Journal: Autophagy

Article Title: TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

doi: 10.1080/15548627.2017.1422850

Figure Lengend Snippet: Intracellular accumulation of TGFB1 large latent complex in Ilk cKO murine fibroblasts. (A) Control and Ilk cKO primary murine fibroblasts were grown for 4 or 7 d in the absence of serum. Ilk ablation abolished formation of FBN1 (fibrillin 1) and LTBP1 fibers and networks (red) in the extracellular space. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (B) Levels of LTBP1 (not bound to the TGFB1 SLC and not anchored to ECM) secreted into the supernatant (SN) were comparable in control and Ilk cKO fibroblasts. By contrast, cell lysates (CL) showed an increased presence of shifted signals representing LTBP1 molecules that are disulfide-linked to the TGFB1 prodomain LAP. This complex is indicated as LLC. Levels of FBN1 in supernatants (SN) and cell lysates (CL) were not affected by absence of ILK. (C) Immunoblotting for the TGFB1 prodomain (LAP) confirmed increased intracellular presence (CL) of TGFB1 linked to LTBP1 in Ilk cKO fibroblasts cultured for 2 d. Upon reduction (+SH), the LAP signal shifted down to its monomeric position. Membranes stained by Ponceau indicate comparable loading. (D) Control and Ilk cKO primary murine fibroblasts were transfected with the double-tagged LAP-TGFB1 construct (shown in Figure S2A). After 24 h, HA-LAP-TGFB1 localization was visualized by detection of the tags. First, nonpermeabilized cells were incubated with an antibody specific for the HA-tag to detect secreted HA-LAP-TGFB1 (green). Cells were then permeabilized and incubated with an anti-FLAG antibody to visualize total HA-LAP-TGFB1-FLAG. Abundant intracellularly located, but hardly any extracellular LAP-TGFB1 was detected in Ilk cKO fibroblasts. Scale bar: 10 μm.

Article Snippet: Antibodies Antibodies directed to the following proteins were used: ACTB/actin (BD Transduction Laboratories, 612656, clone C4); ATG5 (Sigma-Aldrich, A0856); ATG7 (Cell Signaling Technology, 8558P); BECN1 (Novus Biologicals, NB110-87318); cerulean and YFP were detected using a polyclonal antibody against GFP (Abcam, ab290 and ab6556); FLAG M2 (Sigma-Aldrich, F3165); GAPDH (EMD Millipore, ABS16); GFP (Abcam, ab290); GOLGA2/GM130 (BD Transduction Laboratories, 610823); GORASP2 (Proteintech, 10598-1-AP); HA (hemagglutinin; Sigma-Aldrich, 12158167001, clone 3F10); FBN1 (kind gift from the Sakai lab, Shriners Hospital for Children, Portland, OR, USA; 9543); ILK (Sigma-Aldrich, I1907); LAP (R&D Systems, AF-246-NA); LTBP1 (Sakai lab, Shriners Hospital for Children; 8579, for IB); MAP1LC3B (Sigma-Aldrich, L7543); MYC (Santa Cruz Biotechnology, sc40, clone 9E10 and Santa Cruz Biotechnology, sc789, clone A14); RAB8A (Sigma-Aldrich, SAB2500852); RAB8B (Proteintech, 11792-1-AP); SMAD2 (Cell Signaling Technology, 3103, clone L16D3) and phospho-SMAD2 (Cell Signaling Technology, 3101S); SQSTM1 (Enzo Life Sciences, BML-PW9860-0100); TGFB1 (R&D Systems, 3709); V5 (AbD Serotec, MCA1360).

Techniques: Control, Staining, Western Blot, Cell Culture, Transfection, Construct, Incubation

Journal: Autophagy

Article Title: TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

doi: 10.1080/15548627.2017.1422850

Figure Lengend Snippet: Transport of latent TGFB1 to the cell surface requires RAB8A-mediated secretion. (A) Immunofluorescence analysis of human Wi26 fibroblasts expressing HA-LAP-TGFB1 (red), stained for endogenous RAB8A (green) and GOLGA2 (magenta) identified colocalization of HA-LAP with RAB8A at Golgi-derived autophagosomal intermediates (yellow in merged inset). Scale bar: 10 μm. (B) Immunofluorescence analysis of murine control and Ilk cKO fibroblasts, transfected with HA-tagged RAB8A (green; DAPI-stained nuclei appear blue) showed relocalization of RAB8A to perinuclear aggregates. Scale bar: 10 μm. (C and D) Immunoblots illustrating knockdown efficiency of RAB8A (C) and RAB8B (D) in comparison to control (si-Scr). ACTB levels were used to indicate comparable loading. (E and F) Secreted TGFB1 levels were significantly reduced upon knockdown of RAB8A (P<0.001) (E); whereas no change in TGFB1 secretion was detected upon knockdown of RAB8B (F). Each symbol represents one independent transfectant. (G and H) Autophagy was analyzed in human Wi26 fibroblasts following treatment either with DMSO, or with 100 nM BafA, or with 20 μg/ml rapamycin (Rapa) for 4 h. The upper and lower ‘<’ symbols in the immunoblots indicate the position of the MAP1LC3B-I and -II variants, respectively. Autophagy was visualized by the presence of MAP1LC3-II and SQSTM1 bands in immunoblots. Depletion of RAB8A (G) and of RAB8B (H) did not affect basal or stimulated autophagy. Data are representative of 3 experiments. Signal intensities were quantified densitometrically and the ratio of MAP1LC3-II to ACTB is presented below the blots. (I) Immunofluorescence analysis of MEFs derived from atg5 KO or Atg5 WT animals expressing HA-LAP-TGFB1 (red) and stained for endogenous RAB8A (green) identified partial colocalization of HA-LAP with RAB8A at Golgi-derived autophagosomal intermediates (yellow in merged inset), which is not observed in atg5 KO cells. Scale bar: 5 μm. (J) The images of 20 Atg5 WT and 26 atg5 KO cells were used to determine the Pearson correlation coefficient, which reflects the extent of colocalization of TGFB1 with RAB8A (LAP and RAB8A). Values of about 0.15 confirmed selective colocalization.

Article Snippet: Antibodies Antibodies directed to the following proteins were used: ACTB/actin (BD Transduction Laboratories, 612656, clone C4); ATG5 (Sigma-Aldrich, A0856); ATG7 (Cell Signaling Technology, 8558P); BECN1 (Novus Biologicals, NB110-87318); cerulean and YFP were detected using a polyclonal antibody against GFP (Abcam, ab290 and ab6556); FLAG M2 (Sigma-Aldrich, F3165); GAPDH (EMD Millipore, ABS16); GFP (Abcam, ab290); GOLGA2/GM130 (BD Transduction Laboratories, 610823); GORASP2 (Proteintech, 10598-1-AP); HA (hemagglutinin; Sigma-Aldrich, 12158167001, clone 3F10); FBN1 (kind gift from the Sakai lab, Shriners Hospital for Children, Portland, OR, USA; 9543); ILK (Sigma-Aldrich, I1907); LAP (R&D Systems, AF-246-NA); LTBP1 (Sakai lab, Shriners Hospital for Children; 8579, for IB); MAP1LC3B (Sigma-Aldrich, L7543); MYC (Santa Cruz Biotechnology, sc40, clone 9E10 and Santa Cruz Biotechnology, sc789, clone A14); RAB8A (Sigma-Aldrich, SAB2500852); RAB8B (Proteintech, 11792-1-AP); SMAD2 (Cell Signaling Technology, 3103, clone L16D3) and phospho-SMAD2 (Cell Signaling Technology, 3101S); SQSTM1 (Enzo Life Sciences, BML-PW9860-0100); TGFB1 (R&D Systems, 3709); V5 (AbD Serotec, MCA1360).

Techniques: Immunofluorescence, Expressing, Staining, Derivative Assay, Control, Transfection, Western Blot, Knockdown, Comparison

Journal: Cancer Research

Article Title: Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

doi: 10.1158/0008-5472.can-10-1513

Figure Lengend Snippet: Figure 2. TGF-b1 is necessary for flow-enhanced tumor cell invasion only when fibroblasts are present. Invasion results are normalized to the leftmost condition in each graph unless otherwise noted. A, effects of TGF-b1 neutralization on MDA-MB-435S (TC) invasion (left) or fibroblast (Fb) migration (right) using a function-blocking antibody (a-TGF-b1 block). B, total TGF-b1 levels as measured by ELISA after 18 hours culture with 5 105 tumor cells/mL and 2.5 105 fibroblasts/mL. C, active TGF-b1 levels as measured by TMLC luciferase reporter assay, after 18 hours culture with 2.5 106 reporter cells/mL. The first condition contains only TMLC reporter cells; all other conditions contain the cells noted plus TMLCs. D, left, effects of exogenous TGF-b1 on the invasion of tumor cells alone. Middle, effects of an exogenous TGF-b1 gradient on the invasion of tumor cells alone. Right, effects of exogenous TGF-b1 on tumor cell invasion (in the presence of fibroblasts) and fibroblast invasion alone. Tumor cell and fibroblast invasion are normalized to their respective flow, no TGF-b1 condition. *, P < 0.05 versus matching static condition; #, P < 0.05 between indicated groups.

Article Snippet: As appropriate, 0.45 mg/mL heparan sulfate, 0.45 mg/mL chondroitin sulfate A, 0.3 U/mL heparinase III, 100 mmol/L blebbistatin, 10 mmol/L ML-7 (Sigma-Aldrich), 1 mg/mL mouse anti-human TGF-b1 antibody or mouse IgG1 isotype control (R&D Systems), rhTGF-b1 (PeproTech), 10 mmol/L GM6001 (Millipore), and 2 mg/mL cell-permeable C3 transferase (Cytoskeleton Inc.) were added to the matrix and medium.

Techniques: Neutralization, Migration, Blocking Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay

Journal: Cancer Research

Article Title: Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

doi: 10.1158/0008-5472.can-10-1513

Figure Lengend Snippet: Figure 6. Hypothesized mechanism of flow- and fibroblast-enhanced tumor cell migration. Stromal fibroblasts secrete latent TGF-b1 that binds ECM. Interstitial flow enhances activation and availability of TGF-b1, stimulates collagen degradation, and increases fibroblast migration. As the fibroblast moves, it primes the ECM through Rho-dependent contractility. Nearby tumor cells (already invading by CCR7-dependent autologous chemotaxis) take advantage of the primed matrix to enhance their invasion.

Article Snippet: As appropriate, 0.45 mg/mL heparan sulfate, 0.45 mg/mL chondroitin sulfate A, 0.3 U/mL heparinase III, 100 mmol/L blebbistatin, 10 mmol/L ML-7 (Sigma-Aldrich), 1 mg/mL mouse anti-human TGF-b1 antibody or mouse IgG1 isotype control (R&D Systems), rhTGF-b1 (PeproTech), 10 mmol/L GM6001 (Millipore), and 2 mg/mL cell-permeable C3 transferase (Cytoskeleton Inc.) were added to the matrix and medium.

Techniques: Migration, Activation Assay, Chemotaxis Assay