Journal: bioRxiv

Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

doi: 10.1101/2025.09.23.677683

Figure Lengend Snippet: (A) Bright-field images of MDA-MB-231 spheroids embedded in the collagen matrix at 0h and 48h in the presence or absence of HGF or complete media. The ratio of the invaded area to the core area was quantified and plotted. The core has been outlined with green, and the invaded area has been outlined with yellow colour. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, One-way ANOVA with multiple comparisons. **** P < 0.0001, * P < 0.05, Scale bar: 100µm. N=3, n=30. (B) MDA-MB-231 cells were seeded on Alexa568-labeled gelatin-coated coverslip with or without HGF for 3h. Degradation index was quantified and plotted. The arrowhead represents degradation spots. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test, **** P < 0.0001. scale bar: 10µm. N=3, n=200. (C) MDA-MB-231 cells were seeded on a gelatin-coated glass-bottom dish and allowed to attach for 3h, followed by incubation with or without HGF for another 3h. Cells were fixed and immunostained for TKS5 and Cortactin. Images were captured using a TIRF microscope. TKS5 puncta positive for Cortactin were considered as invadopodia. Arrowheads indicate TKS5-Cortactin positive puncta. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Paired t-test, ** P < 0.01. scale bar: 10µm. N = 4, n = 150. (D, E) MDA-MB-231 cells were seeded on gelatin-coated coverslips for 3h, followed by incubation with vehicle or PHA665752 (5 µM) with or without HGF for an additional 3h. Cells were fixed, immunostained with anti-TKS5 antibody, Phalloidin to stain F-actin, and DAPI. Images were captured using a confocal microscope. TKS5 puncta positive for Actin were considered as invadopodia. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm. Inset: 2µm. The error bar represents Mean ±SEM, One-way ANOVA with multiple comparisons. **** P < 0.0001, ** P < 0.01, ns P > 0.05, N=3, n=200. (F) MDA-MB-231 cells were transfected with control SHC002 or MET shRNA clone #3, and after 36h of transfection, cells were seeded on Alexa568-labeled gelatin-coated coverslip for 6h. The cells were fixed and immunostained with anti-MET antibody, Phalloidin, and DAPI. Degradation spots immunostained for Actin were quantified and plotted. The inset shows a magnified view of the region indicated by the box. The Arrowheads indicate the degradation spots that were immunostained for Actin. scale bar: 10µm. Inset: 2µm. The error bar represents Mean ±SEM, Unpaired t-test. *** P < 0.001, N=3, n=200.

Article Snippet: The following antibodies were purchased commercially: rabbit anti-FISH (TKS5) (SantaCruz, sc-30122), 1:500 (IF), mouse anti-cortactin (Millipore, 05–180), 1:300 (IF); (SantaCruz, sc-55579) 1:500 (IF), rabbit anti-MET (CST, D1C2 XP) 1:400 (IF), 1:1000 (IB), mouse anti-MET (CST, L6E7) 1:500 (IF), 1:1000 (IB), rabbit anti-Phospho-Met (Tyr1234/1235) (CST, D26 XP) 1:800 (IF), 1:1000 (IB), mouse anti-V5 (Sigma, SV5-Pk1) 1:400 (IF), 1:1000 (IB), rabbit anti-Rab11Fip1 (Protein technology, 16778-1-AP) 1:1000 (IB), rabbit anti-mouse MT1-MMP (Millipore, MAB3328), 1:1,000 (IB) and 1:400 (IF); mouse anti-MT1-MMP (R&D Systems, MAB9181-SP), 1:200 (IF), mouse anti-Vps35(SantaCruz, sc-374372) 1:300 (IF); rabbit anti-Vps26 (Abcam, ab23892) 1:1000 (IB), rabbit anti-EGFR (CST, D38B1), rabbit anti-Golgin97 (CST, D8P2K) 1: 200 (IF); mouse anti-vinculin (Sigma, V9131), 1:1,000 (IB); rabbit anti-actin (Sigma, A2066), 1:1,000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB), mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-Rab5 (BD Biosciences, 610724), 1:300 (IF); mouse anti-His (ThermoFischer Scientific), 1:1,000 (IB).

Techniques: Labeling, Incubation, Microscopy, Staining, Transfection, Control, shRNA

Journal: bioRxiv

Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

doi: 10.1101/2025.09.23.677683

Figure Lengend Snippet: (A) MDA-MB-231 cells were seeded on gelatin-coated imaging dishes for 6h. Cells were fixed and immunostained for Cortactin and MET. Images were captured using a TIRF microscope. Colocalized Cortactin-MET puncta were quantified and plotted. The arrowheads indicate the MET-positive Cortactin puncta. The error bar represents Mean ±SD, scale bar: 10µm, n= 60. (B) GFP-TKS5-expressing MDA-MB-231 cells were seeded on gelatin-coated imaging dishes for 6h. Cells were fixed, immunostained for MET and imaged with TIRFM. The arrowhead indicates the MET-positive TKS5 puncta. scale bar: 10µm. (C) Cherry-TKS5-expressing MDA-MB-231 cells were seeded on gelatin-coated glass-bottom imaging dishes for 6h. Cells were fixed, immunostained with anti-pY-MET antibody and imaged with TIRFM. The arrowhead indicates the pY-MET-positive TKS5 puncta. scale bar: 10µm. (D) Colocalized TKS5-MET or pY-MET puncta were quantified and plotted. The error bar represents Mean ±SD, N=3, n ≤ 50. (E) The fluorescence intensity of TKS5 and MET at individual invadopodia was quantified using Motiontracking and normalized with their respective mean intensities. The normalized fluorescence intensities of TKS5 (X-axis) and MET (Y-axis) were plotted. The points were fitted with a smoothing spline. n=800. (F) MDA-MB-231 cells were seeded on Alexa568-labeled gelatin-coated coverslips for 12h. Cells were fixed and immunostained for F-actin and MET. The inset shows a magnified view of the region indicated by the box. The white Arrowhead represents Actin at the degradation spots, and the yellow arrowhead represents Actin-MET puncta without degradation. scale bar: 10µm. Inset: 2µm. (G) BT-549 cells were seeded on gelatin-coated coverslips for 3h followed by incubation with or without HGF for 3h. Cells were fixed, immunostained for TKS5, MET and Nucleus. Images were taken using the confocal microscope. Colocalized TKS5-MET puncta were quantified and plotted. The error bar represents Mean ±SEM/SD, Unpaired t-test scale bar: 10µm, *** P < 0.001, N=3, n = 200. (H) BT-549 cells expressing GFP-MET were seeded on glass-bottom dishes. The cells were imaged using a TIRF microscope in the presence or absence of HGF. The GFP-MET vesicles moving in the TIRF field for a minimum of 4 consecutive frames were quantified and plotted as MET tracks. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. ** P < 0.01, N=3, n=30.

Article Snippet: The following antibodies were purchased commercially: rabbit anti-FISH (TKS5) (SantaCruz, sc-30122), 1:500 (IF), mouse anti-cortactin (Millipore, 05–180), 1:300 (IF); (SantaCruz, sc-55579) 1:500 (IF), rabbit anti-MET (CST, D1C2 XP) 1:400 (IF), 1:1000 (IB), mouse anti-MET (CST, L6E7) 1:500 (IF), 1:1000 (IB), rabbit anti-Phospho-Met (Tyr1234/1235) (CST, D26 XP) 1:800 (IF), 1:1000 (IB), mouse anti-V5 (Sigma, SV5-Pk1) 1:400 (IF), 1:1000 (IB), rabbit anti-Rab11Fip1 (Protein technology, 16778-1-AP) 1:1000 (IB), rabbit anti-mouse MT1-MMP (Millipore, MAB3328), 1:1,000 (IB) and 1:400 (IF); mouse anti-MT1-MMP (R&D Systems, MAB9181-SP), 1:200 (IF), mouse anti-Vps35(SantaCruz, sc-374372) 1:300 (IF); rabbit anti-Vps26 (Abcam, ab23892) 1:1000 (IB), rabbit anti-EGFR (CST, D38B1), rabbit anti-Golgin97 (CST, D8P2K) 1: 200 (IF); mouse anti-vinculin (Sigma, V9131), 1:1,000 (IB); rabbit anti-actin (Sigma, A2066), 1:1,000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB), mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-Rab5 (BD Biosciences, 610724), 1:300 (IF); mouse anti-His (ThermoFischer Scientific), 1:1,000 (IB).

Techniques: Imaging, Microscopy, Expressing, Fluorescence, Labeling, Incubation

Journal: bioRxiv

Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

doi: 10.1101/2025.09.23.677683

Figure Lengend Snippet: (A) Graphical representation of WT MET and mutants’ endocytosis and degradation. (B) BT-549 cells expressing WT or the mutants of V5-MET were treated with or without HGF for 2 hours, followed by cell lysis. The lysates were subjected to immunoblotting with anti-V5 and anti-vinculin antibodies. The V5 signal intensity was quantified and normalized with Vinculin. Error bar represents Mean ±SEM. Unpaired t-test. * P < 0.05. (C) BT-549 cells expressing WT or M1250T V5-MET were lysed and incubated with Ni-NTA beads. The pulled-down samples were processed for immunoblotting for MET and pY-MET. (D, D’) WT or mutant V5-MET expressing BT-549 cells were seeded on glass coverslips with or without HGF. Cells were fixed, immunostained for V5 and EEA1. Images were captured with a confocal microscope. V5-MET puncta localizing with EEA1 were quantified and plotted. scale bar: 10µm. The error bar represents Mean ±SEM. ** P < 0.01. ns P > 0.05 N=3, n=60. (E, E’) BT-549 cells expressing V5-MET WT or mutants were seeded on Alexa568-labeled gelatin-coated coverslips with or without HGF for 6h. Cells were fixed, then immunostained for V5 and the Nucleus and imaged with the confocal microscope. Gelatin degradation by the V5-MET overexpressing cells was calculated and plotted. scale bar: 10µm. The error bar represents Mean ±SEM, Unpaired t-test. ** P < 0.01, ns P > 0.05, N=3, n=120. (F) V5-MET WT or M1250T mutant expressing BT-549 cells were seeded on a gelatin-coated coverslip for 6h and fixed. Cells were immunostained for V5, TKS5, and images were captured with the confocal microscope. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. ** P < 0.01, N=3, n=120 (G) Bright-field images of GFP-MET WT or M1250T expressing BT-549 spheroids embedded in the collagen matrix at 0h and 24h. The ratio of the invaded area to the core area was quantified and plotted. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 100µm. **** P < 0.0001, N=3, n=40.

Article Snippet: The following antibodies were purchased commercially: rabbit anti-FISH (TKS5) (SantaCruz, sc-30122), 1:500 (IF), mouse anti-cortactin (Millipore, 05–180), 1:300 (IF); (SantaCruz, sc-55579) 1:500 (IF), rabbit anti-MET (CST, D1C2 XP) 1:400 (IF), 1:1000 (IB), mouse anti-MET (CST, L6E7) 1:500 (IF), 1:1000 (IB), rabbit anti-Phospho-Met (Tyr1234/1235) (CST, D26 XP) 1:800 (IF), 1:1000 (IB), mouse anti-V5 (Sigma, SV5-Pk1) 1:400 (IF), 1:1000 (IB), rabbit anti-Rab11Fip1 (Protein technology, 16778-1-AP) 1:1000 (IB), rabbit anti-mouse MT1-MMP (Millipore, MAB3328), 1:1,000 (IB) and 1:400 (IF); mouse anti-MT1-MMP (R&D Systems, MAB9181-SP), 1:200 (IF), mouse anti-Vps35(SantaCruz, sc-374372) 1:300 (IF); rabbit anti-Vps26 (Abcam, ab23892) 1:1000 (IB), rabbit anti-EGFR (CST, D38B1), rabbit anti-Golgin97 (CST, D8P2K) 1: 200 (IF); mouse anti-vinculin (Sigma, V9131), 1:1,000 (IB); rabbit anti-actin (Sigma, A2066), 1:1,000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB), mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-Rab5 (BD Biosciences, 610724), 1:300 (IF); mouse anti-His (ThermoFischer Scientific), 1:1,000 (IB).

Techniques: Expressing, Lysis, Western Blot, Incubation, Mutagenesis, Microscopy, Labeling

Journal: bioRxiv

Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

doi: 10.1101/2025.09.23.677683

Figure Lengend Snippet: MDA-MB-231 cells, seeded on glass coverslips, were transfected with (A) GFP-RAB4 or (B) Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. scale bar: 10µm. (C) Graph showing the percent colocalization of MET with GFP-RAB4 and Cherry-RAB14 in the presence of absence of HGF treatment. The error bar represents Mean ±SD, One-way ANOVA. ** P< 0.01. N=3, n=200 (D) BT-549 cells were transfected with siRNA against RAB4, RAB14, RAB22, or control siRNA. After 60 hours, cells were transfected with GFP-MET and seeded on the gelatin-coated glass-bottom dish. The GFP-MET-transfected cells were imaged in the presence of HGF using a TIRF microscope for 30 frames without an interval. scale bar: 10µm (E) The GFP-MET vesicles forming the track were identified as mentioned in the Method section. The numbers of GFP-MET tracks moving for at least 4 consecutive frames were quantified for each condition and plotted. In addition, the total number of GFP-MET vesicles was quantified. The error bars represent Mean±SD. Unpaired t-test. *** P< 0.001, ** P< 0.01, ns P > 0.05, N=3, n = 30 (F) BT-549 cells were depleted with RAB4 or RAB14, and after 60h, cells were seeded on gelatin-coated coverslips for 6h with HGF stimulation. Cells were fixed and immunostained for MET, TKS5, and F-actin. Images were captured in a confocal microscope. MET colocalizing with invadopodia was quantified and plotted. The number of cells forming lamellipodia was quantified. scale bar: 10µm. The error bar represents Mean ±SEM, One-way ANOVA, *** P< 0.001, ** P< 0.01. N=3, n=200.

Article Snippet: The following antibodies were purchased commercially: rabbit anti-FISH (TKS5) (SantaCruz, sc-30122), 1:500 (IF), mouse anti-cortactin (Millipore, 05–180), 1:300 (IF); (SantaCruz, sc-55579) 1:500 (IF), rabbit anti-MET (CST, D1C2 XP) 1:400 (IF), 1:1000 (IB), mouse anti-MET (CST, L6E7) 1:500 (IF), 1:1000 (IB), rabbit anti-Phospho-Met (Tyr1234/1235) (CST, D26 XP) 1:800 (IF), 1:1000 (IB), mouse anti-V5 (Sigma, SV5-Pk1) 1:400 (IF), 1:1000 (IB), rabbit anti-Rab11Fip1 (Protein technology, 16778-1-AP) 1:1000 (IB), rabbit anti-mouse MT1-MMP (Millipore, MAB3328), 1:1,000 (IB) and 1:400 (IF); mouse anti-MT1-MMP (R&D Systems, MAB9181-SP), 1:200 (IF), mouse anti-Vps35(SantaCruz, sc-374372) 1:300 (IF); rabbit anti-Vps26 (Abcam, ab23892) 1:1000 (IB), rabbit anti-EGFR (CST, D38B1), rabbit anti-Golgin97 (CST, D8P2K) 1: 200 (IF); mouse anti-vinculin (Sigma, V9131), 1:1,000 (IB); rabbit anti-actin (Sigma, A2066), 1:1,000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB), mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-Rab5 (BD Biosciences, 610724), 1:300 (IF); mouse anti-His (ThermoFischer Scientific), 1:1,000 (IB).

Techniques: Transfection, Incubation, Microscopy, Control

Journal: bioRxiv

Article Title: RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

doi: 10.1101/2025.09.23.677683

Figure Lengend Snippet: (A) MDA-MB-231 cells seeded on coverslips, surface labeled with MT1-MMP antibody at 4°C for 1 h Complete media was added, and cells were shifted to 37°C for 30 minutes to allow endocytosis. To remove surface-bound antibodies, an acid wash was given. After washing with PBS, serum-free media was added with or without HGF, and the cells were shifted to 37°C for 10 minutes to allow recycling from the endocytosed pool. Cells were fixed and immunostained for EEA1. The integral intensity of the MT1-MMP and the percentage of MT1-MMP antibody recycling were calculated. N = 3, n= 300; scale bars = 10 µm. The error bar represents means ± SD. Paired t-test, *** P< 0.001, Unpaired t-test, * P < 0.05. (B) MDA-MB-231 cells transfected with pHluorin MT1-MMP were seeded on gelatin-coated glass-bottom dishes. Time-lapse imaging was performed in the presence or absence of HGF stimulation using a Nikon TIRF microscope without any intervals for 300 frames. The flashes of pHluorin MT1-MMP, which represent exocytic events, were calculated using Motiontracking. scale bars = 10 µm. The error bar represents Means ± SEM. Unpaired t-test, ** P < 0.001. N = 4, n= 35. (C) MDA-MB-231 cells were seeded on gelatin-coated glass-bottom dishes and allowed to attach for 3h, followed by incubation with or without HGF for another 3h. Cells were fixed and immunostained for TKS5, MT1-MMP, and F-actin. Images were captured using a Nikon TIRF microscope. TKS5, MT1-MMP, and Actin-positive structures were identified using Motiontracking. The error bar represents Mean ±SEM, Unpaired t-test, * P < 0.01. scale bar: 10µm. N=3, n=120. (D) GFP-MT1-MMP expressing MDA-MB-231 cells were fixed and immunostained for MET. Images were captured using an LSM900 Airyscan super-resolution microscope. Images were processed using the Airyscan Joint deconvolution (jDCV) method. The image was converted to Maximum Intensity projection for representation. The inset shows a magnified view of the region indicated by the box. Scale bar: 10 µm, inset: 2 µm. (E) GFP or GFP-MT1-MMP expressing BT-549 cells were lysed and incubated with GST agarose beads bound to 25µg of GFP binding protein (GBP). Next washes were given to remove unbound proteins and proceeded for immunoblotting with GFP and MET. WCL: whole-cell lysates (F) MDA-MB-231 cells co-expressing GFP-MET and Cherry MT1-MMP were subjected to live-cell TIRF imaging. The inset represents an event at different time points. Scale bar: 10 µm, Inset: 2 µm. (G) BT-549 cells expressing MT1-MMP WT or a catalytic mutant alone or with V5-MET WT or M1250T mutant were seeded on gelatin-coated coverlips for 6h and fixed. Cells were immunostained for V5 and TKS5. Images were captured with the confocal microscope. The number of TKS5 puncta in the MT1-MMP and/or MET expressing cells was calculated and plotted. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. ** P<0.01, * P < 0.05, N=3, n=150

Article Snippet: The following antibodies were purchased commercially: rabbit anti-FISH (TKS5) (SantaCruz, sc-30122), 1:500 (IF), mouse anti-cortactin (Millipore, 05–180), 1:300 (IF); (SantaCruz, sc-55579) 1:500 (IF), rabbit anti-MET (CST, D1C2 XP) 1:400 (IF), 1:1000 (IB), mouse anti-MET (CST, L6E7) 1:500 (IF), 1:1000 (IB), rabbit anti-Phospho-Met (Tyr1234/1235) (CST, D26 XP) 1:800 (IF), 1:1000 (IB), mouse anti-V5 (Sigma, SV5-Pk1) 1:400 (IF), 1:1000 (IB), rabbit anti-Rab11Fip1 (Protein technology, 16778-1-AP) 1:1000 (IB), rabbit anti-mouse MT1-MMP (Millipore, MAB3328), 1:1,000 (IB) and 1:400 (IF); mouse anti-MT1-MMP (R&D Systems, MAB9181-SP), 1:200 (IF), mouse anti-Vps35(SantaCruz, sc-374372) 1:300 (IF); rabbit anti-Vps26 (Abcam, ab23892) 1:1000 (IB), rabbit anti-EGFR (CST, D38B1), rabbit anti-Golgin97 (CST, D8P2K) 1: 200 (IF); mouse anti-vinculin (Sigma, V9131), 1:1,000 (IB); rabbit anti-actin (Sigma, A2066), 1:1,000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB), mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-Rab5 (BD Biosciences, 610724), 1:300 (IF); mouse anti-His (ThermoFischer Scientific), 1:1,000 (IB).

Techniques: Labeling, Transfection, Imaging, Microscopy, Incubation, Expressing, Super-Resolution Microscopy, Binding Assay, Western Blot, Mutagenesis