Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Relative CLC3 mRNA expression levels in various CVC cell lines (SiHa, MS751, HeLa, ME180, Hela229, CaSKi) compared to normal cervical epithelial cells (NCCs). * P < 0.05, ** P < 0.001. vs. NCCs. B Western blot analysis showing CLC3 protein expression in NCCs and different CVC cell lines (SiHa, MS751, HeLa, ME180, Hela229, CaSKi), with GAPDH as a loading control. * P < 0.05, ** P < 0.001. vs. NCCs. C CLC3 mRNA expression in CVC cell lines (SiHa, MS751, HeLa, ME180, Hela229, and CaSKi) following CLC3 overexpression (CLC3 High ) or knockdown (CLC3 Low ) compared to control. * P < 0.05, ** P < 0.001. vs. Control. D Western blot analysis of CLC3 protein levels in CLC3 High , CLC3 Low , and control cells across the same cell lines, with quantification shown in the accompanying graphs. * P < 0.05, ** P < 0.001. vs. Control. E IC50 values for cisplatin, paclitaxel, and 5-FU in CLC3 High , CLC3 Low , and control groups in SiHa and CaSKi cells, showing CLC3 modulation’s effect on chemoresistance. * P < 0.05 vs. Control.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Over Expression, Knockdown

Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Fluorescence images and bar graphs showing cytosolic pH in SiHa and CaSKi cells with CLC3 overexpression (CLC3 High ), knockdown (CLC3 Low ), and control, using SypHer3s as the pH probe at 60- and 120-min post-treatment. B Representative images and quantification of lysosomal pH in SiHa and CaSKi cells with CLC3 modulation, analyzed using LysoSensor Yellow/Blue DND-160. C Lysosomal pH in cells treated with V-ATPase inhibitor Baf A1 combined with CLC3 High and CLC3 Low modulation. D Immunofluorescence images showing the fluorescence intensity of cathepsin B (B MR) and cathepsin L (L MR) in cells treated with Baf A1 and CLC3-modulated conditions, indicating lysosomal degradation capacity. * P < 0.05, ** P < 0.001. vs. Control.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Fluorescence, Over Expression, Knockdown, Control, Immunofluorescence

Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Western blot analysis of autophagy and apoptosis markers, including P62, LC3 (LC3I and LC3II), and cleaved caspases-3 and -8 in CLC3 High , CLC3 Low , and control cells. Bar graphs quantify the relative expression levels of these proteins, showing increased autophagic flux and apoptotic activity in CLC3 Low cells. B Immunofluorescence staining of LC3 and P62 in CLC3-modulated cells, with quantification of relative fluorescence intensity in CLC3 High , CLC3 Low , and control groups. * P < 0.05, ** P < 0.001. vs. Control. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA with post hoc tests; p < 0.05, p < 0.01, p < 0.001.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Heatmap showing the expression of various V-ATPase subunits and companion genes across control, a2v-mAb-treated, CLC3 Low with a2v-mAb, and CLC3 High with a2v-mAb groups in CVC cells. B Lysosomal pH analysis in SiHa and CaSKi cells under the same experimental conditions, with images from LysoSensor Yellow/Blue DND-160 assays and quantitative analysis of pH changes. * P < 0.05, ** P < 0.001. vs. Control. # P < 0.05, ## P < 0.001. vs. a2v-mAb.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Cell viability assays in SiHa and CaSKi cells under various treatment conditions, including a2v-mAb treatment, CLC3 High , and CLC3 Low . B Colony formation assay results, quantifying cell colony numbers to evaluate the proliferative capacity of each treatment group. C Flow cytometry analysis of apoptosis rates in SiHa and CaSKi cells with CLC3 modulation and a2v-mAb treatment, with quantification of apoptotic cells. D RT-PCR analysis of tumor stemness markers (NANOG, OCT4, KLF4, ABCG2, BMI1, MYC, TIP30, MAD2, STAT3) and CLC3 expression levels in CLC3-modulated cells treated with a2v-mAb, demonstrating the effect of CLC3 on maintaining stemness characteristics. E IC50 values for cisplatin, paclitaxel, and 5-FU in SiHa and CaSKi cells under each treatment condition. * P < 0.05 vs. Control. # P < 0.05 vs. a2v-mAb.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Colony Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: Cell Death Discovery

Article Title: CLC3 regulates V-ATPase to enhance lysosomal degradation and cisplatin resistance in cervical cancer cells

doi: 10.1038/s41420-025-02876-0

Figure Lengend Snippet: A Tumor volume and weight measurements in mice treated with cisplatin, CLC3 Low with cisplatin, CLC3 High with cisplatin, and control. B Hematoxylin and eosin (H&E) staining, along with immunohistochemical (IHC) staining for CLC3, cyclin D1, cyclin D3, and Ki67 in tumor sections from each group, with quantification of positive cells. C TUNEL assay showing apoptosis levels in tumors from each treatment condition, with quantification of TUNEL-positive cells. D Histological analysis of lung tissue for metastases, comparing the number of metastatic lung nodules across different treatment groups, highlighting the anti-metastatic effect of CLC3 knockdown with cisplatin. * P < 0.05, ** P < 0.001. vs. Control. # P < 0.05, ## P < 0.001. vs. Cisplatin.

Article Snippet: For immunoprecipitation, equal amounts of total protein (500–800 μg) were incubated overnight at 4 °C with 2–3 μg of anti-CLC3 antibody or normal rabbit IgG as a negative control, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology).

Techniques: Control, Staining, Immunohistochemical staining, Immunohistochemistry, TUNEL Assay, Knockdown

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: CLC-3 is highly expressed in gastric cancer. Notes: (A) Heat map of QOE 3.1 software ( http://www.qlucore.com/ ) represents the levels of CLC-3 mRNA expression of GSE63089 and GSE56807 datasets. The results show that CLC-3 was significantly higher in T than N ( P <0.05 and P <0.01, respectively). (B) The relative mRNA levels of CLC-3 were detected by qRT-PCR in gastric cancer cell lines and GES-1. The data are presented as the mean±SD. (C) Western blot analysis of CLC-3 protein expression in gastric cancer cell lines and GES-1. (** P <0.01). GAPDH served as a loading control. Abbreviations: CLC-3, chloride channel-3; QOE, Qlucore Omics Explorer; qRT-PCR, quantitative real-time PCR; T, gastric cancer tumors; N, non-tumor tissues; GES-1, gastric epithelial cell line.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Software, Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: CLC-3 is elevated in gastric cancer tissues and predicts a poor prognosis. Notes: (A) Immunohistochemical staining revealed CLC-3 protein’s high expression (a, b) and low expression (c) in gastric cancer tissues, and low expression in adjacent non-tumor tissues (d). (a, scoring index=12; b, scoring index=8; c, d, scoring index=0). The lower panels display representative immunohistochemical staining patterns in selected zones with an enlarged view. Scale bars=100 μm; magnification of the upper images ×40, and the lower images ×100. (B) Survival curve for 353 gastric cancer patients according to CLC-3 protein expression status (log-rank test). Gastric cancer patients with a high expression of CLC-3 protein had a significantly shorter OS time than those patients with a low expression of CLC-3 ( P =0.000). Abbreviations: CLC-3, chloride channel-3; OS, overall survival.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: Knockdown of CLC-3 inhibits gastric cancer cells’ proliferation, migration and invasion. Notes: MTT (right panel) and cell count assay (left panel) indicate that reduced CLC-3 expression markedly abrogated cell viability or the number cells of (A) BGC-823 and (B) SGC-7901 cells compared with those of the control group (** P <0.01 vs. NC group). Experiments were performed in triplicate. Bars: SD. Transwell assay showed that inhibition of CLC-3 reduced the number of migrated and invasive (C) BGC-823 cells and (D) SGC-7901 cells compared with those of the control group (** P <0.01). Experiments were performed in triplicate. Bars: SD. Magnification ×100. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA; SiCLC-3, CLC-3 siRNA.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Migration, Cell Counting, Expressing, Transwell Assay, Inhibition

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: Overexpression of CLC-3 promotes gastric cancer cells’ proliferation, migration and invasion. Notes: (A) MTT and cell count assay indicated that cell proliferation in N87 cells was notably enhanced by upregulating CLC-3 expression. (B) Transwell assay revealed that overexpression of CLC-3 significantly increased migratory and invasive abilities of N87 cells. ** P <0.01. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Over Expression, Migration, Cell Counting, Expressing, Transwell Assay

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: TGF-β/Smad signaling pathway is involved in CLC-3–induced gastric cancer cell migration and invasion. Notes: (A) qRT-PCR implies that inhibition of CLC-3 prohibited the expression of TGF-β1 and TGF-β2 mRNA in BGC-823 and SGC-7901 cells (** P <0.01). (B) Western blot analysis reveals that inhibition of CLC-3 suppressed the protein expression level of TGF-β1, TGF-β2, p-Smad2 and p-Smad3 (** P <0.01). GAPDH was used as an internal standard for protein loading. The values are presented as the mean±SD. (C) The downstream genes of TGF-β/Smad signaling pathway, such as Snail, ZEB1, Slug, except for Twist1, were significantly decreased in gastric cancer cells when CLC-3 was knocked down. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA; p-Smad2, phospho-Smad2; p-Smad3, phospho-Smad3; qRT-PCR, quantitative real-time PCR; SiCLC-3, CLC-3 siRNA.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Migration, Quantitative RT-PCR, Inhibition, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

doi: 10.2147/CMAR.S159790

Figure Lengend Snippet: Supplement of TGF-β reverses the suppressed proliferation, migration and invasion induced by SiCLC-3. Notes: rTGF effectively reversed the suppressed (A) proliferation, (B) migration and invasion induced by inhibition of CLC-3 both in BGC-823 and SGC-7901 cells (* P <0.05; ** P <0.01). Bars: SD. All of the experiments were performed in triplicate. Abbreviation: CLC-3, chloride channel-3; rTGF, recombinant purified TGF-β; NC, nonspecific control siRNA; SiCLC-3, CLC-3 siRNA; TGF, transforming growth factor.

Article Snippet: The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam).

Techniques: Migration, Inhibition, Recombinant, Purification