anti clic1  (Santa Cruz Biotechnology)

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Silencing CLIC1 suppresses glycolysis in gastric cancer cells. ( A ) KEGG pathway enrichment analysis of CLIC1-associated genes. ( B ) Correlation analysis between CLIC1 expression and glycolytic pathway activity. ( C ) qPCR validation of CLIC1 silencing in AGS and HGC27 cells. ( D - E ) Western blot analysis confirmed CLIC1 knockdown efficiency in AGS and HGC27 cells. ( F - G ) ECAR assays showed that CLIC1 silencing decreases glycolytic levels and glycolytic capacity in both cell lines. ( H ) Glucose consumption in CLIC1-silenced AGS and HGC27 cells. ( I ) Intracellular ATP production upon CLIC1 knockdown in both cell lines. ( J ) Lactate secretion in CLIC1-silenced gastric cancer cells. Two-sided unpaired Student’s t-test between two groups, and one-way ANOVA among three or more groups. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Activity Assay, Biomarker Discovery, Western Blot, Knockdown

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promoted gastric cancer progression through glycolysis-dependent mechanisms. ( A ) WB confirmed the efficiency of CLIC1 overexpression in AGS and HGC27 gastric cancer cells. ( B – C ) CLIC1 overexpression increased ( B ) glucose consumption and ( C ) lactate secretion, effects that were attenuated by 2-DG. ( D – G ) Wound-healing assays showed that CLIC1 overexpression enhanced cell migration, which was suppressed by 2-DG. ( H – I ) CCK-8 assays demonstrated that CLIC1 overexpression promoted cell proliferation, an effect partially reversed by 2-DG treatment. ( J – M ) Transwell assays revealed that CLIC1 enhanced both migratory and invasive capacities in a glycolysis-dependent manner. ( N ) Representative images of xenograft tumors from each treatment group at the study endpoint. ( O ) Final tumor weights at sacrifice. ( P ) Tumor growth curves showed tumor volume changes over time. One-way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Over Expression, Migration, CCK-8 Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 interacts directly with PKM2 in gastric cancer cells. ( A ) Venn diagram showed the overlap among CLIC1-interacting proteins identified by mass spectrometry, glycolysis-related genes, and CLIC1-associated genes from GeneCards and LinkedOmics databases. Eleven overlapping candidates were identified. ( B – C ) Exogenous Co-IP assays in 293T cells demonstrated that CLIC1 immunoprecipitated PKM2 and vice versa, confirming their reciprocal interaction. ( D – E ) Endogenous Co-IP assays in AGS and HGC27 gastric cancer cells further verified the specific interaction between CLIC1 and PKM2. ( F – G ) No interaction was detected between CLIC1 and the PKM1 isoform. ( H ) Immunofluorescence staining showed cytoplasmic co-localization of CLIC1 and PKM2 in AGS and HGC27 cells. ( I ) GST pull-down assay revealed that GST-tagged CLIC1 directly bound to PKM2, whereas the GST control did not. ( J ) Schematic representation of CLIC1 truncation mutants. ( K ) Co-IP assays demonstrated that full-length CLIC1 and two truncation mutants retained PKM2-binding ability

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Immunoprecipitation, Immunofluorescence, Staining, Pull Down Assay, Control, Binding Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Mapping of the CLIC1–PKM2 interaction domain and analysis of CLIC1’s effect on PKM2 expression and stability. ( A ) Diagram of PKM2 truncation mutants covering different structural domains. ( B ) Co-IP assays showed that deletion of the C-terminal domain abolished the interaction between PKM2 and CLIC1. ( C – F ) Western blot analysis showing that neither CLIC1 overexpression nor knockdown altered PKM2 protein expression levels in AGS and HGC27 gastric cancer cells.( G - H ) CHX chase assay revealed that CLIC1 depletion did not affect PKM2 protein stability. Two-sided unpaired Student’s t-test between two groups, and one-way ANOVA among three or more groups. (* p < 0.05, ** p < 0.01, *** p < 0.001, ns. not significant)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 regulates the oligomeric state and nuclear localization of PKM2. ( A – B ) Protein cross-linking and Western blot analyses showed that CLIC1 overexpression promoted PKM2 dimer formation while reducing tetramer formation in AGS and HGC27 cells. ( C – D ) Silencing of CLIC1 led to decreasing PKM2 dimers and increasing tetramers. ( E – F ) Subcellular fractionation and WB analyses demonstrating that CLIC1 overexpression enhanced the nuclear localization of PKM2. ( G ) Immunofluorescence staining confirmed that CLIC1 overexpression promoted PKM2 accumulation in the nucleus. ( H ) Molecular docking analysis indicated that the binding interface between CLIC1 and PKM2 covered both truncation regions of CLIC1, whereas the interaction site on PKM2 was primarily located within its C-terminal domain

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Western Blot, Over Expression, Fractionation, Immunofluorescence, Staining, Binding Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promotes gastric cancer progression by modulating PKM2-mediated glycolysis. ( A ) WB analysis demonstrated PKM2 protein expression following PKM2-IN-1 treatment in AGS and HGC27 cells. ( B – C ) Quantification of lactate production ( B ) and glucose consumption ( C ) in control and CLIC1-overexpressing AGS and HGC27 cells with or without PKM2-IN-1 treatment. ( D ) CCK-8 assay showed that CLIC1 overexpression enhanced gastric cancer cell proliferation, whereas PKM2-IN-1 partially reversed this effect. ( E – H ) Representative images and quantitative analysis of wound-healing assays. ( I – L ) Representative images and statistical quantification of transwell migration and invasion assays. One-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Control, CCK-8 Assay, Over Expression, Migration

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promotes gastric tumor growth through PKM2-dependent glycolysis in vivo. ( A ) Representative xenograft tumors from each group at the endpoint. ( B ) Tumor weights showed that PKM2-IN-1 treatment attenuated CLIC1-induced tumor growth. ( C ) Tumor growth curves depicted the kinetics of tumor volume expansion over time in each experimental group. ( D ) Representative IHC staining of CLIC1 and PKM2 in tumor tissues. One-way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: In Vivo, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Expression pattern and clinical relevance of PKM2 and CLIC1 in gastric cancer. ( A – B ) Differential expression analysis of CLIC1 ( A ) and PKM2 ( B ) between tumor and adjacent normal tissues in the TCGA-STAD cohort. ( C – D ) Expression levels of CLIC1 ( C ) and PKM2 ( D ) in gastric cancer and normal tissues from the GSE66229 dataset. ( E ) Correlation analysis of CLIC1 and PKM2 expression in the TCGA-STAD cohort. ( F – G ) Kaplan–Meier survival curves showing that high CLIC1 ( F ) and PKM2 ( G ) expression were associated with poor overall survival in gastric cancer patients from the GSE62254 cohort. ( H – J ) Kaplan–Meier plots illustrated that high PKM2 expression correlated with reduced OS, PFS, and PPS. ( K ) RT-qPCR analysis of PKM2 mRNA expression in paired gastric cancer and adjacent normal tissues. ( L ) Western blot analysis of PKM2 protein expression in representative paired GC and normal tissues. ( M – N ) Representative IHC staining and quantification of PKM2 ( M ) and CLIC1 ( N ) in paired gastric cancer specimens. ( O ) Correlation analysis between CLIC1 and PKM2 protein expression levels in gastric cancer tissues. Two-sided unpaired Student’s t-test. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemistry

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