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Shank3 knockdown in rat hippocampal neurons. Hippocampal neurons were transduced with GFP‐tagged Shank3 lentiviral shRNA particles on DIV1, then treated with vehicle or 100 μM p ‐Cresol from DIV8 to DIV14. Cells were immunostained with anti‐GFP, ‐Shank3, ‐VGLUT, and ‐VGAT Abs at the end of treatment. (A–F) Representative images of GFP + neurons (green) stained with anti‐Shank3 (magenta) in Scr (full expression of Shank3) and KD conditions (reduced expression of Shank3). Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (G) Transfection efficiency of Shank3 KD lentiviral particles expressed as Shank3 puncta/100 μm. (H–W) Representative images of GFP + (green) Shank3 Scr and KD neurons stained with VGLUT (red) and VGAT (cyan) in ctrl and 100 μM p ‐Cresol conditions. Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (X) VGLUT + and VGAT + puncta / 100 μm in Scr and Shank3 KD conditions, treated with vehicle or 100 μM p ‐Cresol. (Y) Effect of Shank3 KD combined with p ‐Cresol treatment on neuronal dendrite length (μm). All data were expressed as mean ± SEM values and analyzed by two‐way ANOVA followed by Tukey's post hoc comparisons: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Scr ctrl; £ p < 0.05; ££ p < 0.01; £££ p < 0.001 vs. Shank3 KD Ctrl; +++ p < 0.001 vs. Scr p ‐Cres 100 μM; #0.10 > p > 0.05.

Journal: Journal of Neurochemistry

Article Title: Unveiling the Molecular Mechanism of Intestinal Metabolite para ‐Cresol in Modulating Neuroinflammation and Synaptic Dysfunction: Implications for Autism Spectrum Disorder

doi: 10.1111/jnc.70457

Figure Lengend Snippet: Shank3 knockdown in rat hippocampal neurons. Hippocampal neurons were transduced with GFP‐tagged Shank3 lentiviral shRNA particles on DIV1, then treated with vehicle or 100 μM p ‐Cresol from DIV8 to DIV14. Cells were immunostained with anti‐GFP, ‐Shank3, ‐VGLUT, and ‐VGAT Abs at the end of treatment. (A–F) Representative images of GFP + neurons (green) stained with anti‐Shank3 (magenta) in Scr (full expression of Shank3) and KD conditions (reduced expression of Shank3). Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (G) Transfection efficiency of Shank3 KD lentiviral particles expressed as Shank3 puncta/100 μm. (H–W) Representative images of GFP + (green) Shank3 Scr and KD neurons stained with VGLUT (red) and VGAT (cyan) in ctrl and 100 μM p ‐Cresol conditions. Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (X) VGLUT + and VGAT + puncta / 100 μm in Scr and Shank3 KD conditions, treated with vehicle or 100 μM p ‐Cresol. (Y) Effect of Shank3 KD combined with p ‐Cresol treatment on neuronal dendrite length (μm). All data were expressed as mean ± SEM values and analyzed by two‐way ANOVA followed by Tukey's post hoc comparisons: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Scr ctrl; £ p < 0.05; ££ p < 0.01; £££ p < 0.001 vs. Shank3 KD Ctrl; +++ p < 0.001 vs. Scr p ‐Cres 100 μM; #0.10 > p > 0.05.

Article Snippet: Cells were transduced with green fluorescent protein (GFP)‐tagged SH3 and multiple ankyrin repeat domains 3 (Shank3) rat shRNA lentiviral particles (ORIGENE, #TL710469V) on day in vitro (DIV) 1, with a multiplicity of infection of 5 for 20 h (hr).

Techniques: Knockdown, Transduction, shRNA, Staining, Expressing, Transfection