Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Mouse hippocampal neurons (HT22 cells) are subjected to OGD/R to mimic CIRI. OGD/R triggers upregulation of CLIC1, which leads to suppression of the Nrf2/HO-1 antioxidant signaling pathway. This results in increased ROS accumulation and disturbance of redox balance, promoting apoptosis by increased Bax and decreased Bcl-2 expression. Additionally, elevated CLIC1 activates the NLRP3 inflammasome and caspase-1 via inhibiting the Nrf2/HO-1 pathway, inducing pyroptosis via upregulation of pyroptosis markers, such as caspase-3, GSDMD, IL-1β, and IL-18. Collectively, these processes contribute to neuronal cell damage after CIRI. ARE, antioxidant response element; CIRI, cerebral ischemia-reperfusion injury; CLIC1, chloride intracellular channel 1; GSDMD, gasdermin D; HO-1, heme oxygenase 1; IL, interleukin; NLRP3, NOD-like receptor family pyrin domain containing 3; Nrf2, nuclear factor erythroid 2–related factor 2; OGD/R, oxygen-glucose deprivation/reoxygenation; ROS, reactive oxygen species.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Expressing

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a) Real-time PCR analysis of CLIC1 gene expression showed significantly higher expression in the OGD/R group compared to the control group, with a marked reduction upon CLIC1 silencing. (b, c) Western blot analysis of CLIC1 protein levels demonstrated increased CLIC1 expression in the OGD/R group and its suppression following CLIC1 silencing. (d-f) Flow cytometry analysis shows that elevated CLIC1 expression in OGD/R-treated HT22 cells led to an increased apoptosis rate (d, e) and higher ROS mean fluorescence intensity (MFI) values (d, f). These effects were reversed by silencing CLIC1. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Control, Western Blot, Flow Cytometry, Fluorescence

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) The activities of SOD (a), CAT (b), and GSH-Px (c) were significantly reduced in OGD/R-treated HT22 cells compared to the control group, with the restoration of these antioxidant enzyme activities upon CLIC1 silencing. (d) The LDH level was elevated in the OGD/R group and decreased following CLIC1 silencing. (e) The CCK-8 OD value was reduced in the OGD/R group and increased with CLIC1 silencing. (f-h) Real-time PCR (f) and WB (g,h) analyses showed that OGD/R treatment decreased anti-apoptotic Bcl-2 expression and increased pro-apoptotic Bax expression, with these effects reversed by CLIC1 silencing. Data are presented as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Control, CCK-8 Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Real-time PCR, WB, and/or ELISA analyses show that the expression levels of inflammation- and pyroptosis-related indicators were all upregulated after the increased expression of CLIC1 in OGD/R-treated HT22 cells, via inhibiting the Nrf2/HO-1 pathway. (a-c) Real-time PCR (a) and WB (b, c) analyses showed upregulation of NLRP3 and caspase-1 expression in OGD/R-treated HT22 cells, with significant downregulation upon CLIC1 silencing. Inhibition of Nrf2/HO-1 signalling or activation of NLRP3 partly reversed the effects of CLIC1 silencing. (d-f) Real-time PCR (d) and WB (e, f) analyses of caspase-3 and GSDMD protein expression showed similar trends with NLRP3 and caspase-1. (g-j) Real-time PCR (g), WB (h,i), and ELISA (j) analyses revealed elevated levels of IL-1β and IL-18 in OGD/R-treated HT22 cells, which were reduced upon CLIC1 silencing. These cytokines were further upregulated with Nrf2/HO-1 inhibition or NLRP3 activation after CLIC1 silencing. Quantitative data were presented as mean ± SEM with three replicate experiments. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Inhibition, Activation Assay, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) Real-time PCR (a) and WB (b, c) analyses showed that Nrf2 and HO-1 expression levels were significantly downregulated in OGD/R-treated HT22 cells compared to the control group. CLIC1 silencing partially restored Nrf2 and HO-1 expression levels, although normal levels were not fully achieved in any group following OGD/R treatment. Data are shown as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Flow cytometry analysis shows that inhibition of the Nrf2/HO-1 signalling pathway increased both the apoptosis rate (a, b) and ROS mean fluorescence intensity (MFI) value (a, c) in OGD/R-treated HT22 cells. These levels were higher in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Flow Cytometry, Inhibition, Fluorescence

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) Activity of oxidative stress markers of SOD (a), CAT (b), and GSH-Px (c) was reduced following inhibition of the Nrf2/HO-1 signalling pathway. These reductions were more pronounced in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group. (d) CCK-8 OD values were decreased after Nrf2/HO-1 inhibition, with lower values observed in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group than the OGD/R+CLIC1 silencing group. (e) The LDH levels were increased after Nrf2/HO-1 inhibition and were significantly higher in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group. (f-h) Real-time PCR (f) and WB (g, h) analyses showed that Bcl-2 expression was downregulated, while Bax expression was upregulated following Nrf2/HO-1 inhibition. These effects were more pronounced in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group than the OGD/R+CLIC1 silencing group. Data were presented as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Activity Assay, Inhibition, CCK-8 Assay, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a) Gene expression heatmap illustrates the relative expression levels of key genes (detected by real-time PCR) across all the six groups [the control, oxygen and glucose deprivation/reoxygenation (OGD/R), OGD/R+ negative control (NC), OGD/R+CLIC1 silencing, OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition, and OGD/R+CLIC1 silencing+NLRP3 activation]. Each row represents a specific gene, and each column represents a sample. (b) Correlation heatmap showing the Spearman correlation coefficients among the genes detected by real-time PCR. Each cell represents the correlation between two genes, with the colour gradient indicating the strength and direction of the correlation. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were immersed in a blocking solution and then treated with primary antibodies targeting CLIC1 (1:1000, 14545–1-AP, Proteintech, China), Nrf2 (1:500, WL02135, Wanleibio, China), HO-1 (1:500, WL02400, Wanleibio, China), B-cell lymphoma-2 (Bcl-2: 1:400, WL01556, Wanleibio, China), Bax (1:1000, WL01637, Wanleibio, China), caspase-3/cleaved caspase-3 (1:400, WL02117, Wanleibio, China), NLRP3 (1:500, WL02635, Wanleibio, China), pro caspase-1/ cleaved caspase-1 (1:500, WL03450,Wanleibio,China), IL-1β (1:1000, WL00891, Wanleibio, China), IL-18 (1:500, WL01127, Wanleibio, China), or GSDMD (1:500, AF4012, Affinity, China) overnight at 4°C.

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Negative Control, Inhibition, Activation Assay

Journal: Alzheimer's research & therapy

Article Title: Circular RNA APP contributes to Alzheimer's disease pathogenesis by modulating microglial polarization via miR-1906/CLIC1 axis.

doi: 10.1186/s13195-025-01698-7

Figure Lengend Snippet: Fig. 2 CircAPP knockdown in microglia modulates microglial polarization, improves AD pathology and inhibits CLIC1 expression and channel activity. (A ~ G) The effects of circAPP knockdown on the expression of TNF-α, Pro- and cleaved IL-1β, CD16, Arg1, IL-10, Aβ42 and p-tau in the hippocampus of APP/PS1 mice were evaluated using Western blot assay, n = 4. (H) The effect of circAPP knockdown on Aβ phagocytosis of BV-2 cells was assessed using flow cytometry assay, n = 3. (I) Golgi staining assay was performed to assess the effect of circAPP knockdown on spine density in the hippocampus of APP/PS1 mice, n = 3 mice. (J ~ L) The effects of circAPP knockdown on total CLIC1 expression in Aβ-treated BV-2 cells (J) and the hippocampus of APP/ PS1 mice (K) and CLIC1 membrane expression in Aβ-treated BV-2 cells (L) were evaluated using Western blot assay, n = 4. (M) The effect of circAPP knock down on CLIC1 channel activity in Aβ-treated BV-2 cells was assessed using functional chloride channel assay, n = 6. All data in the figure are presented as mean ± SEM. Student’s t test (B ~ I, K) and one-way ANOVA (J, L, M) were used to assess statistically significant differences. *P < 0.05, **P < 0.01

Article Snippet: After the protein samples were separated and transferred, primary antibodies against Aβ42 (Biolegend, San Diego, USA, dilution: 1:1000), p-tau S396 (ABclone, Wuhan, China, dilution: 1:1000), pro-and cleaved IL-1β (Affinity Biosciences, dilution: 1:1000), TNF-α (Proteintech, Wuhan, China, dilution: 1:1000), IL-10 (Proteintech, Wuhan, China, dilution: 1:800), CD16 (Proteintech, Wuhan, China, dilution: 1:1000), Arg1 (Proteintech, Wuhan, China, dilution: 1:1000), CLIC1 (Proteintech, Wuhan, China, dilution: 1:1000), Iba-1 (Proteintech, Wuhan, China, dilution: 1:1000), Gapdh (Proteintech, Wuhan, China, dilution: 1:3000) and β-actin (ABclonal, Wuhan, China, dilution: 1:8000) were incubated at 4 °C overnight.

Techniques: Knockdown, Expressing, Activity Assay, Western Blot, Flow Cytometry, Staining, Membrane, Functional Assay

Journal: Alzheimer's research & therapy

Article Title: Circular RNA APP contributes to Alzheimer's disease pathogenesis by modulating microglial polarization via miR-1906/CLIC1 axis.

doi: 10.1186/s13195-025-01698-7

Figure Lengend Snippet: Fig. 6 CircAPP regulates microglial polarization through miR-1906/CLIC1 axis in vitro.(A ~ F) The effects of miR-1906 knockdown on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by circAPP knockdown in Aβ-treated BV-2 cells were assessed via Western blot assay, n = 4. (G) The effect of miR-1906 knockdown on Aβ phagocytosis of BV-2 cells regulated by circAPP knockdown was assessed using flow cytometer assay, n = 3 ~ 4. (H ~ M) The effects of CLIC1 overexpression on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by circAPP knockdown in Aβ-treated BV-2 cells were assessed using Western blot assay, n = 4. (N) The effect of CLIC1 overexpression on Aβ phagocytosis of BV-2 cells regulated by circAPP knockdown was assessed using flow cytometry assay, n = 4 ~ 5. (O ~ T) The effects of CLIC1 overexpression on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by miR-1906 overexpression in Aβ-treated BV-2 cells were assessed using Western blot assay, n = 4. (U) The effect of CLIC1 overexpression on Aβ phagocytosis of BV-2 cells regulated by miR-1906 overexpression was assessed using flow cytometry assay, n = 3 ~ 4. (V) Schematic representation of proposed mechanism of circAPP in AD microglial polarization, pathology and cognitive func tion. CircAPP was upregulated in Aβ-treated microglial cells and the hippocampus of APP/PS1 mice. CircAPP knockdown inhibited its interaction with miR-1906 and increased miR-1906 expression, which in turn retarded CLIC1 expression and channel activity, thereby regulating microglial polarization and ameliorating AD pathology and cognitive function. All data in the figure are presented as mean ± SEM. One-way ANOVA was used to assess statisti cally significant differences. *P < 0.05, **P < 0.01

Article Snippet: After the protein samples were separated and transferred, primary antibodies against Aβ42 (Biolegend, San Diego, USA, dilution: 1:1000), p-tau S396 (ABclone, Wuhan, China, dilution: 1:1000), pro-and cleaved IL-1β (Affinity Biosciences, dilution: 1:1000), TNF-α (Proteintech, Wuhan, China, dilution: 1:1000), IL-10 (Proteintech, Wuhan, China, dilution: 1:800), CD16 (Proteintech, Wuhan, China, dilution: 1:1000), Arg1 (Proteintech, Wuhan, China, dilution: 1:1000), CLIC1 (Proteintech, Wuhan, China, dilution: 1:1000), Iba-1 (Proteintech, Wuhan, China, dilution: 1:1000), Gapdh (Proteintech, Wuhan, China, dilution: 1:3000) and β-actin (ABclonal, Wuhan, China, dilution: 1:8000) were incubated at 4 °C overnight.

Techniques: In Vitro, Knockdown, Expressing, Western Blot, Flow Cytometry, Over Expression, Activity Assay

Journal: Alzheimer's research & therapy

Article Title: Circular RNA APP contributes to Alzheimer's disease pathogenesis by modulating microglial polarization via miR-1906/CLIC1 axis.

doi: 10.1186/s13195-025-01698-7

Figure Lengend Snippet: Fig. 2 CircAPP knockdown in microglia modulates microglial polarization, improves AD pathology and inhibits CLIC1 expression and channel activity. (A ~ G) The effects of circAPP knockdown on the expression of TNF-α, Pro- and cleaved IL-1β, CD16, Arg1, IL-10, Aβ42 and p-tau in the hippocampus of APP/PS1 mice were evaluated using Western blot assay, n = 4. (H) The effect of circAPP knockdown on Aβ phagocytosis of BV-2 cells was assessed using flow cytometry assay, n = 3. (I) Golgi staining assay was performed to assess the effect of circAPP knockdown on spine density in the hippocampus of APP/PS1 mice, n = 3 mice. (J ~ L) The effects of circAPP knockdown on total CLIC1 expression in Aβ-treated BV-2 cells (J) and the hippocampus of APP/ PS1 mice (K) and CLIC1 membrane expression in Aβ-treated BV-2 cells (L) were evaluated using Western blot assay, n = 4. (M) The effect of circAPP knock down on CLIC1 channel activity in Aβ-treated BV-2 cells was assessed using functional chloride channel assay, n = 6. All data in the figure are presented as mean ± SEM. Student’s t test (B ~ I, K) and one-way ANOVA (J, L, M) were used to assess statistically significant differences. *P < 0.05, **P < 0.01

Article Snippet: After the protein samples were separated and transferred, primary antibodies against Aβ42 (Biolegend, San Diego, USA, dilution: 1:1000), p-tau S396 (ABclone, Wuhan, China, dilution: 1:1000), pro-and cleaved IL-1β (Affinity Biosciences, dilution: 1:1000), TNF-α (Proteintech, Wuhan, China, dilution: 1:1000), IL-10 (Proteintech, Wuhan, China, dilution: 1:800), CD16 (Proteintech, Wuhan, China, dilution: 1:1000), Arg1 (Proteintech, Wuhan, China, dilution: 1:1000), CLIC1 (Proteintech, Wuhan, China, dilution: 1:1000), Iba-1 (Proteintech, Wuhan, China, dilution: 1:1000), Gapdh (Proteintech, Wuhan, China, dilution: 1:3000) and β-actin (ABclonal, Wuhan, China, dilution: 1:8000) were incubated at 4 °C overnight.

Techniques: Knockdown, Expressing, Activity Assay, Western Blot, Flow Cytometry, Staining, Membrane, Functional Assay

Journal: Alzheimer's research & therapy

Article Title: Circular RNA APP contributes to Alzheimer's disease pathogenesis by modulating microglial polarization via miR-1906/CLIC1 axis.

doi: 10.1186/s13195-025-01698-7

Figure Lengend Snippet: Fig. 6 CircAPP regulates microglial polarization through miR-1906/CLIC1 axis in vitro.(A ~ F) The effects of miR-1906 knockdown on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by circAPP knockdown in Aβ-treated BV-2 cells were assessed via Western blot assay, n = 4. (G) The effect of miR-1906 knockdown on Aβ phagocytosis of BV-2 cells regulated by circAPP knockdown was assessed using flow cytometer assay, n = 3 ~ 4. (H ~ M) The effects of CLIC1 overexpression on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by circAPP knockdown in Aβ-treated BV-2 cells were assessed using Western blot assay, n = 4. (N) The effect of CLIC1 overexpression on Aβ phagocytosis of BV-2 cells regulated by circAPP knockdown was assessed using flow cytometry assay, n = 4 ~ 5. (O ~ T) The effects of CLIC1 overexpression on the expression of Iba-1, TNF-α, Pro- and cleaved IL-1β, CD16, Arg1 and IL-10 regulated by miR-1906 overexpression in Aβ-treated BV-2 cells were assessed using Western blot assay, n = 4. (U) The effect of CLIC1 overexpression on Aβ phagocytosis of BV-2 cells regulated by miR-1906 overexpression was assessed using flow cytometry assay, n = 3 ~ 4. (V) Schematic representation of proposed mechanism of circAPP in AD microglial polarization, pathology and cognitive func tion. CircAPP was upregulated in Aβ-treated microglial cells and the hippocampus of APP/PS1 mice. CircAPP knockdown inhibited its interaction with miR-1906 and increased miR-1906 expression, which in turn retarded CLIC1 expression and channel activity, thereby regulating microglial polarization and ameliorating AD pathology and cognitive function. All data in the figure are presented as mean ± SEM. One-way ANOVA was used to assess statisti cally significant differences. *P < 0.05, **P < 0.01

Article Snippet: After the protein samples were separated and transferred, primary antibodies against Aβ42 (Biolegend, San Diego, USA, dilution: 1:1000), p-tau S396 (ABclone, Wuhan, China, dilution: 1:1000), pro-and cleaved IL-1β (Affinity Biosciences, dilution: 1:1000), TNF-α (Proteintech, Wuhan, China, dilution: 1:1000), IL-10 (Proteintech, Wuhan, China, dilution: 1:800), CD16 (Proteintech, Wuhan, China, dilution: 1:1000), Arg1 (Proteintech, Wuhan, China, dilution: 1:1000), CLIC1 (Proteintech, Wuhan, China, dilution: 1:1000), Iba-1 (Proteintech, Wuhan, China, dilution: 1:1000), Gapdh (Proteintech, Wuhan, China, dilution: 1:3000) and β-actin (ABclonal, Wuhan, China, dilution: 1:8000) were incubated at 4 °C overnight.

Techniques: In Vitro, Knockdown, Expressing, Western Blot, Flow Cytometry, Over Expression, Activity Assay

Journal: Science Advances

Article Title: Identification of an ionic mechanism for ERα-mediated rapid excitation in neurons

doi: 10.1126/sciadv.adp0696

Figure Lengend Snippet: ( A ) BioID to screen for ERα-interacting proteins. ( B ) ERα-interacting membrane proteins, with ERα (encoded by the ESR1 gene) and CLIC1 highlighted by the red circles. ERα interactors are classified on the basis of their subcellular localization. The same BioID-MS were performed in two independent biological experiments. ( C ) Co-immunoprecipitation showing the interaction of recombinant human ERα and Clic1 proteins in the test tube. The same results were repeated in three independent biological experiments. ( D ) Co-immunoprecipitation showing the interaction of overexpressed human ERα-GFP and Clic1-Flag proteins in HEK293T cells. Cells coexpressing Clic1-Flag and ERα-EGFP were treated with or without E 2 for 30 min. The Clic1-Flag protein complex was then pulled down using Flag-M2 magnetic beads. Clic1 and ERα were identified by Western blotting. The same results were repeated in three independent biological experiments. ( E ) Co-immunoprecipitation showing the interaction of ERα-WT or ERα-C451A and Clic1 proteins in HEK293 cells. Cells coexpressing Clic1-Flag and ERα-WT-EGFP (or ERα-C451A-EGFP) were subjected to immunoprecipitation with Flag-M2 beads. Identification of Clic1 and ERα in the immunoprecipitation samples was performed by Western blotting. Data are quantified from four independent biological experiments. Data are presented as means ± SEM. **** P < 0.0001 in two-sided unpaired t test. IP, immunoprecipitation. IgG, immunoglobulin G; WT, wild type; LC-MS, liquid chromatography tandem mass spectrometry.

Article Snippet: Subsequently, the antibody against Clic1 (Cell Signaling Technology, 53424S) was introduced to precipitate the Clic1 protein complex.

Techniques: Membrane, Immunoprecipitation, Recombinant, Magnetic Beads, Western Blot, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry

Journal: Science Advances

Article Title: Identification of an ionic mechanism for ERα-mediated rapid excitation in neurons

doi: 10.1126/sciadv.adp0696

Figure Lengend Snippet: ( A ) An illustration of Clic1 genetic disruption in ERα neurons from one side of the vlVMH and using ERα neurons from the other side of vlVMH as controls. ( B ) Peak inward and outward IAA-94–senstive currents in ERα vlVMH neuron from Clic1 disrupted side (Cas9) versus the control side (GFP). N = 5 neurons from three mice per group. * P < 0.05 and *** P < 0.001 in two-sided unpaired t tests. ( C ) Resting membrane potential (left) and firing frequency (right) of ERα vlVMH neurons from Clic1 disrupted side (Cas9; N = 14) versus the control side (GFP, N = 21) at the baseline and after E 2 treatment (100 nM). **** P < 0.0001 in two-way ANOVA analysis followed by Šídák comparisons. Note that some neurons did not have spontaneous action potential firing and therefore were not included in the firing frequency analysis. Data are presented as means ± SEM with individual data points.

Article Snippet: Subsequently, the antibody against Clic1 (Cell Signaling Technology, 53424S) was introduced to precipitate the Clic1 protein complex.

Techniques: Disruption, Control, Membrane

Journal: Science Advances

Article Title: Identification of an ionic mechanism for ERα-mediated rapid excitation in neurons

doi: 10.1126/sciadv.adp0696

Figure Lengend Snippet: ( A ) A schematic illustration of Clic1 genetic disruption in ERα vlVMH and ERα ARH neurons in ERα-Cre mice and using WT littermates as controls. ( B and C ) Temporal changes in body weight in HFD-fed WT (B) and ERα-Cre (C) female mice in response to OVX + V versus OVX + E 2 treatment. Data are presented as means ± SEM. N = 6, 7, or 8 mice per group. **** P < 0.0001 in two-way ANOVA analysis. ( D ) Body weight changes on day 48 in mice described in (B and C). ( E ) Averaged daily food intake in mice described in (B and C). ( F and G ) Fat (F) and lean (G) mass of mice described in (B and C). Data are presented as means ± SEM. N = 6, 7, or 8 mice per group. ** P < 0.01 or *** P < 0.001 between OVX + V and OVX + E 2 groups; # P < 0.05 or #### P < 0.0001 between WT and ERα-Cre mice in two-way ANOVA analysis followed by Šídák comparisons.

Article Snippet: Subsequently, the antibody against Clic1 (Cell Signaling Technology, 53424S) was introduced to precipitate the Clic1 protein complex.

Techniques: Disruption