Fig 1: Western blot analysis of SHANK2 expression in genome-edited cell lines (6A4, 6H6) versus the non-edited controls (1F11, WT). SHANK2-expression was normalized against the expression of GAPDH (reference) in undifferentiated cells (UN) and in cells after 7 days of neuronal differentiation (DIF7). Cropped western blot membrane images are shown and full-length blots are presented in Supplementary Fig. 2. n = 4 experiments. One-way ANOVA was performed and the two different mutant cell lines as well as the WT control were compared to the control 1F11 (ANOVA results: SHANK2-UN F = 266.8, P < 0.0001; SHANK2-DIF7 F = 229.8, P < 0.0001). Correction for multiple testing with Dunnett’s test (n = 3 tests for each time point). Data were presented as box-plots and the corrected P-values are indicated. ***P = 0.001.
Fig 2: Analysis of apoptosis and cell proliferation in SHANK2 mutant cell lines. A TUNEL assay was carried out to determine the rate of apoptotic cells and TUNEL-positive cells were quantified relative to the total number of nuclei (Hoechst), revealing significantly lower numbers of TUNEL-positive cells for both SHANK2 mutant lines compared to the control 1F11 (one-way ANOVA, F = 22.74, P < 0.0001, P-values after correction for multiple testing with Dunnett’s test 6A4 P < 0.0001, 6H6 P = 0.0004, WT P = 0.326) after 7 days of differentiation (DIF7). In addition, immunofluorescence microscopy was carried out with the proliferation marker Ki67 followed by quantification of the Ki67 positive cells versus the total number of nuclei. A significantly increased number of Ki67-positive cells was identified in cell line 6A4 with bi-allelic SHANK2 mutation in comparison to the control 1F11 (one-way ANOVA, F = 20.97, P < 0.0001, P-values after correction for multiple testing with Dunnett´s test 6A4 P < 0.0001, 6H6 P = 0.49, WT P = 0.327) at DIF7. The pictures illustrate the expression after 7 days of differentiation. No difference was observed between the two control lines WT and 1F11. Undifferentiated cell lines showed equally low apoptosis levels (one-way ANOVA, F = 2.285, P = 0.09, corrected P-values Dunnett´s test 6A4 P = 0.078, 6H6 P = 0.98, WT P = 0.99) and equal levels of cell proliferation (one-way ANOVA, F = 1.12, P = 0.3478, corrected P-values Dunnett´s test 6A4 P = 0.30, 6H6 P = 0.99, WT P = 0.40). Data were presented in box plots. n = 3 experiments. ***P = 0.001.
Fig 3: Immunofluorescence microscopy of SHANK2 mutant cells. (a) ß3-Tubulin expression after 7 days of differentiation (DIF7) illustrating the morphology of control and SHANK2 mutant cell lines. (b) Nestin and ß3-Tubulin co-staining using immunofluorescence microscopy. During 16 days of neuronal differentiation, the number of cells showing Nestin expression decreased in the control line (1F11), and nearly all cells developed neuronal morphology. In comparison, cells expressing Nestin could be detected throughout the differentiation period in the genome edited cell line (6A4). UN undifferentiated, DIF7 7 days of differentiation, DIF16 16 days of differentiation.
Fig 4: Analysis of cell morphology. Immunofluorescence microscopy was carried out with ß3-Tubulin and the length of neurites after 4 days of differentiation was measured, revealing significantly shorter neurites for the cell line with compound heterozygous SHANK2 mutation (6A4) in comparison to the control 1F11. n = 3 experiments, one-way ANOVA (F = 31.1, P < 0.0001), corrected P-values from Dunnett’s test (6A4 P < 0.0001, 6H6 P = 0.36, WT P = 0.48). No difference was observed between the two control lines WT and 1F11. Data were presented in box plots. ***P = 0.001.
Fig 5: Quantification of protein expression in SH-SY5Y cells (7 days after differentiation). Western blot analysis with protein from two control and two SHANK2 mutant cell lines. The protein expression levels of NESTIN (NES), GRIN2B, PSD95, AKT, ß3-Tubulin (TUBB3) and SYP were normalized against the expression of GAPDH (reference). GAPDH expression was analyzed against total protein expression and a differential expression of GAPDH between the different cell lines was ruled out. No differences between the two control lines WT and 1F11 could be detected. Differential expression levels between mutant and control cell lines were found for NES, PSD95 and SYP. Cropped western blot membrane images are shown and full-length blots are presented in Supplementary Fig. 8. n = 5 experiments, one-way ANOVA was carried out and protein expression of the two mutant cell lines (6A4, 6H6) and the wildtype cell line (WT) was compared against the control 1F11 (ANOVA results: NES F = 11.57, P = 0.0007; GRIN2B F = 1.21, P = 0.348; PSD95 F = 63,51, P < 0.0001; AKT F = 1.91, P = 0.181; TUBB3 F = 0.28, P = 0.837; SYP F = 6.48, P = 0.0074; GAPDH F = 0.93, P = 0.458). n.s.—not significant. Correction for multiple testing with Dunnett´s test (n = 3 tests for each protein). Data were presented as box-plots and the corrected P-values are indicated, *P = 0.05, ***P = 0.001.
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