Oregon green 488 gelatin

Coverslips were coated with either Oregon Green-488 gelatin (for IF analysis) or gelatin labeled using an Alexa594 protein labeling kit (for live imaging analysis) (Thermo Fisher) and used to analyze ECM degradation as per Martin et al. [42 (link)]. Immunofluorescence conditions are as described above.

Glass coverslips (Fisher Scientific, 12-545-82) or glass-bottom dishes (MatTek, P35G-1.5–14 .C-GRID) were coated with 1 mg/ml Oregon Green 488-gelatin or FITC-gelatin (Thermo Fisher Scientific, G13186 or G13187) for 10 min and followed by cross-linking with 0.5% glutaraldehyde (Sigma, G5882) in PBS for 15 min. After that, gelatin-coated coverslips or glass-bottom dishes were treated with 5 mg/ml NaBH₄ (Sigma, 452882) for 3 min. Dissociated cells were plated on fluorescent gelatin-coated coverslips or glass-bottom dishes and were maintained for different time periods with or without treatment, as specified. All images were taken using the same acquisition settings across different experimental groups. The extent of gelatin degradation was quantified using ImageJ (NIH), by which the mean intensity of fluorescent gelatin signals within the regions of aneural AChR clusters or nerve-muscle contact sites was measured, and then normalized with the mean intensity at the adjacent region in the same cell (except in MT1-MMP-mCherry overexpression experiments, the mean intensity at the adjacent region without cell attachment was used for normalization).

Rabbit polyclonal antibodies (pAbs) for ERC1, FLAG, FN, LL5β (recognizing human and monkey LL5β, but not mouse LL5β), and mouse monoclonal antibodies (mAbs) for ERC1a (clone ELKS-30), tubulin, FLAG clone M2 (Sigma-Aldrich); rabbit pAb for GFP (Life Technologies); chicken pAb for GFP and rabbit pAb (Abcam); rabbit pAb anti DS-RED (Clontech); rabbit pAb for Liprin-α1 (Protein Tech); mouse mAb for filamin A (Millipore), paxillin (clone 349, BD Biosciences), GST (Amersham Biosciences), His-tag (Qiagen), LL5α/β (clone 1H12) [19 (link)]; hamster mAb for LL5α [5 (link)] was kindly provided by dr. Yuko Mimori-Kiyosue (RIKEN Center for Biosystems Dynamics Research, Kobe, Japan). Secondary Abs Alexa-488, Alexa-568, Alexa-546 and Alexa-647, phalloidin Alexa-568 and Alexa-647, and Oregon green 488-gelatin (Life Technologies); HRP-conjugated anti-rabbit and anti-mouse secondary Abs (Jackson and Amersham Biosciences). Other reagents included: FN (Corning); poly-L-lysine hydrobromide.

The matrix degradation assay was conducted as described previously [30] (link). Glass-bottom collagen I coated dishes (35 mm, MatTek Corporation, USA, Ashland) were further coated with Oregon green 488 gelatin (Life Technologies) according to the manufacturer’s protocol. Next, 3×10⁴ cells were seeded on each plate and cultured O/N. Cells were fixed with 3.7% PFA, permeabilized with 0.05% Triton-X-100, then blocked with 5% FBS and probed for F-actin (TRITC-phalloidin; Sigma Aldrich). Images of the cells were collected using a confocal microscope Leica SP8 using a 63×/NA 1.4 Plan-Neofluar objective. Invadopodia were manually counted as actin-positive dots associated with gelatin degradation. Matrix degradation areas were calculated as the total area covered by degradation holes/field in thresholded images using the Analyze Particles tool in the NIH ImageJ software and normalized to the cells area in each field.

For collagen degradation assays, 12-well plates were coated with rat tail type I collagen (Life Technologies) for 30 min at 37 °C, 5% CO₂ in a humidified incubator as described for invasion assays. For invadopodia formation on gelatin, glass coverslips were coated with Oregon Green 488-gelatin (Life Technologies) or unlabelled gelatin as described previously.^{67 (link)} Briefly, coverslips were coated with 50 μg/ml poly-L-lysine followed by 0.5% gluteraldehyde, and inverted on a 80 μl drop of gelatin for 10 min. Coated coverslips were then incubated with 5 mg/ml NaBH₄ and washed extensively with PBS. For invadopodia formation on DQ collagen, glass coverslips were coated with 400 μl of 10 μg/ml DQ Collagen, type I (Life technologies), incubated for 1 h at 37 °C and washed extensively with PBS.

Coverslips were washed clean and sterilized in advance and put into 6-well plates. Next day, 50 μL of 1 mg/mL Oregon green 488-gelatin (Life Technologies, MA) was diluted with 200 μL of cold 2% sucrose solution to make the 0.2 mg/mL gelatin solution, and then warmed in a water bath at 37 °C. Then the coverslips were all coated with the gelatin solution. After incubating each coverslip with 200 μL, 0.5% ice-cold glutaraldehyde in PBS, the plates were placed at 4 °C for 15 min. Coverslips were washed with PBS and incubated with 5 mg/mL sodium borohydride in PBS for 3 min. After washing with PBS for 3 times, sterilizing with 70% alcohol for 20 min and washing with PBS for another 3 times, the coverslips were incubated with serum-free culture media for 1 hour at 37 °C. Then 1 × 10⁵ cells were seeded into each well and cultured at 37 °C for 24 h. The next day, coverslips were washed with PBS and fixed with 1 mL 4% paraformaldehyde for 15 minutes. After washing with PBS again, the coverslips were incubated with 1 mL 0.05% Triton-X 100 for 10 min. After washing with PBS, the coverslips were mounted onto slides with Prolong Gold anti-fade reagent with DAPI (Invitrogen, MA). After drying the slides for 1–2 days, the coverslips were observed and imaged using a fluorescence microscope. All the procedures were performed in the dark and at room temperature.

Sterile cover-slips (12 mm, #1 Menzel Microscope Coverslips) were coated with a thin layer of 0.2 mg/ml Oregon Green 488-gelatin (Life Technologies, G13186) for 20 min, fixed in 0.5% glutaraldehyde for 40 min and washed three times with sterile PBS. Cover-slips were then transferred to a 24-well plate and incubated in complete growth medium for 30 min at 37°C. BT549 cells transfected with either Ctrl-siRNA or GOLGB1-siRNA (Dharmacon) for 48h were seeded on top of the cover-slip at a density of 7.5x10⁴ cells/well and incubated overnight. Cells were then processed for anti-GM130 immunofluorescence staining. Images were randomly acquired on Andor Dragonfly spinning disk using a Nikon Ti2 inverted optical microscope equipped with a 60 × TIRF objective (Plan-APOCHROMAT 60 × /1.49 Oil). Fluorescence was collected using an EMCCD camera (iXon Ultra 888, Andor). To quantify the gelatin degraded areas per image, appropriate functions of ImageJ were used. The measured area was divided by the number of cells in the corresponding image. Kruskal-Wallis rank sum test was used to compare the gelatin degraded areas between the two conditions.