Glycergel

Animal experiments were performed as previously described [22 (link)]. Expressions of SDC2 protein were measured using immunochemical analysis on 5-mm slices of formalin fixed paraffin-embedded tumor xenografts in nude mice. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 W for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counterstaining with Mayer hematoxylin (Invitrogen), slides were mounted in aqueous mounting medium (glycergel, Dako). Pictures were taken using a LEICA DM 4000B microscope, while the relative level of each protein was calculated using LEICA software, percentage of the mock over the chemotherapeutic treated tumors was calculated and plotted.

Samples were washed with PBS, fixed with 4% (w/v) paraformaldehyde in PBS (PFA, Sigma-Aldrich, Switzerland) for 15 min and then immersed in 0.1 M Glycine (Thermo Fisher Scientific, Switzerland) for another 15 min. Next, samples were immersed with the primary antibody, sheep polyclonal hyaluronic acid (ab53842, Abcam, United Kingdom, at 50 μg/mL) for 2 h at room temperature. Samples were then washed thoroughly, and the secondary antibody, donkey polyclonal anti-sheep Alexa Fluor^® 488 (ab150177, Abcam, United Kingdom, at 20 μg/mL), was added. Additionally, F-actin and nuclei were stained using rhodamine-phalloidin (R415, Thermo Fisher Scientific, Switzerland) and DAPI (4’,6-diamidino-2-phenylindole, 10236276001, Sigma-Aldrich, Switzerland), respectively. The samples were then washed, mounted on microscope slides using Glycergel^® (Dako, Denmark), and stored in the dark at 4°C. Images (1024 x 1024 pixels) were acquired using LSM (LSM 710 meta, Zeiss, Germany) and processed using Fiji (ImageJ, NIH, US).

In situ hybridization was performed as previously described (Stylianopoulou et al., 2016 (link)). Slides were mounted in Glycergel (DAKO) and photographed with a Leica DM5500 B (Leica Microsystems) microscope equipped with a DFC7000T or a DFC310FX digital camera (Leica Microsystems). Images were captured using the camera software (LAS v4.13, Leica Microsystems); image panels and schemata were created with the GIMP (gimp.org).

The Nuclear Actin ChromobodyTagGFP plasmid (pnACTagGFP) was purchased from Chromotek. Cells were plated on sterile cover slips and transfected 24 h later with Lipofectamine 3000 (Thermo Fischer Scientific). The chemotherapy agents (cisplatin/5FU) were added 36 h after transfection for 12 and 24 h. After PBS washes, samples were fixed with 4% paraformaldehyde for 5 min at room temperature, counterstained with Hoescht and then mounted with Glycergel (Dako) supplemented with 2.5% DABCO (Sigma-Aldrich). For co-immunostaining of EdU with p-H3 and nuclear actin chromobody–GFP, cells grown onto round coverslips were treated as described in the figures and incubated with 10 μM EdU for the last 45 min. The coverslips were then washed three times with PBS and fixed with 4% paraformaldehyde for 5 min at room temperature. Permeabilization steps and detection of EdU were performed using click-iT Plus EdU cell proliferation kit, Alexa Fluor 647 dye (Thermo Fischer Scientific, C10640), according to the manufacturer’s protocol. The coverslips were then stained with p-H3 and Hoescht before proceeding to mounting as previously described.

Cell monolayers were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde in PBS, permeabilized using 0.05% Saponin in PBS, incubated for 1 h in PBS supplemented with 5% of goat serum for blocking, and then with the appropriate primary antibodies. The cells were washed 3 times in PBS Tween 20 0.1%, and then incubated with appropriate secondary antibodies. Coverslips were mounted in Glycergel (Dako, C0563) and examined using a Zeiss Axiovert 200 M epifluorescence microscope (Zeiss instruments) or a Zeiss ApoTome 2 microscope piloted with Zen software 2012 or with a confocal Leica TCS SP5 (LAS AF version 2.6.0; laser wavelengths 488 nm, 561 nm and 633 nm) (SFR Necker, Cell Imaging Platform). Images were resized, organized, and labeled using Photoshop software.

To confirm adipogenic differentiation capacity, 3a6 cells without treatment (3a6-wt) and 3a6 Rho-0 cells were cultured in adipogenic differentiation medium and the cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at 4°C, followed by double-staining for 30 minutes with a 1:10000 dilution of a neutral lipid dye, 4,4-Difluoro-2.3,5.6-bis-tetramethylene-4-bora-3a,4a-diaza-s-indacene (LD540), kindly provided by Dr Thiele [35 (link)]. This step was followed by 5 minutes incubation with the nuclear dye 2′-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33258. Sigma-Aldrich). After washing with PBS, cover slips were mounted on microscopy chamber slides using Glycergel (Dako). Fluorescence was visualized and photographed under fluorescence microscopy at 20X (Olympus BX61).

Cells (2x10⁴) were cultured on 12-mm diameter glass cover slips in 24-well plates for 48 hr, washed with PBS, fixed with 4% fresh paraformaldehyde in PBS, blocked with 1% BSA/0.1% NaN₃/PBS for 1hr, stained with primary antibody at 4°C overnight, and then with FITC-conjugated secondary antibody for 1 hr at room temperature. Cell nuclei were stained with Hoechst 33342 (Invitrogen, Paisley, UK). Cells were mounted with Glycergel (Dako Cytomation, Carpinteria, CA) and observed by laser confocal fluorescence microscopy (model Eclipse Ti-U; Nikon, Tokyo, Japan) at 600× magnification.