Formaldehyde

For investigations of the TLR4 and/or nestin-positive cells, freshly isolated and cultivated (6 days) enteric NSPCs were fixed with 4% formaldehyde (Applichem, Darmstadt, Germany) prior immunostaining. The amount of TLR4+/nestin+, TLR4+/nestin−, TLR4−/nestin+ and TLR4−/nestin− cells was determined for 1000 cells using the image-processing software ImageJ (National Institutes of Health, freeware).

mESCs were cultured in GS, after inhibitor withdrawal (DM) and after inhibitor withdrawal plus supplementation of 250 μM DETA/NO (NO) (SIGMA ALDRICH®). At 24 h, cross-linking with 1% formaldehyde (Applichem) and quenching with 10× Glycine (Millipore) as well as nuclear extraction were performed. Then, chromatin solution was sheared by Bioruptor® Plus (Diagenode) to obtain about 500 bp fragments for qRT-PCR detection or 200 bp fragments for Sequencing. Immunoprecipitation protocol was performed after overnight incubation at 4 °C with 1 μg of p53 (Pab240 from Santa Cruz), 8 μg of Zeb1 (H-102 from Santa Cruz), 10 μg of Hdac2 (GeneTex) or 4 μg of Hdac1 (Abcam) antibody with Magna-ChIP™ A/G (Millipore) kit according to manufacturer’s instruction. Details related to used antibodies are provided in Supplementary Table 4. DNA fragments were recovered and analyzed either by Sequencing or by qRT-PCR (0.5–1 μL of immunoprecipitated chromatin) using the primers listed in Supplementary Table 6.
The qRT-PCR analyses were performed in duplicate and the data obtained were normalized to the corresponding DNA input control. Data are represented as relative enrichment (with values for IgG being subtracted from those with antibody).

RM-1 murine prostate-cancer cells were diluted in PBS at a concentration of 1.5 × 10⁶ cells/mL and 100 µL were subcutaneously injected in the right flank of mice. Then, animals were randomly divided into 4 groups of 7 mice so as to start the different treatments on the following day. Control group received a vehicle solution. The second group of mice was treated with an oral dose of Ocoxin (100 µL) daily, the third one received intraperitoneal injections of 5 mg/kg bodyweight of Docetaxel every other day and, finally, the fourth group received the combination of both treatments, that is, a daily dose of Ocoxin and an intraperitoneal injection of Docetaxel on alternate days. After 12 days of treatment, mice were sacrificed and tumors were either frozen in O.C.T^TM Compound (Sakura Finetek, Alphen aan den Rijn, The Netherlands) or fixed in 3.7–4% formaldehyde (AppliChem, Darmstadt, Germany) for 18 h at 4 °C and embedded in paraffin for histological analyses.

For visualization of the osteogenic differentiation the calcium-phosphate deposits were stained by the von Kossa staining. For this the samples were fixed with 10% (v/v) formaldehyde (Applichem, Darmstadt, Germany) for 15 min. Afterwards the cells were washed with ddH₂O and incubated in 5% (w/v) AgNO₃ (Merck, Darmstadt) in ddH₂O for 15 min. The cells were washed again and incubated with 1% (w/v) pyrogallol (Merck) in ddH₂O for 2 min in a dark chamber. After a washing step, the cells were fixed with 5% (w/v) sodium thiosulfate (Sigma-Aldrich) in ddH₂O. The samples were visualized with an Axiovert A1 light microscope adapted with a camera (Axiocam Cc1 60N-C1″1.0x camera, Carl Zeiss).

Mv1Lu or MDA-MB-231 cells were seeded in 24-well plates until they reached 100% confluence, using the appropriate medium for each line with all supplements. At that time, medium was changed to FBS-free medium for 24 h. At the initial time (T0), a cross-shaped scratch was made on the cell monolayer using a sterile p-200 µl pipette tip. After replacing FBS-free culture medium to wash out released cells, OA (indicated concentration at each experiment) or an equivalent volume of DMSO was added. Additionally, EGFR pharmacological inhibitor PD153035 (EGFRi) 2.5 µM, was added to OA sample. After a 24-h incubation period, cells were fixed with 4% formaldehyde (Applichem GmbH, Darmstadt, Germany) in PBS (Biowest, Nuaillé, France) for 10 min. Finally, well plates were washed twice with PBS. Pictures were taken at 10× magnification using an optical microscope equipped with a digital camera (Motic Optic AE31, Motic Spain, Barcelona, Spain). To quantify cell migration, the areas of the gaps in the wounds were measured by ImageJ software. The initial cell‐free surface was subtracted from the endpoint cell‐free surface and plotted in a graph^{66 (link)}.