Fig 1: Representative images of SVCT2 immunohistochemistry and their corresponding H&E staining (a, Right) and statistical graph (b, Left). Immunohistochemistry shows that the expression of SVCT2 (transporter of AA into cells) in SW620 tumor cells is significantly higher than that in HCT8 tumor cells. Scale bar = 50 µm. Data are shown as mean ± SD (n = 5). **P < 0.01, ns = not significant.
Fig 2: SVCT2 over-expression benefits functional recovery induced by AA treatment. (A) Quantitative data of behavioral tests: latency to fall (s) in rotarod (left), percentage of turn right (%) in corner teat (middle) and the slip ratio (%) of the contralateral limbs within 50 steps in beam walking test (right) on days 3, 7, and 14 after ischemia in different groups: Sham + AAV-Control, MCAO + AAV-Control, MCAO + 250 mg/Kg AA, MCAO + AAV-SVCT2, MCAO + AAV-SVCT2 + 250 mg/Kg AA. Data represented mean ± SEM, n = 4; *P < 0.05, **P < 0.01, MCAO + AAV-SVCT2 vs. MCAO + AAV-Control group; #P < 0.05, ##P < 0.01, MCAO + AAV-SVCT2 + 250 mg/Kg AA vs. MCAO + 250 mg/Kg AA and MCAO + AAV-SVCT2. Two-way ANOVA followed by Tukey’s post hoc test. (B) Representative TTC staining images. White area with black dotted was infarct area. Scale bar: 2 mm. (C) Quantitative data of infract volume after ischemia in different groups: Sham + AAV-Control, MCAO + AAV-Control, MCAO + 250 mg/Kg AA, MCAO + AAV-SVCT2, MCAO + AAV-SVCT2 + 250 mg/Kg AA. Data represented mean ± SEM., n = 3; *P < 0.05, **P < 0.01. One-way ANOVA followed by Tukey’s post hoc test.
Fig 3: SVCT2 over-expression facilitates NSPCs migration in vivo. (A) Emblematic immunostaining of DCX+ and BrdU+ NSPCs along RMS (area between two white lines) without ischemia on day 7 in various groups: Sham, 250 mg/Kg AA, AAV-shRNA (SVCT2) or MCAO + LV-SVCT2. DAPI was used to label the nuclei. Scale bar: 200 µm. (B) Number of BrdU+ NSPCs along RMS per field in four groups. One-way ANOVA followed by Tukey’s post hoc test. ns, not significant. (C) Percentage of BrdU+ NSPCs along RMS at distances of 0–500, 500–1000, 1000–1500, and 1500–2000 µm away from V-SVZ. Data were expressed in mean ± SEM, n = 3; *P < 0.05, **P < 0.01, Two-way ANOVA followed by Tukey’s post hoc test. (D) Schematic image shows the migration route of NSPCs (red arrows) in coronal brain section after brain injury. (E) Representative immunostaining of DCX+ and BrdU+ NSPCs in cortex and striatum (yellow dotted grid indicates the observation area, and white dotted lines distinguish the infarct core and the penumbra) on day 7 after ischemia in group MCAO + AAV-Control and MCAO + AAV-SVCT2. Scale bar: 100 µm. (F) Quantitative analysis of BrdU+ and DCX+ cells around penumbra in cortex in group MCAO + AAV-Control and MCAO + AAV-SVCT2. Data were shown as mean ± SEM, n = 4; *P < 0.05, Student’s t-test. (G) Quantitative analysis of BrdU+ and DCX+ cells around penumbra in striatum in group MCAO + AAV-Control and MCAO + AAV-SVCT2. Data were shown as mean ± SEM, n = 4; *P < 0.05, Student’s t-tests. V-SVZ, ventricular-subventricular zone; RMS, rostral migratory stream; OB, olfactory bulb.
Fig 4: SVCT2 down-regulation might attenuate the regenerative ability resulting from NSPCs after ischemia. (A) Representative immunostaining of co-labeled DCX and SVCT2 at SVZ on day 7 in group Sham and MCAO. DAPI was used to label the nuclei. White arrows indicated co-labeled DCX+ and SVCT2+ NSPCs. Scale bar: 20 µm. (B) Representative immunoblotting images and quantitative analysis of SVCT2 at SVZ on days 1, 3, 7, and 14 in group Sham and MCAO. Data presented mean ± SEM, n = 3; *P < 0.05, **P < 0.01, significantly different from Sham. One-way ANOVA followed by Tukey’s post hoc test. SVZ, subventricular zone.
Fig 5: SVCT2 furthers F-actin assembling to potentiate NSPCs migration via up-regulating CDC42 expression. (A) Representative immunostaining of a-tubulin and phalloidin after neurospheres migration for 24 h in various groups: Control, OGD, OGD + 400 µm AA, OGD + LV-SVCT2, and OGD + LV-SVCT2 + 400 µm AA. Insets were magnified images from each photograph. Scale bar: 50 µm. (B) Bar graph summarized the percent of F-actin assembling after different treatments. Quantitative analysis of average number of primary leading processes (C) and second branches (D) after above treatments, respectively. (E) Bands showed expression of SVCT2, CDC42 and F-actin after above treatments and ß-actin was served as an internal control. Data were shown as mean ± SEM, n = 6; *P < 0.05, **P < 0.01. One-way ANOVA followed by Tukey’s post hoc test; ns, not significant.
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