Fig 2: Implication of CD36 and TGM2 in the effect of retinoic acid (RA) on myelin phagocytosis. To test the involvement of CD36 and TGM2, myelin endocytosis was measured with the pHrodo assay using 0.5 µM all‐transRA (tRA), combined with either 25 µM sulfo‐N‐succinimidyl oleate (SSO), 0.5 µM cystamine hydrochloride, or 50 µM ERW1041E. This was followed by 8 h myelin exposure. (a) Quantification of myelin phagocytosis in Raw264.7 cells showed a significant increase after RA treatment (***p < .001), which was abolished in the presence of the CD36 inhibitor (# p < .05) and reduced, though not significantly when TGM2 activity was inhibitied (n = 6). (b) The same tendency was observed in bone marrow–derived macrophages, although the effect of RA (**p < .01) was smaller (effect of inhibitors: # p < .05, ## p < .01 in comparison with RA treatment only; n = 4). Data are normalized to the control condition without RA treatment

Fig 3: TG2 modulation of Wnt pathway in the zTg2b zebrafish model.A Knockdown of the zTg2b was performed in Tg(7xTCFX.lasiam:GFP)ia4 Wnt-dependent reporter fishes by injecting three increasing dosages of morpholinos (0.05, 0.1, and 0.2 pmol). Zebrafish at 48hpf were observed by bight field and epifluorescence microscopy. The images are representative for the groups of animals and the GFP quantification is reported in the graph (n = 50; means±SEM; ***p < 0.001). Bars: 1 mm. B Zebrafish TG2 protein and zebrafish β-catenin protein from KD and control larvae’s protein extracts were compared by western blot. The quantification is reported in the graph below. The values are indicated as the percentage of the control samples set as 100% (n = 3; means±SEM; **p < 0.01; ***p > 0.001). C GFP fluorescent zebrafish Tg(7xTCFX.lasiam:EGFP)ia4 Wnt-dependent reporter fishes were treated for 15 h with the following compounds: DMSO 0.2%, 40 μM ZDON, 1 mM Cysteamine. Epifluorescence microscopy images are reported, whereas fluorescence quantification is shown in the graph (n = 50; means±SEM; ***p < 0.001). Bars: 1.5 mm.

Fig 4: TG2 affects the expression profile and regulates the HS response.A Volcano plots for whole transcriptomes of differentially expressed genes between KO and WT MEFs. B Venn diagrams illustrating the overlap of the differentially expressed genes (down- or upregulated genes, FC = -1,5 or =1,5, pval_adj=0.05) between KO and WT MEFs, under heat shock. C Gene set enrichment analysis barcode plot of transcriptomic changes of “Cellular response to heat stress” and “regulation of HSF1 mediated heat shock” (from Reactome database) in KO MEFs compared with WT MEFs. Red, blue, and pink shaded rectangles mark upregulated, downregulated, and genes that are unchanged, respectively. D Transient knockdown of the putative TG2 paralogous was performed with three independent morpholinos against zTg2a, zTg2b, and zTg2l; a standard control morpholino was also used. Bright-field images of representative morphants are reported. (n = 50). Bars: 1 mm. E Double transgenic Tg(-1.5hsp70l:GAL4(+/-); UAS:Kaede(+/-)) were injected at one-cell stage either with Tg2bMO or CtrlMO, heat-shock inductions were performed at 24 hpf by incubation of larvae for 1 h at 37 °C, whereas images were acquired between 29 and 30 hpf. Heat-shock protein activation was followed by an increase in the fluorescence. Bright-field and epifluorescent images are reported. The images are representative of different experiments. GFP epifluorescence quantification is reported in the graph on the right. Dots represent treated embryos (n of embryos >40). Thapsigargin was used as a positive control. (n = 3; means±SEM; *p < 0.05; ***p < 0.001). Bars: 1 mm.

Fig 5: Overall visit frequency detected in the PhenoCube system as a function of genotype, age and light cycle phase in animals from the zQ175×TG2 KO line.*Significant HD genotype differences regardless of the light phase (in the mean path length the differences were detected between HET and WT animals); ##significant differences due to light phase in the cycle independently of genotype; ∧significant TG2 genotype differences (see results method for details). WT: wild-type, TG2−/−: homozygous TG2 knockout, HET: zQ175 HET, HOM: zQ175 HOM.