Fish gelatin

The FGelMa used in this study was fabricated according to the protocols reported in our previous study [26 (link)]. Firstly, fish gelatin (Sigma-Aldrich, CAT#SLCJ6149, St. Louis, MO, USA) was dissolved in 250 mL of cold distilled water at a concentration of 15 wt% and stirred at 50 °C for 2 h at 100 rpm. A ratio of 0.6 g of methacrylic anhydride (Sigma-Aldrich, Lot#STBK0716, St. Louis, MO, USA): 1 g of gelatin was then added to the fish gelatin solution and stirred at 50 °C for 2 h at 100 rpm in a dark environment. Then, 250 mL of distilled water was added and the FGelMa solution was centrifuged to remove the unreacted methacrylic anhydride. After this, the FGelMa was dialyzed at 40 °C in deionized water for 3 days in order to further remove the unreacted methacrylic anhydride. The pH of the FGelMa solution was adjusted to 7.4, lyophilized, and stored at −20 °C in a refrigerator until further usage.

HGSC cell lines, OCKI patient-derived cancer cells and CAFs, as well as NFs were characterized by IB for COL6A1, PAX8, and FSP1. Cells were lysed with RIPA (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% Igepal CA630, 0.5% sodium deoxycholate) buffer together with 5 mM EDTA, protease inhibitor (cOmplete ULTRA, Sigma), and phosphatase inhibitor (phosSTOP, Sigma) and incubated for 20 min on ice. Cell lysates were centrifuged at 21,300 × g  for 15 min at +4 °C. After mixing the lysates with 5× sample buffer (0.3 M Tris-HCl pH 6.8, 50% glycerol, 10% sodium dodecyl sulfate, 0.05% bromophenol blue; 0.5 M dithiothreitol), heat denaturation was done by incubation at +95 °C for 10 min. Lysates were then separated in 4–20% Mini‐PROTEAN TGX Precast Protein Gels (Bio‐Rad) and transferred to Trans‐Blot Turbo Mini Nitrocellulose Transfer Packs (Bio‐Rad). Membranes were blocked for 45 min with 3% fish gelatin (Sigma) in Tris‐buffered saline (TBS; 10 mM Tris, pH 7.6, 150 mM NaCl) and probed with primary antibody in TBS 0.1% Tween‐20 (TBS‐T) with 3% fish gelatin at the recommended dilutions at +4 °C overnight. Membranes were incubated with IRDye Subclass‐Specific Antibodies (LI‐COR Biosciences) diluted in TBS‐T for 1 h at RT, and the signal was detected using Odyssey Imaging System equipped with the software Image Studio Lite Ver 5.2 (LI‐COR Biosciences).

Proteins were resolved on 4-12% gradient Nu-PAGE gels (Life Technologies) and transferred to nitrocellulose membrane. Membranes were blocked in 2% v/v fish gelatin (Sigma) in PBS-T for 30 min and incubated with the anti BANF1 antibody diluted in 1% fish gelatin in PBS-T overnight at 4°C. Membranes were washed in PBS-T, incubated with secondary antibodies (LiCor) and scanned on an Odyssey infrared imaging system (LiCor). Where necessary, membranes were stripped using a mild stripping buffer (15 g L^-1 glycine pH 2.2, 1 g L^-1 SDS, 1% Tween20) and reprobed with the appropriate antibodies.

Cervical 5-μm cryosections were dried at RT, fixed in 3.7% formaldehyde (Sigma Aldrich) diluted in PHEM buffer as previously described in (34 (link)), permeated with 0.2% Triton X-100 (Sigma Aldrich) in PHEM buffer and blocked with 0.2% cold fish gelatin (Sigma Aldrich), 0.1% Triton X-100 and 10% normal goat serum (Gibco) diluted in PBS. Sections were first incubated with the following primary mAbs: mouse anti-Siglec-1 7-239 Ab (Abcam), rabbit anti-CD11c EP1347Y Ab (Abcam) or rabbit anti-CD14 EPR3653 Ab (Abcam). Samples were then washed extensively with PBS and incubated with the secondary mAbs Alexa 488-conjugated donkey anti-rabbit or Alexa 647-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). Sections were covered with mounting medium (ProLong™ Gold Antifade Mountant with DAPI, Life Technologies, Invitrogen) and a coverslip. Images were obtained by confocal microscopy using a Zeiss LSM 710 microscope and the Zen Blue Image acquisition software.

Embryoid bodies at day 9 were collected and embedded in OCT compound (Bio-Optica). Frozen tissue blocks were sectioned (10 μm) with a CM3050S Leica cryostat (Leica Mycrosystems) and kept at −80 °C. For immunostaining, slides were thawed and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at room temperature (RT). Samples were washed three times in PBS for 5 min and incubated in Gelatin Blocking (GB) solution (2% fish gelatin (Sigma Aldrich), 5% foetal bovin serum (Thermo Fisher), 1% BSA (Sigma Aldrich), 0.3% Triton X-100 (Sigma Aldrich) in PBS) for 1 h at RT. Sections were again washed three times with PBS. The following primary antibodies were resuspended in GB solution and incubated overnight at 4 °C: anti-Sox2 (clone 245610) (Mouse IgG2a, 1:200, BD Biosciences_#560291); anti-Brachyury (Goat IgG, 1:200, Biotechne_#AF2085); anti-TUJ1 (Rabbit IgG, 1:500, BioLegend_#MRB_435P). After 15 min of PBS washes, samples were incubated with secondary antibodies 1 h at RT. Alexa Fluor-568- or 488- conjugated secondary antibodies (Life Technologies) were used at 1:1000 dilution. 6-diamidine-20-phenylindole dihydrochloride (DAPI) (Thermo-Fisher, diluted 1 μg/mL) was used for nuclear staining and incubated in the dark for 5 min at RT. Slides were mounted with ProLong Glass Antifade Mountant (Invitrogen).

L224, Bdf2^KO, or Bdf3^kd trypanosomes were treated for 2–3 d with I-BET151 and Dox, respectively. Cells were stained with unconjugated anti-VSG3 (L224) or anti-VSG2 (Bdf2^KO or Bdf3^kd) primary antibody and washed. Cells were subjected to a 5 min incubation at either 4°C (controls) or at 37°C to allow internalization to take place. Cells were immediately fixed in 1% formaldehyde and stained with a FITC-conjugated secondary antibody (BD 349031) to measure the remaining amount of primary antibody on the surface. Cells were then analyzed by flow cytometry. For immunofluorescence, cells were adhered to poly-L-lysine coverslips following fixation, blocked in PBS with 0.2% cold water fish gelatin (Sigma G7765) and 0.5% (w/v) BSA and stained with anti-mouse IgG FITC (BD 349031) at 1:1,000 for 1 h. Cells were washed, counterstained with DAPI and imaged on a DeltaVision Image Restoration inverted Olympus IX-70 microscope (Applied Precision).

96‐well microplates (Thermo Scientific™ MaxiSorp™, cat. no. 442404, Waltham, MA, USA) were coated with 100 μL of solutions containing human recombinant BAG3 protein (1 μg·mL⁻¹ in PBS1X) or with specific BAG3 peptides and incubated overnight at 4 °C. The day after, wells were washed with PBS 1X‐0.05% Tween and the blocking of nonspecific sites was performed for 1 h at room temperature in PBS 1X containing 0.5% fish gelatin (Sigma‐Aldrich, Saint Louis, MO, USA). Hence, plates were washed five times with the washing buffer and loaded with hybridoma's supernatants, murine anti‐BAG3 clone AC‐2, humanized mAbs, or mouse sera. Plates were then extensively washed and incubated 30 minutes at room temperature with HRP‐conjugated anti‐mouse IgGs 1 : 2000 (115‐035‐205, Jackson ImmunoResearch, Cambridgeshire, UK) or anti‐human IgG 1 : 20 000 (A0170, Sigma‐Aldrich). Subsequently, TMB solution 1X (eBioscience, San Diego, CA, USA) was added to the wells for the analyte detection. The chromogenic reaction was blocked by acidification with 0.5 m H₂SO₄, and the optical density (O.D.) was measured at 450 nm.