Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Example of Shank3 expression in WT, Shank3Δ11-/- (KO) and Shank3 fullKO (fKO) cortical synaptosomal fraction (5 µg) and in peripheral blood mononuclear cells (40 µg) from WT and Shank3Δ11-/- (KO) adult mice. B Representative western blot images and relative SHANK3 quantification from peripheral blood mononuclear cells isolated from Shank3 WT, HT and KO adult mice. Protein expression was normalized to the respective total protein levels. Data were analyzed by one-way ANOVA; n = 7 WT, n = 3 Shank3∆11 + /−, n = 7 Shank3∆11-/-. ** p ≤ 0.01; **** p ≤ 0.0001. C Shank3 mRNA expression from PBMCs of WT and Shank3∆11-/- adult mice. Data were analyzed by two-tailed Mann-Whitney test; n = 6 WT, n = 6 Shank3∆11-/-. ** p ≤ 0.01. D Representative western blot images and relative protein quantification from peripheral blood mononuclear cells isolated from adult mice. Protein expression was normalized to the respective total protein levels. Data obtained from the quantification of DAP12 were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; TREM2 was analyzed by unpaired, two-tailed Student’s t-test; n = 7 WT, n = 7 Shank3∆11-/-. **** p ≤ 0.0001.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Expressing, Western Blot, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Representative western blot images and relative protein quantification from peripheral blood mononuclear cells isolated from healthy donor and PMS patients. Protein expression was normalized to the respective total protein levels. Data obtained from the quantification of SHANK3 were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; n = 24 controls, n = 20 patients; TREM2 was analyzed by unpaired, two-tailed Student’s t-test; n = 12 controls, n = 11 patients; DAP12 data were analyzed by two-tailed Mann-Whitney test; n = 7 controls, n = 7 patients; CD3 was analyzed by unpaired, two-tailed Student’s t-test; n = 14 controls, n = 10 patients. **** p ≤ 0.0001. B Immunocytochemistry of SHANK3 (red), CD3 (green) and DAPI (blue) in a peripheral blood smear of healthy donor sample. Scale bar: 10 µm. C SHANK3 mRNA expression from PBMC of controls and PMS patients. Data were analyzed by unpaired, two-tailed Student’s t-test; n = 8 controls, n = 7 patients. **** p ≤ 0.0001. D Plasma concentration of beta-synuclein in healthy donors and PMS patient samples. Data were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; n = 22 controls, n = 22 patients; * p ≤ 0.05.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Western Blot, Isolation, Expressing, Two Tailed Test, MANN-WHITNEY, Immunocytochemistry, Clinical Proteomics, Concentration Assay

Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Level of beta-synuclein isolated from plasma of WT and Shank3 Δ11-/- adult mice after pharmacological treatment. Data were analyzed by unpaired, two-tailed Student’s t-test; n = 6 WT vehicle, n = 8 Shank3 ∆11-/- vehicle, n = 9 Shank3 ∆11-/- VU. * p ≤ 0.05. B Display of results of SHANK3 (unstandardized) and the occurrence of regression (left; Welch’s t = 2.8, p = 0.01, |Cohen’s d | = 1.20) and beta-synuclein levels in PMS individuals with different degrees of speech impairment (right; Welch’s t = 3.36, p = 0.0041, |Cohen’s d | = 1.43). Data were analyzed by a Welch’s t-test. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Isolation, Clinical Proteomics, Two Tailed Test

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: The spatiotemporal expression of Shank3 in cerebral ischemia-reperfusion injury. (A) Western blot analysis of Shank3 protein levels in tissue extracts of mouse hippocampus after 60 min tMCAO followed by 3, 6, 12, and 24 h of reperfusion. (B) Quantification of Shank3 gray value relative to β-actin [n = 6, F [ , ] = 141.7, P < 0.0001]. (C) The qRT-PCR analysis of Shank3 mRNA levels isolated from the hippocampus after ischemia-reperfusion [n = 6, F [ , ] = 104.1, P < 0.0001]. (D) Representative immunofluorescence staining of Shank3 (Red) and NeuN (Green) in hippocampus regions after ischemia and reperfusion, Scale bar = 50 μm. (E) Fluorescence intensity analysis of Shank3 staining [n = 6, F [ , ] = 196.0, P < 0.0001]. (F) Western blot analysis of Shank3 protein levels in HT22 cells subjected to OGD for 8 h and reperfusion for 24 h. (G) Quantification of Shank3 gray value relative to β-actin [n = 6, F [ , ] = 218.9, P < 0.0001]. (H) The qRT-PCR analysis of Shank3 mRNA levels in HT22 cells after OGD/R [n = 6, F [ , ] = 252.3, P < 0.0001]. (I) Immunofluorescence staining of Shank3 (Red) and NeuN (Green) in HT22 cells after OGD and reperfusion, Scale bar = 100 μm. (J) Fluorescence intensity analysis of Shank3 staining [n = 6, F [ , ] = 159.2, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Isolation, Immunofluorescence, Staining, Fluorescence

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 knockout exacerbates ischemic brain injuries. (A) Construction of conditional knockout mice of Shank3. (B, C) General [n = 12, F [ , ] = 159.4, P < 0.0001] and focal deficits [n = 12, F [ , ] = 456.9, P < 0.0001] in the Clark score after I/R. (D) Quantification of brain edema [n = 6, F [ , ] = 50.29, P < 0.0001]. (E, F) TTC staining indicated the infarction (white) of the brains [n = 7, F [ , ] = 1570, P < 0.0001]. (G–I) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 359.6, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 236.9, P < 0.0001] levels in tissue extracts of mouse hippocampus. (J) Representative confocal images of NeuN (Green) and TUNEL (Red) in hippocampus regions, Scale bar = 100 μm. (K) Quantitative analysis of TUNEL-positive cells [n = 6, F [ , ] = 310.4, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Knock-Out, Staining, Western Blot, TUNEL Assay

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 deficiency aggravates oxidative stress and inflammation after I/R. (A–I) The hippocampus tissues after I/R were collected and homogenized to determine the levels of ROS [n = 6, F [ , ] = 231.2, P < 0.0001], MDA [n = 6, F [ , ] = 218.5, P < 0.0001], GSH-Px [n = 6, F [ , ] = 279.6, P < 0.0001], GSH [n = 6, F [ , ] = 98.82, P < 0.0001], SOD [n = 6, F [ , ] = 240.8, P < 0.0001], CAT [n = 6, F [ , ] = 237.2, P < 0.0001], TNF [n = 6, F [ , ] = 326.6, P < 0.0001], IL-1β [n = 6, F [ , ] = 319.7, P < 0.0001], and IL-6 [n = 6, F [ , ] = 77.59, P < 0.0001] using assay kits. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques:

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Effects of Shank3 deficiency on the cytotoxicity and apoptosis in HT22 cells after OGD/R. (A–C) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 175.9, P < 0.0001], and cleaved-caspase3 [F [ , ] = 13.7, P < 0.0001] levels in HT22 cells affected by Shank3 after OGD/R. (D, E) Representative confocal images of the effect of Shank3 on TUNEL staining, Scale bar = 100 μm [n = 6, F [ , ] = 145.4, P < 0.0001]. (F) HT22 cells viability [n = 6, F [ , ] = 151.8, P < 0.0001]. (G, H) Representative Fluorescence staining images (Scale bar = 100 μm) and quantitative analysis of ROS levels [n = 6, F [ , ] = 179.6, P < 0.0001]. (I–O) Effects of Shank3 on MDA [n = 6, F [ , ] = 322.5, P < 0.0001], GSH-Px [n = 6, F [ , ] = 333.8, P < 0.0001], GSH [n = 6, F [ , ] = 124.5, P < 0.0001], SOD [n = 6, F [ , ] = 137.5, P < 0.0001], TNF [n = 6, F [ , ] = 166.7, P < 0.0001], IL-1β [n = 6, F [ , ] = 534.9, P < 0.0001], and IL-6 [n = 6, F [ , ] = 109.4, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Western Blot, TUNEL Assay, Staining, Fluorescence

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Regulatory effects of Shank3 on STIM1. (A, B) Overview of Shank3 ANK domain (Green) binding to SMIT1 SOAR domain (Violet). (C, D) Predictive analysis of the binding mode of Shank3 ANK domain to SMIT1 SOAR domain in 2D and 3D. (E) Double fluorescent staining of Shank3 (Green) and STIM1 (Red) in the hippocampus, Scale bar = 100 μm. (F) Co-immunoprecipitation (Co-IP) assay suggested protein-protein interactions between Shank3 and STIM1. (G) GST pull-down assay revealed Shank3 directly bound with STIM1 (n = 3, t = 18.65, df = 4, P < 0.0001). (H, I) Shank3 mutant significantly reduced the protein-protein interactions between Shank3 and STIM1 [n = 3, t = 18.41, P < 0.0001]. (J, K) Western blot analysis of Shank3 protein levels [n = 6, F [ , ] = 259.1, P < 0.0001] and STIM1 protein levels [n = 6, F [ , ] = 269.9, P < 0.0001] in modified HT22 cells. (L, M) STIM1 ubiquitination levels in Shank3 knockdown HT22 cells and hippocampus of Shank3 cko mice. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons and unpaired t -test between two groups. Values are expressed as mean ± SD.* P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Binding Assay, Staining, Co-Immunoprecipitation Assay, Protein-Protein interactions, Pull Down Assay, Mutagenesis, Western Blot, Modification, Ubiquitin Proteomics, Knockdown

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Downregulation of STIM1 mitigates OGD/R-induced cell injury after Shank3 knockdown. (A–E) Western blot analysis of Shank3 [n = 6, F [ , ] = 248.6, P < 0.0001], STIM1 [n = 6, F [ , ] = 405.9, P < 0.0001], Bcl-2/Bax [n = 6, F [ , ] = 140.0, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 165.5 P < 0.0001] levels in Shank3-knockdown cells affected by silencing STIM1 after OGD/R. (F, G) Effects of silencing STIM1 on TUNEL staining (Scale bar = 100 μm) [n = 6, F [ , ] = 76.09, P < 0.0001]. (H) Cells viability [n = 6, F [ , ] = 45.52, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Knockdown, Western Blot, TUNEL Assay, Staining

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: The Shank3/STIM1/Nrf2 signaling pathway is involved in Nrf2 nuclear accumulation and anti-oxidative stress activity after OGD/R. (A–F) Western blot analysis of nuclear Nrf2 [n = 6, F [ , ] = 179.1, P < 0.0001], calcineurin [n = 6, F [ , ] = 137.5, P < 0.0001], p-Nrf2 [n = 6, F [ , ] = 262.6, P < 0.0001], NQO1 [n = 6, F [ , ] = 278.8, P < 0.0001] and HO-1 [n = 6, F [ , ] = 262.6, P < 0.0001] levels in modified cells after OGD/R. (G, H) Representative fluorescence staining images (Scale bar = 100 μm) and quantitative analysis of ROS levels [n = 6, F [ , ] = 60.28, P < 0.0001]. (I–L) Analysis of antioxidant system including MDA [n = 6, F [ , ] = 93.55, P < 0.0001], GSH-Px [n = 6, F [ , ] = 73.16, P < 0.0001], GSH [n = 6, F [ , ] = 51.18, P < 0.0001], and SOD [n = 6, F [ , ] = 134.9, P < 0.0001]. (M−O) Effects of knockdown STIM1 on TNF [n = 6, F [ , ] = 72.57, P < 0.0001], IL-1β [n = 6, F [ , ] = 170.9, P < 0.0001], and IL-6 [n = 6, F [ , ] = 49.04, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Activity Assay, Western Blot, Modification, Fluorescence, Staining, Knockdown

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Silencing STIM1 alleviates Shank3 deficiency-induced ischemia-reperfusion brain injuries. (A, B) General [n = 12, F [ , ] = 96.78, P < 0.0001] and focal [n = 12, F [ , ] = 253.1, P < 0.0001] deficits in the Clark score after I/R. (C, D) The infarct volume was measured with TTC staining at 24 h after tMCAO/R [n = 7, F [ , ] = 504.2, P < 0.0001]. (E–G) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 288.6, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 84.04, P < 0.0001] levels. (H) Representative confocal images of NeuN (Green) and TUNEL (Red), Scale bar = 100 μm. (I) Quantitative analysis of TUNEL-positive cells [n = 6, F [ , ] = 173.1, P < 0.0001]. (J–P) The levels of ROS [n = 6, F [ , ] = 68.41, P < 0.0001], MDA [n = 6, F [ , ] = 170.0, P < 0.0001], GSH-Px [n = 6, F [ , ] = 114.2, P < 0.0001], SOD [n = 6, F [ , ] = 188.4, P < 0.0001], TNF [n = 6, F [ , ] = 101.2, P < 0.0001], IL-1β [n = 6, F [ , ] = 100.3, P < 0.0001], and IL-6 [n = 6, F [ , ] = 68.64, P < 0.0001] using assay kits. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD for n = 6. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Staining, Western Blot, TUNEL Assay

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 interacts with STIM1 and regulates post-I/R neuronal oxidative stress and inflammatory injuries through the Nrf2 pathway.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques:

Journal: Nature communications

Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers.

doi: 10.1038/s41467-024-52326-1

Figure Lengend Snippet: Fig. 1 | SHANK3 depletion inhibits cell proliferation in vitro and in vivo in different cancer types driven by distinct KRAS mutations. a A cell proliferation screen following control (siCTRL, grey) or SHANK3 silencing (siSHANK3_2 (red) or siSHANK3_7 (blue)) in wild-type (WT) or KRAS-mutant pancreatic (PDAC), lung (NSCLC) and colorectal (CRC) cancer cell lines. ARPE-19, non-transformed retinal epithelial cells. Shown are the individual data points relative to control [the mean of the control is set to 1.0 by definition; data are mean ± s.d.; n = 3 (Panc10.05 siCTRL and H226 siSHANK3_2) or 4 (other samples) individually silenced wells; two-way ANOVA with Dunnett’s multiple comparisons test]. b Spheroid growth of siCTRL or siSHANK3 PANC-1 or A549 cells. Shown are representative images and quantifica- tion of spheroid area (mean ± s.d.; n = 3 independent experiments; statistical

Article Snippet: All primary antibodies were used at 1:1000 dilution, except the SHANK3 antibody, which was used at a 1:100 (Cat. no. sc-30193, Santa Cruz) or 1:500 (Cat. No. HPA003446, Atlas antibodies) dilution.

Techniques: In Vitro, In Vivo, Control, Mutagenesis, Transformation Assay