Dextran alexafluor 568

The dye-coupling assay to test the functionality of GJs was performed using carboxyfluorescein (2187, Sigma-Aldrich, USA) and Dextran AlexaFluor 568 (10,000 MW, D22912, Invitrogen, USA) was used as a control. Chondrocytes were seeded on plates (28 cm² culture plate) and cultured until the confluence reached 100%. Cells were rinsed two times with PBS and then two scrapes were made using a scalpel in the presence of 0.5% (w/v) carboxyfluorescein and 0.5% (w/v) Dextran AlexaFluor 568 (D22912, Invitrogen, USA) in DMEM at room temperature. Cells were incubated for 2 min at room temperature. After washing with a DMEM, dye transfer was captured using AXIOCAM MRm ZEISS - HBO100 (Zeiss, Germany) with Axioplan 2 (Zeiis, Germany) with Axiovision 4.6 (Zeiss, Germany) software. The number of dye-positive cells (carboxyfluorescein transfer) from the cutting site (red/green cells) to the farthest visual uptake of carboxyfluorescein (only green cells) indicates the GJ connectivity between cells. The score was calculated as previously reported [3 ].

Dechorionated stage 16 sns-nGFP or hand-GFP embryos were covered with oil 10S (VWR) and injected with 0.5 mg/ml Dextran-Alexa Fluor 568; 10,000 MW (Invitrogen D22912) diluted in injection buffer (5 mM KCl and 10 mM sodium phosphate buffer, pH 7.2). Embryos were kept at 25°C for 1 h after injection and then visualised using a LSM710 (Zeiss) confocal microscope.

Antibodies used in this work are commercially available, as follows: from Abcam (Cambridge, UK), anti-H. pylori (ab20459) [24 (link)], rabbit anti-Lamp-1 (ab25030-100), mouse anti-EEA1 (ab2900), rabbit anti-Rab7 (ab50533), rabbit anti-Na⁺K⁺ ATPase (ab76020), rabbit anti-cathepsin D (ab75852), and rabbit anti-histone H3 (ab1791); from Sigma-Aldrich (Missouri, USA), rabbit anti-LC3B (L7543). Additionally, the following reagents were purchased: LysoTracker^® Red DND-99 (Invitrogen, California, USA; L7528), cholera toxin B staining kit (Invitrogen; V34404), Magic Red™ cathepsin B staining kit (Bio-Rad, California, USA; ICT937), Oregon Green™ 488 BAPTA-5N (Invitrogen California, USA; O6812), Dextran Alexa Fluor™ 568 (Invitrogen California, USA; D22912), LysoSensor™ Yellow/Blue DND-160 (Invitrogen California, USA; L7545), 3-methyl adenine (Sigma; M9281), chloroquine (Sigma; C6628), rapamycin (Sigma; R8781), bafilomycin-A1 (Sigma; B1793), and concanamycin A (Sigma; C9705).