Journal: Cell reports

Article Title: TM4SF5-dependent crosstalk between hepatocytes and macrophages to reprogram the inflammatory environment.

doi: 10.1016/j.celrep.2021.110018

Figure Lengend Snippet: Figure 2. TM4SF5-positive hepatocytes affect TM4SF5 expression and activation of MFs (A) THP-1 cells were differentiated to M1- or M2-type MFs for 24 h before determination of indicated mRNA levels. (B) TM4SF5 mRNA and protein levels were determined in THP-1 monocytes and M0-, M1-, and M2-type MFs. (C) Relative mRNA levels of the indicated molecules were determined in THP-1 cells infected with EV or TM4SF5-lentivirus. (D and E) CM of SNU449 or SNU449-TM4SF5 cells was used to treat THP-1-derived M1- or M2-type MFs for 24 h before determination of TM4SF5 (D) or CD11b (E) mRNA levels.

Article Snippet: Five-week-old WT or Tm4sf5 / male C57BL/6 mice were randomly assigned (nR 7 per group) and maintained on an ad lib control chow (Teklad AIN-93G purified diet or normal chow diet, NCD) or high-fat diet (Teklad 60 Kcal% fat, Orient.

Techniques: Expressing, Activation Assay, Infection, Derivative Assay

Journal: Cell reports

Article Title: TM4SF5-dependent crosstalk between hepatocytes and macrophages to reprogram the inflammatory environment.

doi: 10.1016/j.celrep.2021.110018

Figure Lengend Snippet: Figure 4. IL-6 secreted by active M1 MFs promotes CCL20 and CXCL10 expression in TM4SF5-positive hepatocytes (A and B) Primary hepatocytes from WT or KO mice were treated with the CM from THP-1-derived M1- or M2-type MFs (A) or co-cultured with primary WT Kupffer cells by using a Tranell chamber system (B) for 24 h, before determination of Ccl20 or Cxcl10 mRNA levels in the hepatocytes. (C) IL-6 mRNA levels were determined from M0, M1, or M2 MFs differentiated from THP-1 cells for 24 h. (D) Primary hepatocytes from WT or KO mice were treated with IL-6 for 24 h, before Ccl20 or Cxcl10 mRNA level determination.

Article Snippet: Five-week-old WT or Tm4sf5 / male C57BL/6 mice were randomly assigned (nR 7 per group) and maintained on an ad lib control chow (Teklad AIN-93G purified diet or normal chow diet, NCD) or high-fat diet (Teklad 60 Kcal% fat, Orient.

Techniques: Expressing, Derivative Assay, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: siRNA or shRNA targeting sequences against TM4SF5 or GLUTs

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: shRNA, Sequencing, Negative Control

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: TM4SF5 mediates the differential modulation of blood glucose levels and BW depending on age. (A, B) BW changes (A) and survival rates (B) of WT and Tm4sf5 −/− homozygote KO mice ( n = 25). (C) BWs of 12‐month‐old WT or KO mice ( n = 7 or 9). (D–F) IPGTT (left) or IPITT (right) using 3‐ (D), 6‐ (E), 12‐ or 18‐month‐old (F) WT or Tm4sf5 −/− KO mice ( n = 9). Data are shown as the mean ± standard deviation (SD). * P < 0.05. # P < 0.05 or ## P < 0.01 depicts a statistically significant difference in blood glucose levels between the animal groups at 0 or 30 min, respectively. * indicates P < 0.05 for the statistical comparison between WT and KO mice based on the AUC calculations for 0–120 min, whereas ns depicts no significance. (G) Serum insulin levels of 3‐month‐old WT, KO or Alb ‐Tm4sf5 TG mice ( n = 10). ns, no significance. (H) Liver tissues from WT mice at the indicated ages were processed to determine Tm4sf5 mRNA levels. (I) The primary hepatocytes or BAT from 3‐month‐old WT or KO mice were analysed for Tm4sf5 mRNA. See also Figure .

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Standard Deviation, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: Tm4sf5 −/− KO mice fed an HCD are glucose intolerant irrespective of insulin resistance. (A) Analysis of TM4SF5‐binding proteins in hepatocarcinoma SNU761 cells ectopically expressing TM4SF5‐Strep. (B, C) Immunoblot of HEK293FT (B) and SNU449 (C) for the indicated molecules. Because TM4SF5 can be multimerized (Lee et al., ) and palmitoylated or N ‐glycosylated (Kim et al., ), the immunoblots showed blurry bands at 20∼35 kD and multimers at higher molecular weights. (D) Confocal fluorescence images of SNU449 cells stably transfected with TM4SF5‐FLAG stained for TM4SF5‐FLAG (red), GLUT1‐HA (green) and DAPI (blue). (E–H) 2‐DG uptake and TM4SF5 expression in TM4SF5‐suppressed Huh7 cells (E, F) and Tm4sf5‐overexpressing AML12 cells (G, H). *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. (I, J) Glycolytic stress test (I) and glucose sensitivity assay (J) of normal AML12 hepatocytes. **** P < 0.0001. # P < 0.05 for the ECAR values significantly different between control and Tm4sf5‐HA‐psotive AML12 cells at the indicated times (min). Data shown represent three independent experiments. (K–M) IPGTT (K) and IPITT (L) in WT and KO mice ( n = 10) fed an HCD for 10 weeks. Changes in BW were calculated (M). ns, no significance. *, **, *** and **** for P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively. # depicts for statistically significant difference ( P < 0.05) for Y ‐axis values between the sample groups at the indicated time point. See also Figure .

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Binding Assay, Expressing, Western Blot, Fluorescence, Stable Transfection, Transfection, Staining, Sensitive Assay, Control

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: TM4SF5 is recruited into hepatic sEVs. (A) TEM image of Huh7 cells transfected with TM4SF5‐APEX2. Multi‐vesicular body, MVB. (B–E) Western blot analysis for sEV markers and control (B), MALDI‐TOF peptide peak sequence alignment (C), heatmap of common peptide peak intensity profiles (D), and PCA (E) of sEVs from control and TM4SF5‐suppressed Huh7 cells. (F–H) Western blots for v5, sEV markers and controls (F), negative‐stain TEM images (G) and cryo‐EM (H) of sEVs from Huh7 cells transfected with TM4SF5‐APEX2. (I, J) Histogram (I) and heatmap with particle diameters of certain ranges in percentages (J) of sEVs from Huh7 cells stably transfected with shNS or shTM4SF5. (K) SNU449 cells expressing control vector (Cont), Strep‐tagged TM4SF5 WT, N ‐glycosylation‐deficient (N138A/N155Q, Gly − ) mutant or palmitoylation‐deficient (C2/6/9/74/75/79/80/84/189A, Pal − ) mutant were used for sEVs and blotted with Strep‐MAB‐classic‐HRP antibody. The data shown represent three isolated experiments. See also Figure

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Transfection, Western Blot, Control, Sequencing, Staining, Cryo-EM Sample Prep, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Isolation

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: Extracellular glucose causes secretion of TM4SF5‐loaded sEVs from hepatocytes. (A, B) sEVs from cells expressing control HA vector (Cont) or HA‐TM4SF5 were immunoblotted for HA. (C–E) sEVs from cells stably infected or transfected for control vector, indicated cDNAs, or shRNAs were immunoblotted. (F) Induced extracellular vesicles (IEVs) or sEVs from SNU449 cells stably expressing control vector (Cont) or HA‐TM4SF5 were immunoblotted for HA. (G–K) sEVs (G–I and K) or whole‐cell lysates (J) from control Huh7 cells and cells stably transfected with shNS or shTM4SF5 #4 (K) were immunoblotted (G–J) or subjected to NTA analysis (K). ** P < 0.01. ns depicts no significance. (L, M) Whole‐cell lysates or sEVs from Huh7 cells transfected with diverse plasmids for empty control vector (Cont), WT, or mutant TM4SF5‐Strep forms were immunoblotted. Data shown present three independent experiments. See also Figure .

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Expressing, Control, Plasmid Preparation, Stable Transfection, Infection, Transfection, Mutagenesis

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: Characteristics of TM4SF5‐loaded sEVs derived from mouse liver or hepatocytes. (A) Schematic diagram of an LCVC system. (B) Liv‐sEVs from Tm4sf5 −/− KO or Alb‐ Tm4sf5 TG C57BL/6N mice or sEVs from primary hepatocytes of KO or TG mice were immunoblotted for the indicated mTm4sf5, FLAG tag and other exosomal markers. (C) Negative‐stain TEM images of liv‐sEVs isolated from LCVC biofluids. (D and E) Size distribution (mean ± SD), heatmap (D) and graphic presentation (E) of liv‐sEVs (D, left and E) from WT, KO and TG mice ( n = 7∼11) or serum‐derived sEVs (D, right) from 6‐month‐old WT mice. * and **** depict for P < 0.05 and P < 0.0001, respectively. (F–J) PCA (F), Venn diagram (G), heatmap (H), MALDI‐TOF peptide fingerprinting (I) and protein–protein interactions (J) of liver‐derived sEVs from WT and Tm4sf5 −/− mice ( n = 3). Circles represent genes, and rectangles represent ontology terms.

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Derivative Assay, FLAG-tag, Staining, Isolation

Journal: Journal of Extracellular Vesicles

Article Title: Liver‐originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance

doi: 10.1002/jev2.12262

Figure Lengend Snippet: Liv‐sEV Tm4sf5 enhance glucose clearance in BAT. (A) IPGTT of WT and Tm4sf5 −/− KO single cohort mice before (left) and after (right) injection of liv‐sEVs from Alb ‐Tm4sf5 TG mice ( n = 10). (B) Age‐matched WT and KO male C57BL/6N mice ( n = 5) without sEV pre‐injection or KO mice ( n = 6) with a tail vein injection of liv‐sEV Tm4sf5‐FLAG were intraperitoneally injected with 14 C‐glucose (40 μCi/kg). The blood 14 C‐glucose levels at various times after glucose injection were assessed using retro‐orbital blood samples for LSC counting. (C) WT or KO mice ( n = 4 or 6, respectively) were subjected to rectal and interscapular BAT temperature measurement simultaneously at every minute (0∼5 min) following cold exposure for 3 h. * P < 0.05. (D) sEV incorporation into WT mice ( n = 10) was imaged 24 h after injection of near‐infrared‐dye‐labelled liv‐sEVs from KO or TG mice. (E–G) Glucose stress test (E) and glucose sensitivity assay (G) of primary WT BAT cells treated with either AML12‐sEV Control or AML12‐sEV HA‐Tm4sf5 and either DMSO or fasentin. (F) Immunoblots of AML12‐Cont or AML12‐HA‐Tm4sf5 cell extracts and sEVs. (H, I) Glucose stress test (H) and glucose sensitivity assay (I) of primary BAT cells whose various Glut forms were knocked‐down in the presence of AML12‐sEV Control or AML12‐sEV HA‐Tm4sf5 . (J) AML12‐sEV Control or AML12‐sEV Tm4sf5‐Strep with or without TSAHC (1 μM) pretreatment were exposed to primary WT BAT. sEV protein extracts precipitated using Strep‐agarose beads were immunoblotted using Strep‐MAB‐classic‐HRP and Glut4 antibodies. (K) Glucose uptake assay of primary BAT after treatment with AML12‐sEVs. (L) Blood glucose levels of mice after IP glucose injection. Single cohort [WT, KO and Alb ‐Tm4sf5 TG ( Alb ‐TG), n = 8∼10] mice were used for AAV8‐ Tbg ‐Tm4sf5 injection. (M) Confocal immunofluorescence of BAT cells treated with AML12‐sEV Tm4sf5‐HA . (N, O) WT or Tm4sf5 −/− KO mice were injected with AAV8‐ Tbg ‐HA‐Tm4sf5, and BAT was analysed by immunohistochemistry for HA/DAB and hematoxylin (N) or by immunoblotting for the indicated molecules (O). (graph) Band intensities normalized with those of α‐tubulin, showing the mean ± SD between groups. * and ** indicate P < 0.05 and P < 0.01, respectively. ns depicts no significance. # and ## depict a statistically significant difference ( P < 0.01 or P < 0.01, respectively) for Y ‐axis values between the sample groups at the indicated time points. Data shown represent three independent experiments. See also Figures .

Article Snippet: Plasmid constructs used included TM4SF5‐STrEP ® , mouse Tm4sf5‐STrEP ® in pEXPR‐IBA‐103 (IBA Lifesciences, Goettingen, Germany), HA‐TM4SF5, HA‐mouse Tm4sf5, HA‐GLUT1, HA‐GLUT2, HA‐GLUT3, HA‐GLUT4 and HA‐GLUT9, which were cloned into the pCMV‐HA‐N vector (Clontech).

Techniques: Injection, Sensitive Assay, Control, Western Blot, Immunofluorescence, Immunohistochemistry

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice led to intramucosal adenocarcinomas in the intestines. We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using hematoxylin and eosin (H&E) staining. Tissues from two representative animals are shown separately in combined images (A, B). We quantified the pathological conditions (C).

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Over Expression, Staining

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice increased β-catenin stabilization and transcriptional activity. (A) We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using immunohistochemistry (IHC) with mouse IgG or anti-β-catenin antibody. β-catenin immunostaining intensities were categorized as explained in the Materials and Methods section or quantitative comparison between conditions. (B) We analyzed HT29 cells via luciferase reporter assay following transfection with the β-catenin-responsive LEF/TCF-1 reporter pTOP-FLASH vector with shRNA plasmids (shControl or shTM4SF5) for 24 h; cells were treated with (+) or without (−) Wnt-3a for 12 h before the analysis. *, **, *** depict P < 0.05, 0.01, and 0.005, respectively. (C-E) HT29 (C, D) or HT116 (E) cells were independently transfected with control or Apc-full (C), shControl (shCon) or shTM4SF5 (D), or Mock-Flag or TM4SF5-Flag (E) plasmids for 24 h, and the cells were treated with recombinant Wnt-3a for 12 h, as explained in (B), prior to whole-cell extract preparation for standard Western blots for the indicated molecules. The data represent three independent experiments.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Over Expression, Activity Assay, Immunohistochemistry, Immunostaining, Comparison, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, shRNA, Control, Recombinant, Western Blot

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Apc Min/+ :Tg TM4SF5 mice showed sinusoidal dilatation, portal hypertension, and extracellular matrix (ECM) deposits in the liver. (A-C) We analyzed liver tissues from wild-type (WT, n = 4), Apc Min/+ ( n = 4), or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using IHC with normal IgG, anti-TM4SF5, or anti-β-catenin antibody (A) and H&E staining or Masson’s trichrome staining (B). The liver tissues were also immunoblotted for the indicated molecules (C). (D) TM4SF5-null SNU449 or endogenously TM4SF5-expressing Hep3B hepatocytes were transfected with control, TM4SF5 WT, shControl, or shTM4SF5 #4 plasmids for 48 h, before whole cell lysate preparation for immunoblots for the indicated molecules. The data represent three independent experiments.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Staining, Expressing, Transfection, Control, Western Blot

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Tg TM4SF5 mice at 1.5 years old showed the phenotypes of portal hypertension associated with steatohepatitis and ECM deposits. We collected and analyzed liver tissues from 1.5-year-old C57BL/6 WT or Tg TM4SF5 male mice (n = 7) using H&E staining and Masson’s trichrome staining. Random representative images are shown. Image magnification is shown at 100× or 400×.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Staining

Journal: Molecular Therapy Oncolytics

Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

doi: 10.1016/j.omto.2022.01.006

Figure Lengend Snippet: Ab27 inhibits cancer cell growth by suppressing TM4SF5-mediated STAT3 phosphorylation (A) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis with rabbit anti-TM4SF5 (in-house) (left) and flow cytometry analysis with Ab27 (right). The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (right). (B) Cells were transfected with siRNA against TM4SF5 for 48 h and then immunostained with Ab27 (5 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) Internalization analysis. HCT-116 cells were incubated with Ab27 (0.3 μg/sample) for 45 min at 4°C, washed to remove unbound antibodies, and then either warmed to 37°C to allow internalization or maintained at 4°C for the indicated periods. Cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. (D) SNU-449Tp cells were treated with DyLight 488, conjugated with Ab27 (green) for 3 h at 37°C, and stained with LysoTracker red DND-99 (red). Cell nuclei were counterstained with DAPI (blue). Arrows indicate signal co-localization. Scale bar, 20 μm. (E) Cells were transfected with siRNA against TM4SF5 for 48 h before lysis for immunoblot analysis. (F) Cells were incubated with Ab27 (250 μg/mL) for 48 h under suspension conditions before lysis for immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed using GAPDH as a loading control except that phosphorylated STAT3 and FAK were normalized against the corresponding total protein (E and F). (G) Anchorage-independent growth assay in the presence of Ab27. Colonies (>0.5 mm for SNU-398 and >0.3 mm for HT-29 cells) were counted in six 100× fields per well. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01.

Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

Techniques: Phospho-proteomics, Transfection, Lysis, Western Blot, Flow Cytometry, Fluorescence, Control, Staining, Binding Assay, Activity Assay, Incubation, Suspension, Growth Assay

Journal: Molecular Therapy Oncolytics

Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

doi: 10.1016/j.omto.2022.01.006

Figure Lengend Snippet: Ab27 inhibits HCC growth in xenograft mouse models (A) SNU-449T7-luc (stably overexpressing TM4SF5 and luciferase) cells (5 × 10 5 ) were injected orthotopically into mouse liver after minimal incision. On day 7, Ab27 (100 μg/mouse) was i.p. injected 2 or 3 times per week for 3 weeks (total of 8 injections). PBS was injected as a negative control. Left: Up to 27 days after cell injection, bioluminescence images were acquired. Right upper: Total bioluminescence flux for 3 weeks of treatment. Right lower: Body weight of injected mice. (B and C) Sorafenib-resistant SNU-449T7 (1 × 10 6 ) cells were mixed with Matrigel and injected subcutaneously into the backs of mice. Ab27 (250 μg/mouse) or sorafenib (400 μg/mouse) was i.p. injected at 2- or 3-day intervals (total of 8 injections). (B) Top: Tumor volume (length × width 2 /2). The minimum value in each group was excluded from the mean calculation. Center: Body weight of injected mice. Bottom: Photographs of dissected tumor masses on day 30. (C) Immunoblot analysis of tumor extracts. Densitometric quantification of bands on the immunoblot was performed using α-tubulin as a loading control, except for phosphorylated proteins, which were normalized against the corresponding total protein. (D and E) SNU-398 cells (1 × 10 7 ) were injected subcutaneously into the flanks of mice. Ab27 (300 μg/mouse), cetuximab (300 μg/mouse), or sorafenib (600 μg/mouse) was i.p. injected into mice (total of 6 injections). Normal human IgG (300 μg/mouse) was injected as a negative control. Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. (E) Ki67 staining of tumor sections was performed to measure the level of cell proliferation. Representative images are shown. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (B and D).

Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

Techniques: Stable Transfection, Luciferase, Injection, Negative Control, Western Blot, Control, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

doi: 10.1016/j.omto.2022.01.006

Figure Lengend Snippet: Cross-reactivity and in vivo toxicity of Ab27 (A) Immunoblot analysis with rabbit anti-TM4SF5 (in-house) and flow cytometry with Ab27 (0.05 μg/sample). (B) CT-26 cells were immunostained with Ab27 (3 μg/mL) (green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (C) PC3 cells were transfected with HA-tagged mouse TM4SF5-expression vector for 48 h. Left, immunoblot analysis with anti-HA and anti-mouse TM4SF5 (in-house) antibodies. Right, flow cytometry with Ab27. (D) ICR mice were i.v. injected with Ab27 (48 mg/kg) or control IgG. Liver function was assessed 28 days post-injection by measuring serum concentrations of ALT, AST, ALP, GGT, Tbil, Dbil, ALB, and T-PRO. ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BW, body weight; Dbil, direct bilirubin; GGT, γ-glutamyl transpeptidase; Tbil, total bilirubin; T-PRO, total protein. Values represent means ± SDs.

Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

Techniques: In Vivo, Western Blot, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection, Control

Journal: Molecular Therapy Oncolytics

Article Title: Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

doi: 10.1016/j.omto.2022.01.006

Figure Lengend Snippet: Target recognition and antitumor activity of humanized antibody Ab27-hz9 (A and B) Affinities of Ab27 and Ab27-hz9 for recombinant human EC2-GST protein were determined using competition ELISA (A) and a BIAcore T200 system (B). k a , association rate; k d , dissociation rate. (C and D) Flow cytometry (C) and immunocytochemistry (D) of SNU-449Cp and SNU-449Tp cells with Ab27 and Ab27-hz9. The extent of a shift in the fluorescence signal compared to control staining, representing binding activity of antibody, is shown as a graph (C). Scale bar, 50 μm. (E) Internalization analysis of SNU-449Tp cells with Ab27 and Ab27-hz9, as described in Figure 1 C. Of note, Ab27 (0.3 μg/mL) and Ab27-hz9 (0.2 μg/mL) were used to maintain a similar extent of initial antibody binding to TM4SF5 on the cell surface. (F and G) SNU-449T 7 cells (3 × 10 6 ) were subcutaneously injected into the flanks of mice. Normal human IgG (negative control), Ab27, Ab27-hz9, or cetuximab (300 μg/mouse) was i.p. injected (total of 12 injections). (F) Top: Tumor volume (length × width 2 /2). Bottom: Body weight of injected mice. Right: Photographs of tumor-bearing mice on day 33. (G) Ki67 staining of tumor sections. Scale bar, 250 μm. Values represent means ± SDs. ∗p < 0.05; ∗∗p < 0.01. p value is shown on the graph (F).

Article Snippet: Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described, , and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5 and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).

Techniques: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunocytochemistry, Fluorescence, Control, Staining, Binding Assay, Injection, Negative Control