EMBO/SourceData huggingface dataset:
Figure 3A, B RNF123 inhibits the activation of the IFN-β (A) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TBK1- and IRF3-5D-induced activation of the IFN-β promoter (A) . HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), IFN-β promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.A, B RNF123 inhibits the activation of the NF-κB (B) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TAB2- and IKKβ-induced activation of the NF-κB promoter (B). HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), NF-κB promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.C Knockdown of RNF123 potentiates the activation of the IFN-β promoter induced by RIG-I, RIG-I CARD, MDA5, and MDA5 CARD, but not that by VISA, TBK1, and cGAS/STING. 293T-GFPsh, shRNF123 #1, #3, and #4 stable cell pools (2×105) were transfected with the indicated plasmids. Luciferase assays were performed 24 h after transfection.D RNF123 interacted with RIG-I and MDA5, but not VISA, TBK1, and cGAS. HEK293T cells (1×106) were transfected with Flag-RNF123 (F-RNF123) together with HA-RIG-I, HA-MDA5, HA-VISA, HA-TBK1, or HA-cGAS plasmids (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-Flag (αF) or anti-HA antibody (αHA). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies to determine the expression levels of transfected plasmids.E Endogenous interaction of RNF123 with RIG-I upon SeV infection. HEK293T cells (3×106) were treated with SeV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-RIG-I antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-RIG-I and anti-RNF123 antibodies.F Endogenous interaction of RNF123 with MDA5 upon EMCV infection. HeLa cells (3×106) were treated with EMCV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-MDA5 antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-MDA5 and anti-RNF123 antibodies. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.
Figure 4B The SPRY and coiled-coil domains are both indispensable for inhibiting IFN-β promoter activation. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for RNF123 and its mutants (100 ng), and the empty vector or RIG-I CARD/MDA5 CARD (100 ng). Reporter assays were performed 24 h after transfection.C The SPRY domain and two coiled-coil domains of RNF123 are both required for interaction with RIG-I CARD. HEK293T cells (1×106) were transfected with HA-RIG-I CARD together with F-RNF123 or its mutants (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (α-F). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-HA (α-HA) or anti-Flag (α-F). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies.D KPC2 has no effect on the activation of the IFN-β reporter induced by RIG-I CARD and MDA5 CARD. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), and the indicated expression plasmids. Reporter assays were performed 24 h after transfection.E Effect of shKPC2 plasmids on endogenous KPC2. RT-PCR was performed with the indicated primers after HEK293T cells were infected with six lentiviruses encoding an shRNA control (GFPsh) and five shRNAs targeting kpc2 (Kpc2 #1-#5).F KPC2 is not required for the inhibitory action of RNF123. HEK293T cells (2×105) with GFPsh or KPC2 sh (#1, #2, or #4) were transfected with the IFN-β reporter plasmid (100 ng) and the pRL-TK Renilla luciferase plasmid (100 ng) with the empty vector or RNF123 (100 ng). Twenty-four hours after transfection, cells were infected with SeV or left uninfected. Reporter assays were performed 18 h after infection.G Knockdown of KPC2 had no effect on SeV-induced IFN-β expression. HEK293T cells with GFPsh or KPC2 sh #4 were infected with SeV for the indicated times, total RNA was isolated, and RT-PCR experiments were performed.
Figure 5A, B RNF123 interacts with RIG-I/MDA5 CARD. HEK293T cells (1×106) were transfected with expression plasmids for Flag-RNF123 together with HA-RIG-I or its truncations (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in the legend of Fig. 3D.C, D RNF123 competes with VISA for RIG-I/MDA5 CARD binding. HEK293T cells (1×106) were transfected with expression plasmids for HA-RIG-I CARD/MDA5 CARD alone or together with Flag-VISA or with Flag-VISA and Flag-RNF123 (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in Fig. 3D.E RNF123 does not compete with REUL for RIG-I CARD binding. The methods of transfection and co-immunoprecipitation were as described in Fig. 5C,D.F Knockdown of RNF123 facilitates the endogenous interaction between RIG-I and VISA in 293T cells. 293T cells stably infected with SeV were treated for the indicated times. Cell lysates were immunoprecipitated with anti-VISA antibody. The immunoprecipitates and lysates were analyzed by western blotting with the indicated antibody.G Knockdown of RNF123 facilitates the endogenous interaction between MDA5 and VISA in HeLa cells. HeLa cells were stably infected with EMCV for the indicated times. Co-immunoprecipitation and whole-cell lysates were analyzed as described in (F).
Figure 1ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166;ELISA assay of secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool.Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (F) *: p=0,0167In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (G) *: p=0,0441; (H) *: p=0,423 ; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.(I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (K) *: p=0,0221 ; (L) *: p=0,0486 . For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.
Figure 3Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.
Figure 4Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12.
Figure 5(A-E) Optical density (600nm) variations during in vitro growth assay of Salmonella enterica Typhimurium (SL1344) co-cultured with stool-purified IgA1 (A) or IgA2 (B) from non-IBD (n=4), CD (n=6) and UC (n=3). Comparison between IgA1 and IgA2 co-culture in non-IBD (C), CD (D), and UC (E). Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (A) UC curve: 2h - p=0,0024, 3h - p=0,003; CD curve: 2h and 3h p<0,0001 (B) UC curve: 4h - p=0,0186, 5h - p=0,0393, 6h - p=0,039, 8h - p<0,0001; CD curve: 4h - p=0,0001, 5h - p=0,0028, 6h - p=0,0024, 8h - p<0,0001. (C) 3h - p=0,0422, 4h - p=0,0422, 6h - p=0,0012, 8h - p<0,0001.(F-I) Optical density (600nm) during in vitro growth assay of Escherichia coli (25922 strain from ATCC) co-cultured with stool-purified IgA1 (F) or IgA2 (G) from non-IBD (n=2) and CD (n=2). All patient samples (biological replicates) have been tested in technical duplicates. Comparison between IgA1 and IgA2 co-culture in non-IBD (H), and CD (I). These IgA samples were from the same patients throughout each experiment. Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (F) Grey curve: 4h - p=0,0004, 5h - p=0,0014; Black curve: 4h and 5h - p<0,0001. (G) De-glycosylated CD vs. De-glycosylated non-IBD: 4h - p=0,0003, 5h - p<0,0001. (H) Native vs. de-glycosylated: 4h and 5h - p<0,0001; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p=0,0012. (H) Native vs. de-glycosylated: 4h - p=0,001 and 5h - p= 0,006; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p<0,0001.
Figure 6(A) Combined FISH-IF staining for each probe. (B) Total fluorescence detected per probe and per biopsy. Erec482 - *: p=0,00233; Ato291 - *: p=0,0268; Bac303 - *: p=0,0196. (C) Percent of co-occurred IgA2 and FISH-bound particles. Erec482 - *: p=0, 0073.Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test. Non-IBD: n = 3; Active CD: n= 6; Inactive CD: n=4. All patient samples (biological replicates) have been tested in technical duplicates.
Figure 1(B) Immunoblots from cytosolic (cyt) and nuclear (nuc) fractionations of RPE-1 cells in early-G1 showing the presence of endogenous ACTN4 in nuclear fractions. Proteins were detected with antibodies against (α) ACTN4, α-Tubulin or Histone 3 as indicated(C) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated.(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.
Figure 3(A) NIH3T3 cells expressing nAC-mCherry were imaged for 120 min. Image frames represent maximum projections of 30 Z planes. Lower panel is a magnification of the indicated area (orange box); scale bars 10 µm.(B) NIH3T3 cells stably expressing H2B-mCherry were transfected with siCtrl or siACTN1 + siACTN4. After mitotic exit z-stacks (30 planes) were acquired every 5 min. Nuclei were 3D-reconstructed and volume was measured by Imaris. Data are mean ± SEM from 3 biological replicates with 22-27 (siCtrl) and 32-42 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by t-test at 90 min.(C) RPE-1 cells were analyzed as in B. Data are mean ± SEM from 5 biological replicates with 32-37 (siCtrl) and 31-39 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by Mann Whitney test at 90 min.(E) NIH3T3 cells stably expressing H2B-mCherry and siRNA-resistant ACTN4-NES were transfected with the indicated siRNAs and analyzed as described in B. Data are mean ± SEM from 5 biological replicates with 63-71 (siCtrl), 37-41 (siACTN1+4), 52-58 (siACTN1) and 52-55 (siACTN4) analyzed nuclei; ****p(siACTN4) < 0.0001; ****p(siACTN1+4) < 0.0001 by One Way ANOVA at 90 min.(F) NIH3T3 cells stably expressing H2B-mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B. Data are mean ± SEM from 3 biological replicates with 63-69 (ACTN4 wt + siACTN4), 63-71 (ACTN4-NES + siCtrl) and 54-58 (ACTN4-NES + siACTN4) analyzed nuclei; ****p(ACTN4-NES + siACTN4) < 0.0001 by One Way ANOVA at 90 min.(G) NIH3T3 cells expressing siRNA-resistant ACTN4-NES were transfected with siCtrl or siACTN4; chromatin density was calculated from H2B-mCherry fluorescence intensities by total nuclear volumes (intensity/μm3) and normalized to t=0 (mitotic exit); Data are mean ± SEM from 5 biological replicates with 44-48 (siCtrl) and 44-47 (siACTN4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and *p < 0.05 by t-test at 90 min.
Figure 4(B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm.(C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA.(D) Number of filaments in the post mitotic nuclei; as in (C); data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei from 5 biological replicates, ***p < 0.001 by t-test. (E) Actin filament widths for ACTN4-wt and ACTN4ΔCH1-NLS in early G1 nuclei; ); data shown as mean ± 2.5-97.5 percentile with 96 filaments (ACTN4-wt) and 36 filaments (ACTN4ΔCH1-NLS) from 5 biological replicates, **p < 0.01 by t-test.
Figure 1(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.(b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment).(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones).(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm.(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed.(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.
Figure 2(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.(c-e) EV and shRAB2AMDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast light microscopy images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).(f-h) Doxycycline-inducible murine RAB2A (mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.
Figure 3Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(a) Images are representative of RAB2A expression scoring according to intensity staining in TMA. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma.Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(b) Correlation of RAB2A expression with clinicopathological parameters. Only for 520 of 622 samples expression data for RAB2A was available. Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma.Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(c) Kaplan-Meier Distant Recurrence Free Interval survival (DRFI [70]) and (d) Univariate and Multivariate Cox-regression analysis, shows that in BC patients' high levels of RAB2A protein predict a poor prognosis, and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence.
Figure 4(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm).(c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm).(d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscopy. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.