Fig 2: Cells harboring UFC1 with the R23Q mutation fail to undergo morphological differentiation. (A) Cells harboring GFP-tagged wild-type (WT) UFC1 or UFC1 (R23Q) were allowed to differentiate for 0 or 5 days. The square fields a and b indicated by the dotted lines in the center panels are magnified in the bottom panels a and b. Cells with widespread membranes were statistically assessed (** p < 0.01; n = 10 fields [a total of 360 cells per experiment]). (B) The lysates of the respective cells were immunoblotted with an antibody against MBP, PLP1, or control actin. Their expression levels were statistically compared to their respective controls (** p < 0.01 or * p < 0.05; n = 3 blots).

Fig 3: Cells harboring mutated TMEM106B show decreased cell differentiation abilities. (A) Cells harboring wild-type (WT) or mutated (D252N) TMEM106B were allowed to differentiate for 0 or 5 days. Cells surrounded with dotted red lines in the middle panels are magnified in the right panels. The cell surrounded by a white dotted line is a typically differentiated one with widespread membranes. (B) Differentiated cells are statistically depicted (** p < 0.01; n = 10 fields). (C) The lysates of cells at 5 days following the induction of differentiation were immunoblotted with the respective antibodies against differentiation markers PLP1 and MBP, cell lineage marker Sox10, and internal control actin. (D) Quantification of immunoreactive bands, using control immunoreactive bands as 100%, is depicted in the respective graphs of PLP1, MBP, Sox10, and actin (** p < 0.01; n = 3 blots).

Fig 4: Hesperetin recovers phenotypes of cells harboring mutated TMEM106B. (A) Cells harboring mutated TMEM106B were allowed to differentiate for 0 or 5 days in the presence or absence of 10 μm hesperetin (DMSO as the vehicle). Cells surrounded with dotted red lines in the middle panels are magnified in the right panels. The cell surrounded by a white dotted line is a typically differentiated one with widespread membranes. (B) Differentiated cells are statistically depicted (** p < 0.01; n = 10 fields). (C) The lysates of cells at 5 days following the induction of differentiation were immunoblotted with the respective antibodies against differentiation markers PLP1 and MBP, cell lineage marker Sox10, and internal control actin. (D) Quantification of immunoreactive bands, using hesperetin plus immunoreactive bands as 100%, is depicted in the respective graphs of PLP1, MBP, Sox10, and actin (** p < 0.01; n = 3 blots).

Fig 5: Hesperetin recovers phenotypes in cells harboring UFC1 with the R23Q mutation. (A) Cells harboring GFP-tagged UFC1 (R23Q) were allowed to differentiate in the presence (+) or absence (−, vehicle) of 5 micromolar of hesperetin (Hes). The square fields a and b indicated by the dotted lines in the center panels are magnified in the bottom panels a and b. Cells with widespread membranes were statistically assessed (** p < 0.01; n = 10 fields [a total of 340 cells per each experiment]). (B) The lysates of the respective cells were immunoblotted with an antibody against MBP, PLP1, or control actin. Their expression levels were statistically compared to their respective controls (** p < 0.01 or * p < 0.05; n = 3 blots).