Journal: Oncology Letters

Article Title: Charged multivesicular body protein 4C promotes the progression of cervical cancer through the HPV E6/miR‑543 axis

doi: 10.3892/ol.2025.15021

Figure Lengend Snippet: Cell line selection and CHMP4C expression validation. (A) Expression of CHMP4C mRNA was assessed using reverse transcription-quantitative PCR in the cervical cancer SiHa, HeLa and Caski cell lines. (B) Western blotting was used to evaluate the protein expression level of CHMP4C in the cervical cancer cell lines HeLa and SiHa, and in the normal cervical H8 cell line. Experiments were repeated three times independently. *P<0.05. CHMP4C, charged multivesicular body protein 4C.

Article Snippet: The HPV16-positive cervical cancer SiHa cell line (National Infrastructure of Cell Line Resource) was cultured in RPMI1640 medium (HyClone; Cytiva), and the human cervical epithelial immortalized H8 (Jennio Biotech Co., Ltd.), HPV16-positive cervical cancer Caski (National Infrastructure of Cell Line Resource) and HPV18-positive cervical cancer HeLa (Pricella ® ; Wuhan Elabscience Biotechnology Co., Ltd.) cell lines were cultured in DMEM medium (HyClone; Cytiva), containing 10% fetal bovine serum (Shanghai Excell Biological Technology Co., Ltd.).

Techniques: Selection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Letters

Article Title: Charged multivesicular body protein 4C promotes the progression of cervical cancer through the HPV E6/miR‑543 axis

doi: 10.3892/ol.2025.15021

Figure Lengend Snippet: Assessment of si-CHMP4C transfection efficiency and the effect of silencing CHMP4C expression on cervical cancer cells. After transfection with si-CHMP4C, reverse transcription-quantitative PCR assessed the expression of CHMP4C in (A) SiHa and (B) HeLa cells. The MTT assay evaluated the effect of CHMP4C silencing on the viability of (C) SiHa and (D) HeLa cells. Flow cytometry was used to assess the effect of CHMP4C silencing on the apoptosis of (E) SiHa and (F) HeLa cells. The wound-healing assay in (G) SiHa and (H) HeLa cells, and the cell invasion assay in (I) SiHa and (J) HeLa cells evaluated cell migration and invasion, respectively, in the presence of si-CHMP4C. Experiments were repeated three times independently. *P<0.05. si, small interfering; CHMP4C, charged multivesicular body protein 4C.

Article Snippet: The HPV16-positive cervical cancer SiHa cell line (National Infrastructure of Cell Line Resource) was cultured in RPMI1640 medium (HyClone; Cytiva), and the human cervical epithelial immortalized H8 (Jennio Biotech Co., Ltd.), HPV16-positive cervical cancer Caski (National Infrastructure of Cell Line Resource) and HPV18-positive cervical cancer HeLa (Pricella ® ; Wuhan Elabscience Biotechnology Co., Ltd.) cell lines were cultured in DMEM medium (HyClone; Cytiva), containing 10% fetal bovine serum (Shanghai Excell Biological Technology Co., Ltd.).

Techniques: Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Wound Healing Assay, Invasion Assay, Migration

Journal: Oncology Letters

Article Title: Charged multivesicular body protein 4C promotes the progression of cervical cancer through the HPV E6/miR‑543 axis

doi: 10.3892/ol.2025.15021

Figure Lengend Snippet: Evaluation of CHMP4C plasmid transfection efficiency and the effect of overexpressed CHMP4C on cervical cancer cells. The overexpression efficiency of CHMP4C in (A) SiHa and (B) HeLa cells was assessed using reverse transcription-quantitative PCR. The effect of CHMP4C overexpression on the proliferation of (C) SiHa and (D) HeLa cells was evaluated using MTT assays. The effect of the overexpression of CHMP4C on the migration of (E) SiHa and (F) HeLa cells was evaluated using wound healing assays. Flow cytometry was used to assess the effect of the overexpression of CHMP4C on the apoptotic rate of (G) SiHa and (H) HeLa cells. Experiments were repeated three times independently, *P<0.05. CHMP4C, charged multivesicular body protein 4C.

Article Snippet: The HPV16-positive cervical cancer SiHa cell line (National Infrastructure of Cell Line Resource) was cultured in RPMI1640 medium (HyClone; Cytiva), and the human cervical epithelial immortalized H8 (Jennio Biotech Co., Ltd.), HPV16-positive cervical cancer Caski (National Infrastructure of Cell Line Resource) and HPV18-positive cervical cancer HeLa (Pricella ® ; Wuhan Elabscience Biotechnology Co., Ltd.) cell lines were cultured in DMEM medium (HyClone; Cytiva), containing 10% fetal bovine serum (Shanghai Excell Biological Technology Co., Ltd.).

Techniques: Plasmid Preparation, Transfection, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Migration, Flow Cytometry

Journal: Oncology Letters

Article Title: Charged multivesicular body protein 4C promotes the progression of cervical cancer through the HPV E6/miR‑543 axis

doi: 10.3892/ol.2025.15021

Figure Lengend Snippet: Effect of CHMP4C and E6 silencing on the expression of Bcl2, Bcl-XL, survivin and Caspase-7 proteins in cervical cancer cells. (A) Western blotting assessed the effect of silencing of CHMP4C expression on the expression of the apoptosis-related proteins Bcl2, Bcl-XL, Survivin and Caspase-7. Evaluation of CHMP4C knockdown using reverse transcription-quantitative PCR in (B) SiHa and (C) HeLa cells. (D) Effect of the knockdown of HPV16 E6 in SiHa cells and HPV18 E6 in HeLa cells on the expression of CHMP4C. The experiments were repeated three times independently. *P<0.05. CHMP4C, charged multivesicular body protein 4C; HPV, human papillomavirus; si, small interfering.

Article Snippet: The HPV16-positive cervical cancer SiHa cell line (National Infrastructure of Cell Line Resource) was cultured in RPMI1640 medium (HyClone; Cytiva), and the human cervical epithelial immortalized H8 (Jennio Biotech Co., Ltd.), HPV16-positive cervical cancer Caski (National Infrastructure of Cell Line Resource) and HPV18-positive cervical cancer HeLa (Pricella ® ; Wuhan Elabscience Biotechnology Co., Ltd.) cell lines were cultured in DMEM medium (HyClone; Cytiva), containing 10% fetal bovine serum (Shanghai Excell Biological Technology Co., Ltd.).

Techniques: Expressing, Western Blot, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Charged multivesicular body protein 4C promotes the progression of cervical cancer through the HPV E6/miR‑543 axis

doi: 10.3892/ol.2025.15021

Figure Lengend Snippet: CHMP4C as a direct target of miR-543. Effect of the knockdown of (A) HPV16 E6 in SiHa cells and (B) HPV18 E6 in HeLa cells on the expression of miR-543. (C) Bioinformatics analysis results for a potential binding site between miR-543 and CHMP4C. (D) The dual-luciferase reporter assay was used to assess the effect of the overexpression of miR-543 on the luciferase activity of 293T cells transfected with wtCHMP4C. The overexpression efficiency of miR-543 mimics was assessed using reverse transcription-quantitative PCR in (E) SiHa and (F) HeLa cells. (G) Effect of the overexpression of miR-543 on the expression of CHMP4C was evaluating using western blotting. The experiments were repeated three times independently. *P<0.05. CHMP4C, charged multivesicular body protein 4C; miR, microRNA; mu, mutant; wt, wild-type; si, small interfering; NC, negative control; HPV, human papillomavirus.

Article Snippet: The HPV16-positive cervical cancer SiHa cell line (National Infrastructure of Cell Line Resource) was cultured in RPMI1640 medium (HyClone; Cytiva), and the human cervical epithelial immortalized H8 (Jennio Biotech Co., Ltd.), HPV16-positive cervical cancer Caski (National Infrastructure of Cell Line Resource) and HPV18-positive cervical cancer HeLa (Pricella ® ; Wuhan Elabscience Biotechnology Co., Ltd.) cell lines were cultured in DMEM medium (HyClone; Cytiva), containing 10% fetal bovine serum (Shanghai Excell Biological Technology Co., Ltd.).

Techniques: Knockdown, Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Activity Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis, Negative Control

Journal: PLoS ONE

Article Title: PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

doi: 10.1371/journal.pone.0218003

Figure Lengend Snippet: (A) PINK1 mRNA levels and mtDNA copies in culture media in A549 cells treated with increasing doses of TM for 24h (n = 3; *p<0.01 vs TM 0μg/ml; two-way ANOVA with multiple comparison). (B) Mitochondrial and nuclear DNA copies detected in culture media in A549 cells treated with TM 1μg/ml for 24h (n = 4; **p<0.0001; two-way ANOVA with multiple comparison). (C) PINK1 mRNA levels in primary human alveolar epithelial cells treated 24h with TM 0.1μg/ml (n = 4; **p<0.0001; unpaired t-test). Mitochondrial and nuclear DNA copies detected in culture media in primary human alveolar epithelial cells treated 24h with TM 0.1μg/ml. (n = 4; **p<0.0001; two-way ANOVA with multiple comparison). (D) MtDNA copies in culture media in PINK1 knock-down A549 cells (n = 3, *p<0.001 vs scramble; unpaired t-test). (E) MtDNA copies detected in the BAL fluid of PINK1 +/+ and -/- mice (n = 4, *p<0.001 vs PINK1 +/+; unpaired t-test). (F) MtDNA copies in culture media in A549 cells overexpressing GFP or PINK1 then treated with increasing doses of TM for 24h (n = 4; *p<0.01 vs TM 0μg/ml, #p<0.001 vs GFP; two-way ANOVA with multiple comparison). Dot plots represent mean ± SEM. See for details about ER stress markers levels after TM treatment and knock-down / overexpression efficiencies.

Article Snippet: Primary human pulmonary alveolar epithelial cells (AECIIs, ScienCell #3200, cryopreserved at passage 0) grew overnight in complete alveolar epithelial cell medium (ScienCell #3201) over a poly‐L‐lysine coated plate (ScienCell #0403) before been treated.

Techniques: Comparison, Knockdown, Over Expression

Journal: PLoS ONE

Article Title: PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses

doi: 10.1371/journal.pone.0218003

Figure Lengend Snippet: (A) Pre-treatment with endocytosis blockers (dynasore 80μM or chloroquine 50μM) for 1h reduced TFG-β released induced by TRL9 agonist ODN M362 (2μM) after 24h (n = 4; *p<0.01 vs PBS, # p<0.01; one-way ANOVA with multiple comparison). TLR9 mRNA transcript levels in total lung lysate from PINK1 -/- mice (B), A549 cells treated with PINK1 siRNA for 48h (C) or A549 cells treated with 1μg/ml of tunicamycin for 24h (D) (n = 4; *p<0.05, **p<0.01; unpaired t-test). (E) TLR9 mRNA transcript levels in primary human lung epithelial cells (E) after 24 of 0.1μg/ml of tunicamycin or 1 μg/mL exogenous mtDNA in the presence or absence of DNAse (1U/ml) (n = 4; *p<0.05 vs unstimulated, **p<0.01; one-way ANOVA with multiple comparison). TGF-β release in A549 cells were treated with PINK1 siRNA (F) or stimulated with 1 μg/mL exogenous mtDNA for 24h (G) with pre-treatment with a TLR9 antagonist (ODN-TAG, 1μM), the NFκB antagonist BAY11-7082 (5μM), or by silencing TLR9 48h prior stimulation (n = 3; *p<0.001 vs PBS, #p<0.001 vs siPINK1 or mtDNA; one-way ANOVA with multiple comparison). (H) TGF-β release in primary human lung epithelial cells after stimulations with extracellular mtDNA (1μg/ml) in the presence of DNAse (1U/ml), pre-treatments with TRL9 antagonist ODN-TAG (1μM) or exposed to 1μg/ml of nDNA after 24h. TRL9 agonist ODN M362 (1μM) (n = 4; *p<0.001 vs PBS, #p<0.001 vs mtDNA; two-way ANOVA with multiple comparison). Dot plots represent mean ± SEM. See and for knock-down efficiencies.

Article Snippet: Primary human pulmonary alveolar epithelial cells (AECIIs, ScienCell #3200, cryopreserved at passage 0) grew overnight in complete alveolar epithelial cell medium (ScienCell #3201) over a poly‐L‐lysine coated plate (ScienCell #0403) before been treated.

Techniques: Comparison, Knockdown