D, and 3 μm in G. See also . 10.7554/eLife.41801.024 Figure 6—source data 1. Source data on the quantification of Pearson’s coefficient for MFSD1 colocalization with different markers , the number of macrophages in the germband and the level of T antigen in macrophages .

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Sequencing, Marker, Quantitation Assay, Expressing, Control, Mutagenesis, Migration, Staining, Activity Assay

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet: ( A ) Alignment of Minerva and mmMFSD1 by BLAST. The first row shows the Minerva sequence in blue type, the second identical (one letter symbol) or similar (+) amino acids in black, and the third the mmMFSD1 sequence in green. Gaps are marked with ‘-‘. The predicted twelve transmembrane domains of Minerva are shown with dark blue lines and numbered above. ( B ) Western blot of control (-) and Doxycycline induced (+) MFSD1-eGFP expression in MC-38 colon carcinoma cells. MFSD1-eGFP was detected with an anti-GFP antibody. GAPDH serves as a loading control. ( C–F ) Representative images from co-immunofluorescence of mouse MFSD1-eGFP (green) and the Golgi marker GRASP65, early endosome marker Rab5 or late endosome marker Rab7 (red) in ( C ) MC-38 colon carcinoma, ( D ) 4T1 breast cancer, ( E ) LLC1 Lewis lung carcinoma, and ( F ) B16 BL6 cells. Nuclei are labeled with DAPI (blue). Scale bars indicate 10 μm in C-F.

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Sequencing, Western Blot, Control, Expressing, Immunofluorescence, Marker, Labeling

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet:

Article Snippet: Anti Rab7 (mouse monoclonal) , DSHB, RRID: SCR_013527 , DSHB Cat# Rab7, RRID: AB_2722471 , (1:5).

Techniques: Amplification, Clone Assay, cDNA Library Assay, Expressing, Plasmid Preparation, Staining, Software

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: RNAi-mediated depletion of Rab7 causes Vps26 dissociation from endosomes. (A) HeLa cells were treated twice at 24-h intervals with a control, inactive siRNA, or siRNAs to Rab7, Rab4 (targeting both the a and b isoforms), or Vps26. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies to Rab7, Rab4, Vps26, or actin (loading control) as indicated in the figure. (B–Q) HeLa cells treated as in A with control (B, C, F, G, J, K, N, and O) or Rab7 siRNA (D, E, H, I, L, M, P, and Q) were immunostained for Vps26 (B, D, F, H, J, L, N, and P) and AP-3 (C and E), AP-1 (G and I), SNX1 (K and M), and SNX2 (O and Q) using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to AP-3–δ, AP-1–γ1, SNX1, or SNX2 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 488–conjugated donkey anti–mouse IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, SDS Page, Western Blot, Bioprocessing, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Specificity and rescue of Vps26 dissociation from membranes in RNAi-treated cells. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A) or siRNAs to both Rab4a and Rab4b (B) or to Rab7 (C–F). At 36 h after the second treatment with siRNA to Rab7, some cells were transfected with plasmids encoding RNAi-resistant (i.e., canine) GFP-Rab7 (C and E) or GFP (D and F). Examples of cells expressing GFP or canine GFP-Rab7 are indicated by arrows. The cellular distribution of Vps26 (A–D) was assessed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Transfection, Expressing, Staining, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Depletion of Rab7 blocks the transport of CI-MPR from endosomes to the TGN. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A, C, E, and G) or siRNA to Rab7 (B, D, F, and H–N). At 48 h after treatment, the steady-state distribution of CI-MPR (A–D) was assessed by indirect immunofluorescent staining of fixed cells using rabbit polyclonal antibody to cytosolic tail of CI-MPR followed by Alexa Fluor 488–conjugated donkey anti–rabbit IgG. C and D correspond to high magnification views of CI-MPR–positive structures from control or Rab7-depleted cells, respectively. Live control cells (E and G) or Rab7-depleted cells (F and H–N) were incubated with an antibody to the luminal domain of CI-MPR for 2 h at 37°C. Cells were washed, fixed, permeabilized, and stained with Alexa Fluor 488–conjugated donkey anti–mouse IgG to detect internalized antibody to CI-MPR (E–H, J, and M). G and H show high magnification views from control (E) or Rab7-depleted cells (F), respectively. Cells in I–N were additionally stained with rabbit polyclonal antibody to TfR (I–K) or giantin (L–N) followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images in A–H were captured using an epifluorescence microscope, and images in I–N were captured with a confocal microscope. (K and N) For merged images, yellow indicates colocalization. Arrows in I–K indicate examples of foci where proteins colocalize. (O) Extracts of HeLa cells treated with siRNAs to the proteins indicated on top were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting (IB) with antibodies to the proteins indicated on the right. Equal amounts of total protein were loaded. Bars: (A, B, E, and F) 15 μm; (C, D, G, and H) 1.5 μm; (I–N) 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Staining, Incubation, Microscopy, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Rab7 depletion impairs processing of cathepsin D. HeLa cells were treated twice at 24-h intervals with inactive siRNA (control; lane 1) or siRNAs to the proteins indicated on top. At 24 h after the second round of siRNA treatment, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid, and the resulting pellets were dissolved in Laemmli sample buffer. Cell extracts and media samples were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting with rabbit polyclonal antibody to cathepsin D. Equal amounts of total protein were loaded on each lane. Blots were also probed with antibody to actin as a loading control. The positions of molecular mass markers (in kilodaltons) and of the precursor (pCatD), intermediate (iCatD), and mature (mCatD) forms of cathepsin D are indicated.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Incubation, SDS Page, Western Blot

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Differential gene expression data (RNA-seq) for the GDI2 / RAB5A / RAB7A / ERBB2 / ITGB6 cluster in normal breast tissue ( n = 403; light gray) and breast invasive carcinoma ( n = 1097; dark gray). Data were extracted from the TNMplot database ( tnmplot.com ). Black lines in violin blots represent the median. Mann-Whitney test. ( B ) Volcano plot showing statistical analysis (ANOVA) of RNA-seq gene expression data of patients with HER2+ breast cancer from the METABRIC cohort expressing high (Right) and low (Left) levels of ITGB6 (Q1 versus Q4). Significant genes (dark gray); nonsignificant genes (light gray); relevant genes are highlighted in purple. ( C ) Visual representation of GO terms analysis (ClueGO, cellular compartment) of genes highly and significantly expressed in tumors expressing high levels of ITGB6 (Q4). Colors represent specific merged GO term groups, node size represents the level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. ( D ) OS of patients with HER2+ breast cancer and with high (above median) expression of ITGB6 , expressing high (red) or low (black) levels of GDI2 , ERBB2 , RAB5A , and RAB7A . ( E and F ) Differential ITGB6 gene expression (gene chip) in patients with HER2+ breast cancer subdivided according to therapeutic response to trastuzumab. (E) Initial pathological complete response (responder) versus residual disease after completing therapy (nonresponder) ( n = 77 patients). (F) RFS at 5 years (responder) versus samples relapsed before 5 years (nonresponder) ( n = 24 patients). Two-sided Student’s t test. [(A), (E), and (F)] Statistical significance: * P < 0.05; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Gene Expression, RNA Sequencing, MANN-WHITNEY, Expressing, Clinical Proteomics

Journal: eLife

Article Title: A fusion peptide in preS1 and the human protein disulfide isomerase ERp57 are involved in hepatitis B virus membrane fusion process

doi: 10.7554/eLife.64507

Figure Lengend Snippet: Huh7-NTCP cells were grown on glass cover slides and fixed 48 hr after seeding. ( A ) Endogenous ERp57 with Rab5, Rab7, Rab11, or Lamp1 were immune-stained, and the colocalization of ERp57 (red channels) with Rab5, Rab7, Rab11, or Lamp1 (green channels) was analyzed by confocal microscopy. Scale bars of panels and zooms from squared area represent 10 µm and 2 µm, respectively. ( B ) The degree of colocalization between ERp57 and the different cell markers was assessed by determining the Pearson’s correlation coefficients with the JACoP plugin of ImageJ. Results are expressed as the mean of six individual cells. Error bars correspond to standard deviations.

Article Snippet: Antibody , Anti-human Rab7 (rabbit monoclonal) , Cell Signaling Technology , (D95F2):9367 , IF (1:100).

Techniques: Staining, Confocal Microscopy

Journal: eLife

Article Title: A fusion peptide in preS1 and the human protein disulfide isomerase ERp57 are involved in hepatitis B virus membrane fusion process

doi: 10.7554/eLife.64507

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human Rab7 (rabbit monoclonal) , Cell Signaling Technology , (D95F2):9367 , IF (1:100).

Techniques: Virus, Produced, Variant Assay, Construct, Generated, Transduction, Retroviral, Plasmid Preparation, Selection, shRNA, Transfection, Expressing, Clone Assay, Luciferase, Isolation, Sequencing, Mutagenesis, Reporter Assay, Activity Assay, cDNA Synthesis, SYBR Green Assay, Membrane, Integrity Assay, Cytotoxicity Assay, Software, Staining, RNA Extraction

Journal: Cell transplantation

Article Title: Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell-Replacement Therapy

doi: 10.3727/096368915X688948

Figure Lengend Snippet: Quantitative PCR Oligonucleotides

Article Snippet: Detection of the RAB7 protein, a housekeeping gene that regulates vesicular transport, was used as a loading control ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Target Protein Isotype Immunogen Species Clonality Dilution Vendor Catalog Number Primary Antibodies AQP1 rabbit IgG human polyclonal 1:1000 Millipore AB3272 ATP1A1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 3010 CD200 Goat IgG human polyclonal 1:1000 R&D Systems AF2724 CDH2 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 4061 COL8A2 rabbit IgG human polyclonal 1:1000 Santa Cruz Biotechnology sc-82843 GPC4 mouse IgG1 K human monoclonal 1:500 Novus Biologicals NBP1-45286 RAB7 rabbit IgG human monoclonal 1:1000 Cell Signaling Technology 9367 SLC4A11 rabbit IgG human polyclonal 1:1000 Abcam Ab93109 TCF4 rabbit IgG human monoclonal 1:400 Cell Signaling Technology 2569 ZEB1 rabbit IgG human monoclonal 1:500 Cell Signaling Technology 3396 ZO1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 5406 Secondary Horseradish Peroxidase Antibodies Rb-IgG goat IgG rabbit polyclonal 1:30,000 Millipore AP132P Ms-IgG goat IgG mouse polyclonal 1:30,000 Millipore AP124P Gt-IgG donkey IgG goat polyclonal 1:30,000 Millipore AB324P Open in a separate window Antibodies Used for Immunoblotting Statistical Analyses The mean and standard error of the mean (SEM) were graphed for each of the transcript abundance values determined by RNA-seq (RPKM) and qPCR (2 −ΔC T).

Techniques: Real-time Polymerase Chain Reaction, Functional Assay

Journal: Cell transplantation

Article Title: Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell-Replacement Therapy

doi: 10.3727/096368915X688948

Figure Lengend Snippet: Antibodies Used for Immunoblotting

Article Snippet: Detection of the RAB7 protein, a housekeeping gene that regulates vesicular transport, was used as a loading control ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Target Protein Isotype Immunogen Species Clonality Dilution Vendor Catalog Number Primary Antibodies AQP1 rabbit IgG human polyclonal 1:1000 Millipore AB3272 ATP1A1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 3010 CD200 Goat IgG human polyclonal 1:1000 R&D Systems AF2724 CDH2 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 4061 COL8A2 rabbit IgG human polyclonal 1:1000 Santa Cruz Biotechnology sc-82843 GPC4 mouse IgG1 K human monoclonal 1:500 Novus Biologicals NBP1-45286 RAB7 rabbit IgG human monoclonal 1:1000 Cell Signaling Technology 9367 SLC4A11 rabbit IgG human polyclonal 1:1000 Abcam Ab93109 TCF4 rabbit IgG human monoclonal 1:400 Cell Signaling Technology 2569 ZEB1 rabbit IgG human monoclonal 1:500 Cell Signaling Technology 3396 ZO1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 5406 Secondary Horseradish Peroxidase Antibodies Rb-IgG goat IgG rabbit polyclonal 1:30,000 Millipore AP132P Ms-IgG goat IgG mouse polyclonal 1:30,000 Millipore AP124P Gt-IgG donkey IgG goat polyclonal 1:30,000 Millipore AB324P Open in a separate window Antibodies Used for Immunoblotting Statistical Analyses The mean and standard error of the mean (SEM) were graphed for each of the transcript abundance values determined by RNA-seq (RPKM) and qPCR (2 −ΔC T).

Techniques:

Journal: Cell transplantation

Article Title: Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell-Replacement Therapy

doi: 10.3727/096368915X688948

Figure Lengend Snippet: Proteins encoded by the genes associated with HCEnC function and corneal endothelial dystrophies were assessed by Western blotting. (A) Proteins from 5/6 (AQP1, ATP1A1, CDH2 and ZO1), 6/6 (AQP1, ATP1A1, CDH2, GPC4 and ZO1) and 3/6 (ATP1A1, CDH2 and ZO1) genes associated with HCEnC function were detected in evHCEnC, pHCEnC and the three HCEnC lines, respectively. (B) Proteins from 4/4 (COL8A2, SLC4A11, TCF4 and ZEB1), 3/4 (COL8A2, SLC4A11 and ZEB1), 1/4 (ZEB1) and 2/4 (COL8A2 and ZEB1) genes associated with corneal endothelial dystrophies were detected in evHCEnC, pHCEnC, HCEnC-21T and the two (HCEC-12 and HCEC-B4G12) related cell lines, respectively. These are representative blots from three independent samples for each HCEnC group (Table 1). The detection of the RAB7 protein was used as a loading control.

Article Snippet: Detection of the RAB7 protein, a housekeeping gene that regulates vesicular transport, was used as a loading control ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Target Protein Isotype Immunogen Species Clonality Dilution Vendor Catalog Number Primary Antibodies AQP1 rabbit IgG human polyclonal 1:1000 Millipore AB3272 ATP1A1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 3010 CD200 Goat IgG human polyclonal 1:1000 R&D Systems AF2724 CDH2 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 4061 COL8A2 rabbit IgG human polyclonal 1:1000 Santa Cruz Biotechnology sc-82843 GPC4 mouse IgG1 K human monoclonal 1:500 Novus Biologicals NBP1-45286 RAB7 rabbit IgG human monoclonal 1:1000 Cell Signaling Technology 9367 SLC4A11 rabbit IgG human polyclonal 1:1000 Abcam Ab93109 TCF4 rabbit IgG human monoclonal 1:400 Cell Signaling Technology 2569 ZEB1 rabbit IgG human monoclonal 1:500 Cell Signaling Technology 3396 ZO1 rabbit IgG human polyclonal 1:1000 Cell Signaling Technology 5406 Secondary Horseradish Peroxidase Antibodies Rb-IgG goat IgG rabbit polyclonal 1:30,000 Millipore AP132P Ms-IgG goat IgG mouse polyclonal 1:30,000 Millipore AP124P Gt-IgG donkey IgG goat polyclonal 1:30,000 Millipore AB324P Open in a separate window Antibodies Used for Immunoblotting Statistical Analyses The mean and standard error of the mean (SEM) were graphed for each of the transcript abundance values determined by RNA-seq (RPKM) and qPCR (2 −ΔC T).

Techniques: Western Blot, Control

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 1. Loss of Rab7 blocked LC3-II flux under fed conditions, but not under starved

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques:

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 2. Nutrient-starvation-induced clearance of accumulated autolysosomes in Rab7-KO

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques:

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 3. Ultrastructure of the accumulated autolysosomes in fed Rab7-KO cells. (A) Typical

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques:

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 4. Kinetics of the starvation-induced LC3-II turnover in Rab7-KO cells. (A) LC3-II

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques:

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 6. Glutamine-starvation-induced autolysosome clearance in Rab7-KO cells. (A)

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques:

Journal: Journal of cell science

Article Title: Rab7 knockout unveils regulated autolysosome maturation induced by glutamine starvation.

doi: 10.1242/jcs.215442

Figure Lengend Snippet: Fig. 7. Glutamine starvation induced autolysosome clearance even in the presence of Rab7.

Article Snippet: The CRISPR/Cas9-mediated Rab7 knockout (single guide RNA [sgRNA] target sequence: 5’-GGAACGGTTCCAGTCCCTTG-3’) in MDCK-II cells was performed as described previously (Mrozowska and Fukuda, 2016), by using pSpCas9 (BB) 2A-Puro vector (Addgene, Cambridge, MA).

Techniques: