Figure 1.
Differential relationships of TM4SF5, CD151, and CD63 expression.
(A to C) TM4SF5-null Chang (normal hepatocyte) and TM4SF5-expressing Chang-TGFβ1 (Chang cells chronically treated with TGFβ1) cells were analyzed for CD9, CD63, CD151, and TM4SF5 expression by flow cytometry (A), by RT-PCR (B), and by Western blot (C). Data represent three independent experiments.
Figure 2.
The relationships of TM4SF5, CD151, and CD63 in the development of murine liver fibrosis and liver cancer.
(A to D) Liver tissues from mice administrated with control vehicle or CCl4 every other day for 2 weeks were used for RT-PCR analysis (A), harvested for whole extracts, prior to Western blots for the indicated proteins (B), used for Masson’s trichrome staining to determine collagen I expression (C), or processed for immunohistochemistry with double-staining for TM4SF5 and CD151 (D). (E) Human normal or liver tumor tissues were processed for Western blots or immunohistochemistry to identify CD151 (top), TM4SF5 (middle), and CD63 (bottom) and the nuclei were stained using DAPI. Scale bars depict 10 µm. Data represent three independent experiments.
Figure 3.
TM4SF5 expression positively or negatively regulated CD151 and CD63 expression levels, respectively, and altered migratory signaling activity.
(A to C) TM4SF5-expressing Chang-TGFβ1 (A and B) or Huh7 cells (C) transfected with shRNA against TM4SF5 (shTM4SF5) or a control-scrambled sequence (shControl) were processed for RT-PCR (A) or Western blots (B and C) against the indicated molecules. (D to F) TM4SF5-null Chang (D and E) or SNU449 (F) cells transfected with TM4SF5 cDNA or control plasmids (Mock) were processed for RT-PCR (D) or Western Blots (E and F). Note that CD63 immunoblots showed multiple bands, presumably due to 3 possible isoforms with N-glycosylations (http://www.uniprot.org/uniprot/P08962). Data represent three different experiments.
Figure 4.
CD151 regulated CD63 expression, but not TM4SF5 expression.
Chang-TGFβ1 cells were transiently transfected with shRNA against CD151 (shCD151) or a control-scrambled sequence (shControl) for 48 h (A and B). Chang cells were transiently transfected with CD151 cDNA or a control plasmid (Mock) for 48 h (C and D). The cells were then processed for RT-PCR (A and C) or for standard Western blots (B and D) for the indicated molecules. Data represent three different experiments.
Figure 5.
CD63 was antagonistic to TM4SF5 and CD151.
(A and B) Chang-TGFβ1 cells (A) or Huh7 cells (B) transfected with CD63 cDNA or a control plasmid (Mock) were processed for RT-PCR or Western blots. (C) Chang cells transfected with pGL3-TM4SF5 promoter DNA without or with CD63 cDNA for 24 h were treated with vehicle or TGFβ1 for additional 24 h, prior to luciferase reporter gene assay. Each value was shown at mean ± standard deviation. * depicts statistical significance (p<0.05) and ** depicts insignificance (p≥0.05). (D and E) Chang and Chang-TGFβ1 cells were immunostained for TM4SF5 (green, D), CD63 (green, E), or CD151 (red, E) in addition to nuclear staining using DAPI. (F) Chang-TGFβ1 cells were immunostained for TM4SF5 (green) and either CD151 (top panel) or CD63 (bottom panel) in addition to DAPI staining. White box depicts area for an enlarged insert. (G) Chang cells transiently transfected with FLAG-TM4SF5 or CD151 were immunostained for CD63 (blue or red) and either FLAG-TM4SF5 (green) or CD151 (green) prior to visualization by confocal microscopy. (H) Chang cells transfected with FLAG-TM4SF5 for 48 h were immunostained using anti-FLAG (green), anti-LAMP2 (a lysosomal marker, blue), and anti-CD63 (red) antibody, prior to visualizations using confocal microscopy. Scale bars depict 10 µm. Data represent three independent experiments.
Figure 6.
TM4SF5 coimmunoprecipitated with CD151.
(A) Whole cell lysates from Chang cells transfected with control Strep or Strep-TM4SF5 plasmid were immunoprecipitated (IP) with anti-streptavidin-coated agarose beads before immunoblotting using anti-CD151, anti-CD63, or anti-streptavidin. (B) HCC827 lung carcinoma cells transfected with FLAG-mock or FLAG-TM4SF5 were harvested for whole cell extracts. The extracts were immunoprecipitated (IP) with anti-CD151 antibody prior to immunoblotting for FLAG and CD151, in parallel with lysates. (C) Whole cells extracts from Chang-TGFβ1 cells were immunoprecipitated with normal (Nor.) IgG or anti-CD151 (α-CD151) antibody, prior to standard Western blots for CD151 or TM4SF5, in parallel with whole cell lysates (WCL). (D) Chang cells transfected with FLAG-TM4SF5 were immunostained for CD151 (red) and FLAG (green), prior to visualization using a confocal microscope. Note that some TM4SF5 co-localized with CD151 at the membrane surface and internally in endosomal regions, but other TM4SF5 localized independently of CD151 even on the membrane surface. The images were presented in an independent duplicate for the same condition. Data represent three independent experiments.
Figure 7.
Different collaborative effects of TM4SF5, CD151, and CD63 on migration and invasive ECM degradation.
Transwell migration analyses (A to D) or ECM-degradation analyses (E to H) were performed using Chang-TGFβ1 cells transfected with the indicated shRNAs or plasmids. (A to D) The bottom chamber was filled with 10% FBS/DMEM-H. After 18 h, cells migrated to the bottom surface of the filter were stained and imaged. Representative images (at least 5 images) were counted to determine migration in each experimental condition. Mean ± standard deviation were graphed (A to C), and representative images of (C) were shown (D). (E to H) Chang-TGFβ1 cells transfected with shRNA or plasmids were reseeded onto coverslips precoated with Oregon Green 488-conjugated gelatin and incubated for 18 h in a CO2 incubator. The dark-spotted ECM-degraded areas from more than 5 random images were counted for graphic presentations using mean ± standard deviation. Representative images in (H) are shown (G). * or ** depict P values less than 0.001 or 0.05 for statistical significance, respectively, whereas *** depicts P values greater than 0.05 for insignificance, and numbers in (B) represent the P values by Student’s t-tests. Data represent three independent experiments.