Fig 2: Cytoskeleton rearrangement and altered cell mechanics in small intestine on deletion of Pggt1b. (A) F-actin fibre staining using AlexaFluor488-phalloidin in ileum tissue (green). High resolution confocal microscopy (Leica Stellaris). Representative pictures of a maximum projection (system optimised z-stack). Yellow arrows indicate apical actin network; orange arrows indicate redistribution of actin fibres. Three independent experiments. (B) Representative pictures of Myosin IIA staining (red) in ileum tissue (five independent experiments). Confocal microscopy (Leica SP8). (C) RT-FDC analysis from small intestine IECs (n=4/group). (D) Expression and redistribution of selected candidate AJC proteins in ileum tissue (claudin-1, claudin-2 and E-cadherin). Immunostaining (top, red signal) and western blot (bottom). Band densitometry quantification (right). Minimum five independent experiments. Data are expressed as box-plots (Min to Max). One-way ANOVA, Dunnett’s multiple comparisons test. *P≤0.050. AJC, apical junction complex; ANOVA, analysis of variance; RT-FDC, real time fluorescence deformability cytometry.

Fig 3: Western blot and immunofluorescence confirmation of Il6ra−/− in isolated Müller glial cells (MGCs). (A) Knockout of Il6ra was assessed by western blot of MGCs isolated from wildtype (WT), floxed littermates (fl/fl), and MGC-specific Il6ra−/− (KO) mice. Purified IL-6Rα protein (70 kDa) was used as a positive control (+ ctrl) for the membrane-bound receptor. Western blots were also probed for β-actin (42 kDa) for normalization. IL-6Rα protein expression was observed in WT (n = 3) and floxed (n = 1) MGC lysates but absent in KO MGCs (n = 5, FC = 0.156). Data is represented as fold control mean ± SD, n = 3–5/group, ****p-value < 0.0001 vs. WT (unpaired T-test). (B) Immunofluorescence staining for IL-6Rα (red) and vimentin (green) in isolated MGCs from WT and KO mice, counterstained with DAPI (blue). Images were captured using a Leica STELLARIS Confocal Microscope at 40× magnification (n = 4 technical replicates/group, n = 5–10 images/well).

Fig 4: Orthogonal images of stained MFUM-RPTEC-1 grown on inserts in the first passage following protocol 1 shown in green and red for (A) CK-18, (B) phalloidin, (C) P-gp, (D) MATE-1, (E) MRP4, and (F) OCT2. A mounting medium with DAPI was used to stain the nuclei (shown in blue) for all samples. The magnification of all images shown is 63× (according to the manufacturer’s microscope specifications, LEICA SP8 Stellaris upright confocal system, Leica Microsystems, Germany).

Fig 5: Orthogonal images of stained MFUM-RPTEC-2 grown on inserts in the first passage following protocol 2 shown in green and red for (A) CK-18, (B) phalloidin, (C) P-gp, (D) MATE-1, (E) MRP4, and (F) OCT2. A mounting medium with DAPI was used to stain the nuclei (shown in blue) for all samples. The magnification of all images shown is 63× (according to the manufacturer’s microscope specifications, LEICA SP8 Stellaris upright confocal system, Leica Microsystems, Germany).