图1 . Cx32优先促进oα-syn的吸收，而不是单体或纤维组合物。α-syn单体或b低聚α-syn (oα-syn)组合物在280 nm吸光度下的SEC分析。c SEC在550 nm吸光度下确认标记的ato -550 oα-syn组件。d α-syn单体、寡聚物或f纤组合体的TEM表征;比例尺分别代表5 μ m、500 nm、2 μ m。g对低表达(15µg)或高表达(50µg) Cx32-mCherry或Cx43-GFP质粒的未转染野生型(WT)或转染HEK-293细胞进行共聚焦图像分析。比例尺代表200 μ m。h用i Western blot检测WT HEK-293细胞或表达Cx32或Cx43的HEK-293细胞中单体、寡聚或纤原α-syn的摄取(n = 4，双向方差分析，多重比较的Tukey事后检验，n =无显著性，F(8,24) = 13.1， ****p < 0.0001)。j分化的SH-SY5Y WT细胞或表达Cx26、Cx32、Cx43或Cx32- ko的SH-SY5Y细胞摄取oα-syn的k个代表性Western blots的密度分析(n = 5，单因素方差分析，多重比较Tukey’s事后检验，n.s;没有意义,F (24) = 25.28, * * * * p < 0.0001)。 l Immunocytochemistry of differentiated SH-SY5Y cells, WT and those expressing Cx26, Cx32, Cx43 or Cx32-KO treated with oα-syn assemblies. Scale bars represent 20 µm. m Densitometric analysis of n representative Western blot of fibrillar α-syn uptake in differentiated SH-SY5Y cells, WT and those expressing Cx26, Cx32, Cx43, or Cx32-KO (n = 3, one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, n.s; no significance, F(4, 12) = 76.16, *p < 0.05, ****p < 0.0001)
图2 Cx32表达与oα-syn摄取相关。对暴露于SB203580 24小时(10和25 μ M)的分化SH-SY5Y细胞中Cx32蛋白水平的密度分析，(n = 5，单向方差分析，随后进行多次比较的Tukey’s post - hoc检验，n.s;差异无统计学意义，F(2,12) = 5.547， *p < 0.05)。b在SB203580浓度增加的分化SH-SY5Y细胞和未处理细胞中进行Cx32 mRNA的qRT-PCR分析(n = 3，单因素方差分析，多次比较采用Tukey’s post - hoc检验，n =无显著性F(3,8) = 1.415)。c暴露于SB203580 24小时的分化细胞中吸收oα-syn的密度分析(n = 5，单向方差分析，随后进行多次比较的Tukey’s事后检验，n.s;无显著性，F(2,12) = 4.562， *p < 0.05)。d SB203580处理后具有代表性的Western blot结果显示Cx32、α-syn和GAPDH蛋白水平。e暴露于异霉素24 h的分化SH-SY5Y细胞中Cx32蛋白表达的密度分析，(n = 5，单向方差分析，随后多次比较的Tukey’s事后检验，n.s;差异无统计学意义，F(3, 11) = 4.553， *p < 0.05)。f qRT-PCR分析异曲霉素浓度增加处理的分化SH-SY5Y细胞和未处理的细胞中Cx32 mRNA的表达(n = 3，单因素方差分析，多次比较的Tukey’s post - hoc检验，n.s; no significance, F(3, 8) = 3.998, *p < 0.05). g Densitometric analysis of oα-syn uptake following anisomycin treatment for 24 h (n = 4, one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, n.s; no significance, F(3, 12) = 5.327, *p < 0.05). h Representative Western blots showing Cx32, α-syn and GAPDH protein levels following anisomycin treatment. i, j Western blot analysis of oα-syn uptake in SH-SY5Y cells expressing Cx32-mCherry (n = 6) or mCherry (n = 4) incubated at 37 °C or 4 °C (one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, n.s; no significance, F(3, 16) = 83.02, ***p < 0.001, ****p < 0.0001). k Densitometric analysis of l Western blots of oAβ uptake in differentiated SH-SY5Y cells expressing mCherry or Cx32-mCherry (n = 4, unpaired, two-tailed t test, t6 = 8.493, ***p = 0.0001). m Confocal image analysis of differentiated SH-SY5Y cells expressing mCherry or Cx32-mCherry (red) labeled with Aβ (6E10, green) and then stained with Hoechst (blue); scale bar represents 200 µm. n Fluorescence intensity quantification of oAβ uptake in differentiated SH-SY5Y cells expressing mCherry or Cx32-mCherry by confocal microscopy (n = 3 independent experiments and each representing the average of three different measurements, unpaired, two-tailed t test, t4 = 11.86 ***p < 0.001)
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