Fig 2: TAX1BP1 is recruited to Mtb-containing phagosomes by both its UBZ domain and its interaction with galectin-8. (A) Quantification of TAX1BP1+ (left) or p62+ (right) Mtb in control galectin-8 knockout RAW 264.7 cell lines at 6 h postinfection. Circles represent data for individual clonally selected cell lines. (B and C) Same as in panel A but in RAW 264.7 cell lines in which all three galectins are knocked out (B) or galectin-8 knockdown (KD) BMDMs (C). (D) Immunofluorescence of control or galectin-8 knockout RAW 264.7 cells stably expressing full-length HA-TAX1BP1 or truncated HA-TAX1BP1ΔUBZ. Cells were infected with WT mCherry-expressing Mtb (MOI = 1) and harvested at 6 h postinfection. Green, LC3; magenta, HA-TAX1BP1 variants; red, mCherry Mtb; blue, DAPI. (E) Quantification of indicated variant HA-TAX1BP1-positive Mtb in indicated genotype. Error bars indicate the SEM of knockout cell lines or the SD for knockdown cells; at least 300 bacteria per cell line were assessed. *, P < 0.05; ***, P < 0.005; n.s., not significant.

Fig 3: miRNA profiling in the spinal cord and hippocampus of an FUS mouse model(A) Immunoblot analyses of human wild-type FUS transgene and mouse endogenous FUS from the hippocampi and spinal cords. Expression levels of FUS were evaluated using an antibody with comparable affinity to both human and mouse FUS. Upper (blue arrow) and lower (red arrow) bands are human and mouse FUS, respectively. Increased FUS level leads to a dose-dependent down-regulation of endogenous FUS in both the hippocampi and spinal cords.(B) Immunofluorescent image of a spinal cord motor neuron (upper panel) and hippocampal CA3 pyramidal neurons (lower panel) co-labeled with HA (for the human FUS transgene) and p62/SQSTM1, an autophagy marker. p62 aggregates were readily observed in motor neuron from the homozygous FUS mouse (homo-FUSWT mice), but not in the other genotypes (non-transgenics, or het-FUSWT mice). By contrast, there were no p62 aggregates in the pyramidal neurons across all three genotypes (non-transgenics, het-FUSWT mice, or homo-FUSWT). Different CNS regions respond differentially to increasing FUS level. Scale bar, 10 μm.(C) Principal component analysis (PCA) of miRNA expression data from the hippocampi and spinal cords of an FUS mouse model. The percentages in the axis labels indicate the percentages of explained variances. miRNA expression successfully segregates samples from the two CNS regions. Homo-FUSWT mice segregate from the two genotypes (non-transgenics, and het-FUSWT mice).(D) Schematic flow chart of miRNA expression analysis in the spinal cords and hippocampi.

Fig 4: Galectin-decorated Mtb-containing phagosomes colocalize with selective autophagy markers. (A) Immunofluorescence of RAW 264.7 cells stably expressing 3×FLAG (FL)-tagged galectin-8 infected with WT mCherry Mtb (MOI = 1) 6 h postinfection costained for indicated selective autophagy marker (ubiquitin, p62, and LC3). Green and magenta, endogenous selective autophagy marker or FL-galectin-8 (as indicated); red, mCherry Mtb; blue, DAPI. (B) Quantification of marker-positive and FL-galectin-8+ Mtb bacilli shown in panel A. Marker-positive Mtb that are also FL-galectin-8+ (top) and FL-galectin-8+ Mtb that are also marker-positive (bottom) are shown. Error bars indicate the SD of three coverslips per marker with at least 100 bacteria assessed. (C and D) Same as in panel A but in BMDMs expressing GFP–galectin-8 (C) or in U937 cells expressing FL-galectin-8 (D).

Fig 5: TAX1BP1 is recruited to Mtb-containing phagosomes by both its UBZ domain and its interaction with galectin-8. (A) Quantification of TAX1BP1+ (left) or p62+ (right) Mtb in control galectin-8 knockout RAW 264.7 cell lines at 6 h postinfection. Circles represent data for individual clonally selected cell lines. (B and C) Same as in panel A but in RAW 264.7 cell lines in which all three galectins are knocked out (B) or galectin-8 knockdown (KD) BMDMs (C). (D) Immunofluorescence of control or galectin-8 knockout RAW 264.7 cells stably expressing full-length HA-TAX1BP1 or truncated HA-TAX1BP1ΔUBZ. Cells were infected with WT mCherry-expressing Mtb (MOI = 1) and harvested at 6 h postinfection. Green, LC3; magenta, HA-TAX1BP1 variants; red, mCherry Mtb; blue, DAPI. (E) Quantification of indicated variant HA-TAX1BP1-positive Mtb in indicated genotype. Error bars indicate the SEM of knockout cell lines or the SD for knockdown cells; at least 300 bacteria per cell line were assessed. *, P < 0.05; ***, P < 0.005; n.s., not significant.