Fig 1: RLIM knockout in OL lineage cells impairs remyelination in adult mouse brains, and GSH administration restores the defect in remyelination in adult RLIM icKO mice. (A) Western blot assays showing the subcellular distribution of endogenous SLC7A11 in the cortical tissues from adult control (n = 3) and RLIM icKO (n = 3) mice with tamoxifen administration. (B‐E) Bar graphs (mean ± SD) (B) shows the normalized protein levels of RLIM in total cell lysates (B) (icontrol: 1.000 ± 0.1318 and RLIM icKO: 0.3930 ± 0.03400) or SLC7A11 in cytomembrane fractions (C) (icontrol: 1.000 ± 0.1719 and RLIM icKO: 0.3350 ± 0.04984), cytoplasmic fractions (D) (icontrol: 1.000 ± 0.1595 and RLIM icKO: 2.980 ± 0.2273), and total cell lysates (E) (icontrol: 1.000 ± 0.08803 and RLIM icKO: 0.9979 ± 0.05217). The protein levels of RLIM or SLC7A11 in total cell lysates, cytoplasmic fractions, or cytomembrane fractions were normalized against the levels of Na+K+ATPase, VINCULIN, or TUBULIN, respectively. The protein levels of RLIM or SLC7A11 in the control groups were set to 1. Unpaired two‐tailed Student's t‐test; n.s., not significant; **, p < 0.01; ***, p < 0.001. (F, G) Analysis of ubiquitinated levels of SLC7A11 in cortical tissues of adult control (n = 3) and the RLIM icKO (n = 3) mice after tamoxifen administration. The bar graph (mean ± SD) shows normalized polyubiquitinated levels of SLC7A11 in icontrol and the RLIM icKO groups (icontrol: 1.000 ± 0.1220 and RLIM icKO: 0.5051 ± 0.07304). The respective expression level in the control group was set to 1. Unpaired two‐tailed Student's t‐test; **, p < 0.01. (H‐J) Immunofluorescence staining of CC1 and SOX10 in lesion areas of brains from icontrol (n = 3) and the RLIM icKO (n = 3) at DPL 14. Bar graphs showing the quantification of the number of CC1+SOX10+ (I, icontrol: 611.8 ± 124.0 /mm2, and RLIM icKO: 202.6 ± 60.77 /mm2) and the ratio of CC1+SOX10+ cells among SOX10+ cells (J, icontrol: 0.5637 ± 0.06430, and RLIM icKO: 0.2656 ± 0.09807) in (H). Scale bar, 75 µm. Unpaired two‐tailed Student's t‐test; ****, p < 0.0001. (K–N) Electron microscopy images show the myelinated axons in the lesion area in the brains of icontrol (n = 3) and RLIM icKO (n = 3) at DPL 21. Scale bar: 4 µm for the upper panels and 1 µm for the lower panels. Bar graphs (mean ± SD) show quantification of the number of myelinated axons (L, icontrol: 1,456,450 ± 213,392 /mm2 and RLIM icKO: 898,144 ± 175,111 /mm2) and the percentage of myelinated axons (M, icontrol: 63.60 ± 3.527% and RLIM icKO: 37.78 ± 6.237%) in (K). Scatterplots (N) show the myelin g‐ratios (diameter of axon/diameter of entire fiber). At least 200 axons from 3 mice were involved in the analysis for each group. Unpaired two‐tailed Student's t test for (L) and (M); General linear regression model and ANCOVA analysis for (N); ***. p < 0.001; ****, p < 0.0001. (O–Q) Immunofluorescence staining of CC1 and SOX10 in lesion areas of brains from icontrol‐PBS (n = 3), RLIM icKO‐PBS (n = 3), icontrol‐GSH (n = 3), and RLIM icKO‐GSH (n = 3) at DPL 14. Bar graphs showing the quantification of the number of CC1+SOX10+ (P, icontrol‐PBS: 632.9 ± 225.7 /mm2, RLIM icKO‐PBS: 352.8 ± 133.3 /mm2, icontrol‐GSH: 588.9 ± 248.0 /mm2, RLIM icKO‐GSH: 440.3 ± 107.3 /mm2) and the ratio of CC1+SOX10+ cells among SOX10+ cells (Q, icontrol‐PBS: 0.6380 ± 0.05346, RLIM icKO‐PBS: 0.4370 ± 0.07134, icontrol‐GSH: 0.6567 ± 0.1049, RLIM icKO‐GSH: 0.5623 ± 0.1179) in (O). One‐way ANOVA with multiple comparisons; **, p < 0.01; ****, p < 0.0001. iCtrl, icontrol; icKO, RLIM icKO.
Fig 2: RLIM targets SLC7A11 for polyubiquitination, by which RLIM facilitates the membrane expression and function of SLC7A11. (A) In vitro co‐immunoprecipitation (co‐IP) analysis of the interaction between HA‐tagged RLIM and Flag‐tagged SLC7A11 in HEK293 cells with transfection as indicated. The whole cell lysates (WCLs) were immunoprecipitated by a Flag (the upper panel) or HA antibody (the lower panel), and the WCLs and immunoprecipitations (IPs) were immunoblotted with the indicated antibodies. (B) In vivo co‐IP analysis of the interaction between endogenous RLIM and SLC7A11 in cortical tissues from P7 control mice. The lysates were immunoprecipitated by an IgG and an RLIM (the upper panel) / SLC7A11 (the lower panel) antibody, and the inputs and IPs were immunoblotted by the indicated antibodies. (C) GST‐pulldown analysis of the direct interaction between RLIM and SLC7A11. Purified GST‐RLIM was incubated with purified His‐SLC7A11 and subjected to GST pulldown followed by immunoblotting with the indicated antibodies (the upper panel). Purified GST‐RLIM and His‐SLC7A11 proteins were analyzed by SDS‐PAGE and Coomassie blue staining (the lower panel). (D) Polyubiquitination analysis of SLC7A11 by co‐expression of wild‐type or the E3 ligase dead mutant (H569/572A) of RLIM in HEK293 cells. (E) In vitro ubiquitination assays of SLC7A11 by RLIM. In vitro purified SLC7A11 proteins were incubated in the presence of purified RLIM, ubiquitin‐activating enzyme E1, ubiquitin‐conjugating enzyme E2, and ubiquitin for the in vitro ubiquitination assay. (F, G) Analysis of ubiquitinated levels of SLC7A11 in cortical tissues of P7 control (n = 3) and the RLIM cKO (n = 3) mice. The bar graph (mean ± SD) shows normalized polyubiquitinated levels of SLC7A11 in Control and the RLIM cKO groups (control: 1.000 ± 0.07162, RLIM cKO: 0.6587 ± 0.06643). The respective expression level in the control group was set to 1. Unpaired two‐tailed Student's t‐test; **, p < 0.01. (H, I) Results of Western blot assays showing the subcellular distribution of endogenous SLC7A11 in the cortical tissues from P7 control (n = 3) and the RLIM cKO (n = 3) mice. Bar graphs (mean ± SD) (I) show the normalized protein levels of SLC7A11 in total cell lysates (the left panel) (control: 1.000 ± 0.1148, RLIM cKO: 0.9686 ± 0.09590), cytoplasmic fractions (the middle panel) (control: 1.000 ± 0.1489, RLIM cKO: 1.382 ± 0.07420), and cyto‐membrane fractions (the right panel) (control: 1.000 ± 0.1080, RLIM cKO: 0.6850 ± 0.06910). The protein levels of SLC7A11 in total cell lysates, cytoplasmic fractions, or cyto‐membrane fractions were normalized against the levels of Na+K+ATPase, VINCULIN, or TUBULIN, respectively. The protein levels of SLC7A11 in the control groups were set to 1. Unpaired two‐tailed Student's t‐test; n.s., not significant; *; p < 0.05. (J‐L) In vivo co‐IP analysis of the interaction between endogenous SPTBN2 and SLC7A11 in MOPC cells. The lysates were immunoprecipitated by an IgG and an SLC7A11 (the right upper panel) / SPTBN2 (the right lower panel) antibody, and the inputs and IPs were immunoblotted by the indicated antibodies. Bar graphs (mean ± SD) (K) shows the normalized protein levels of immunoprecipitated SLC7A11 by SPTBN2 (K) (NC: 1.000 ± 0.07347, siRLIM‐1#: 0.3243 ± 0.04121, siRLIM‐2#: 0.3546 ± 0.03816, siRLIM‐3#: 0.3672 ± 0.03259) or SPTBN2 by SLC7A11 (L) (NC: 1.000 ± 0.07482, siRLIM‐1#: 0.2600 ± 0.01888, siRLIM‐2#: 0.2387 ± 0.01864, siRLIM‐3#: 0.1988 ± 0.01146). The protein level of immunoprecipitated SLC7A11 or SPTBN2 in the NC group was set to 1. One‐way ANOVA with multiple comparisons; *, p < 0.05 (M). The bar graph shows intracellular GSH levels in the PDGFRα+ OPC cells from cortical tissues of P7 Control (n = 4) and the RLIM cKO (n = 4) mice (control: 1.000 ± 0.1840, RLIM cKO: 0.4259 ± 0.08240). The GSH level in the control group was set to 1. Unpaired two‐tailed Student's t‐test; **, p < 0.01. NC, negative control; Ctrl, control; cKO, RLIM cKO; IP, immunoprecipitation; WCL, whole cell lysate; IB, immunoblotting.
Supplier Page from Sino Biological, Inc. for Human SLC7A11 Gene ORF cDNA clone in cloning vector