Agarose type 7

For cell growth assays, vector ctrl or CRC cells overexpressing Flag-WT-YBX1 or R205A constructs were seeded in triplicate at 2 × 10⁴ cells/well in a 6-well plate. Cells were counted at days 3, 5, 7 and 9 post-seeding using a cell counting chamber. For anchorage-independent growth assays, type VII agarose (Sigma) was used to prepare 2.4% and 1.2% bottom and top agar layers, respectively. 2 × 10⁵ cells were resuspended in the top layer and plated onto the bottom layer. Cells were then cultured for 12–14 days at 37 °C and 5% CO2. Images of colonies were captured using a Canon EOS Rebel T3i Digital SLR camera and colony size and number were quantified using ImageJ (https://imagej.nih.gov/ij/).

For soft agar assays, 1 x 10⁵ cells were suspended in 0.6% type VII agarose (Sigma-Aldrich) and plated in triplicate onto a bottom layer of 1.2% agar in 60-mm plates. Medium was changed every 3 days for 2 weeks. To quantify colonies, each plate was scanned using an automated multipanel scanning microscope and the digital images were analyzed using MetaMorph image quantification software. Laminin rich basement membrane (lrBM) (Matrigel; BD Biosciences) cultures were made by overlaying 200μl of lrBM onto 6 well chamber dishes and allowing it to solidify for 30 minutes at 37ºC in a cell culture incubator. 5×10⁵ cells were embedded in 800μl lrBM and allowed to solidify for 1 hour. The solidified cultures were fed with 1ml complete growth medium, and re-fed with 2ml growth medium every 3 days. Brightfield images were taken at day 7 and total acini were quantified by acini counts in 5 random fields per well.

HT29, DLD1, or HCT116 control cells or cells with overexpression or shRNA knockdown of ARMC4 were plated in triplicates at 20,000/well in a 6-well plate with 3 mL of medium, and the medium was changed every 3 days. Cell number was counted on different days using a cell counting chamber. For anchorage-independent growth assays, type VII agarose (Sigma-Aldrich, Burlington, MA, USA) was autoclaved and mixed with RPMI1640 cell growth medium. Cell culture dishes were coated with 1.2% agarose as the bottom layer. Cells were resuspended in 0.6% of soft agarose and plated on top of the bottom layers. Cells were cultured in the semisolid medium for about 2–3 weeks. The colonies formed were checked under a microscope and measured and counted with the help of ImageJ software (ver. 1.47t).

Sprague Dawley rats were born and raised in the animal facility of the University of Valparaiso, held at 20–30 °C under a 12 h photoperiod with water and food ad libitum. Retinal slices were prepared from 3–4 week-old rats irrespective of sex or weight, by procedures described previously13 (link). The experimental protocols were approved by the bioethics committee of the University of Valparaiso and in accordance with the bioethics and biosafety regulation of the Chilean Research Council (CONICYT). Briefly, rats were anesthetized deeply by halothane inhalation and sacrificed by decapitation. Eyes were quickly removed and the retina was carefully separated from the sclera in a chamber with extracellular solution, containing (in mM): 119 NaCl, 23 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 2.5 CaCl₂, 1.5 MgSO₄, 20 glucose and 2 Na⁺ pyruvate, aerated with 95% O₂ and 5% CO₂, reaching a pH of 7.4. A piece of central retina was embedded in type VII agarose (Sigma), and cut with a vibratome (Leica VT1000S) to 200 μm thickness. Retinal slices were transferred to the recording chamber, sustained by a U-shaped platinum wire, and superfused with oxygenated extracellular solution (flow rate 1 ml/min) at room temperature (20 °C) under conditions of low photopic background illumination (100 lux).

50,000 BT474 shS100P or BT474 shGFP cells were suspended in 0.6% type VII agarose (Sigma-Aldrich) in 10% FBS RPMI medium and plated onto a bottom layer of 1.2% agar in 10% FBS RPMI medium. Cells were plated onto 60mm plates in quadruplicate. 1mL of 10% FBS RPMI with 10 μg/mL trastuzumab was added 24 hours after plating. The medium was changed every 2 days, with fresh trastuzumab added, until cells were analyzed after 3 weeks. To quantify colonies, each plate was scanned using an automated multipanel scanning microscope and the digital images were analyzed using MetaMorph image quantification software.

HT29 cells overexpressing WT-YBX1, S165A-YBX1, and shRNA-YBX1 knockdown cell lines were plated at 2 × 10⁴ cells/well in a six-well plate with 3ml RPMI-1640 media. Cells were seeded in triplicates and counted at different time points i.e. day 2, 4, 6, 8 using a cell counting chamber. For soft agar assays, type VII agarose [Sigma] was autoclaved and mixed with RPMI-1640 cell growth medium. Cell culture dishes were coated with 1.2% type VII agarose as the bottom layer. Cells were resuspended in 0.6% of type VII agarose, and plated on top of the bottom layer. Cells were cultured for 2-3 weeks before being checked under a microscope, measured and quantified with the aid of ImageJ software [http://imagej.nih.gov/ij/].