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

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate attenuates ethanol‐induced gastric mucosal hemorrhage and oedema in vivo. (A) Establishment of ethanol‐induced gastric ulcer in rats by intragastric administration of anhydrous ethanol. (B) Flowchart of the animal experimental procedures. (C) Macroscopic appearance of the gastric mucosa in four groups. (D) Quantification of gastric damage expressed as the ulcer index (UI). The UI was calculated as follows: UI = 10 × (ulcerated area/total mucosal area). (E) Representative HE‐stained images of stomach tissues. Scale bar = 50 µm. (F) Histological evaluation of gastric lesions using a microscopic HE score (0–14). The score sums the severity of four features: inflammatory cells (0–3), mucosal edema (0–4), hemorrhage (0–4), and epithelial loss (0–3). (G) Representative CD31 IF staining images in gastric mucosa. Scale bar = 20 µm. (H and I) RT‐qPCR analysis of Ang1 and Et1 mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. (J and K) The nitrate levels of the serum and gastric mucosa in the four groups. Quantitative data are expressed as the mean ± SD. * p < 0.05, *** p < 0.001, and ns denotes no significance. HE, hematoxylin–eosin; Nit, nitrate; IF, immunofluorescence; CD31, platelet endothelial cell adhesion molecule‐1; Ang1, angiotensin‐1; Et1, endothelin‐1; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; SD, standard deviation.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: In Vivo, Staining, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Tff2 knockdown abolishes nitrate protection against ethanol‐induced gastric ulcers in vivo. (A) Establishment of Tff2‐KD rat by tail vein injection of recombinant AAV. (B) The timeline of dietary nitrate administration (3 weeks after injection and 7 days before ethanol gavage). (C and D) The macroscopic appearance and ulcer index of the gastric mucosa in Tff2‐KD and scramble groups with ethanol gavage. The UI was calculated as follows: UI = 10 × (ulcerated area/total mucosal area). (E and F) Representative histology images of stomach tissue and a histopathologic score of HE staining in Tff2‐KD and scramble groups. The HE score sums the severity of four features: inflammatory cells (0–3), mucosal edema (0–4), hemorrhage (0–4), and epithelial loss (0–3). Scale bar = 50 µm. (G and H) Representative gastric tissue images of AB–PAS staining and mucin histochemical analysis of Tff2‐KD and scramble groups. The mucin area was expressed as fold change relative to the scramble + EtOH group. Scale bar = 50 µm. Quantitative data are expressed as the mean ± SD. *** p < 0.001, and ns denotes no significance. AAV, adeno‐associated virus; HE, hematoxylin–eosin; AB–PAS, Alcian blue and periodic acid‐Schiff; EtOH, ethanol; KD, knockdown; Nit, nitrate; Tff2; trefoil factor 2.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Knockdown, In Vivo, Injection, Recombinant, Staining, Virus

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate promotes migration by TFF2 upregulation in vitro. (A) Images of the scratch healing process of GES‐1 cells in Ibidi culture inserts. Scale bar = 500 µm. (B and C) Quantitative analysis of the migration rate at 24 h and 48 h in (A). (D and E) Representative immunoblotting band of the TFF2 protein and analysis of band gray values. (F) IF staining of pMLC (pink) and DAPI (blue). Scale bar = 40 µm. (G) IF analysis of pMLC with MFI. (H) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts. Scale bar = 500 µm. (I and J) Quantitative analysis of the migration rate at 24 h and 48 h. (K) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts containing ethanol. Scale bar = 500 µm. (L and M) Quantitative analysis of the migration rate at 24 h and 48 h. Migration rate is quantified by the percentage of closed area to the initial scratch area. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and ns denotes no significance. TFF2, trefoil factor 2; GES‐1, human gastric epithelial; EtOH, ethanol; Nit, nitrate; Ctrl, control; MLC, myosin light chain 2; pMLC, phosphorylated myosin light chain 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; NC, negative control; SD, standard deviation.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Migration, In Vitro, Western Blot, Staining, Transfection, Negative Control, Control, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: Primary human gastric mucosal epithelial (CP‐H048) cells and primary human gastric mucosal epithelial cell complete medium (CM‐H048) were purchased from Procell (Wuhan, Hubei, China).

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay

Journal: Pulmonary Circulation

Article Title: MiR‐24 Attenuates Oxidative Stress and Mitochondrial Apoptosis in Ventilator‐Induced Lung Injury by Targeting Bcl‐2‐related Ovarian Killer

doi: 10.1002/pul2.70171

Figure Lengend Snippet: MiR‐24 overexpression alleviated cyclic stretch‐induced injury in alveolar epithelial cells in vitro. (A) RT‐qPCR analysis was used to detect the miR‐24 expression in human alveolar epithelial cells treated with CS and transfected with miR‐24 mimic. (B) CCK‐8 assays were performed to detect the viability of alveolar epithelial cells in each group. (C) RT‐qPCR and (D) western blot were conducted to determine the mRNA and protein expression of proinflammatory cytokines (TNF‐α, IL‐6, and IL‐1β) in alveolar epithelial cells. (E) Intracellular ROS levels, (F) MDA, and (G) SOD levels in each group. (H) Western blot analysis was used to detect the cellular levels of apoptotic proteins (Bcl‐2, Bax, cleaved caspase‐3) in each group. (I) Flow cytometry was performed to detect the cell apoptotic rate. (J) JC‐1 staining examined the MMP of alveolar epithelial cells after CS exposure and miR‐24 overexpression (scale bar = 50 um). (K) The ATP level in alveolar epithelial cells after indicated treatment. *** p < 0.001.

Article Snippet: Primary human alveolar epithelial cells (Passage 2) were sourced from Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Western Blot, Flow Cytometry, Staining

Journal: Pulmonary Circulation

Article Title: MiR‐24 Attenuates Oxidative Stress and Mitochondrial Apoptosis in Ventilator‐Induced Lung Injury by Targeting Bcl‐2‐related Ovarian Killer

doi: 10.1002/pul2.70171

Figure Lengend Snippet: BOK is directly targeted by miR‐24. (A) RT‐qPCR analysis detected the levels of the top 5 targets potentially binding to miR‐24 via the miRDB database in alveolar epithelial cells with or without CS exposure. (B) The binding sequences between miR‐24 and BOK. (C) Luciferase reporter assays evaluated miR‐24 and BOK binding. (D) RNA‐pull‐down assays evaluated the interaction between BOK and miR‐24 in alveolar epithelial cells. (E) IHC and (F) western blot analyzes revealed protein levels of BOK in rat lung tissues in each group. (G) RT‐qPCR and western blot analyzes examined BOK levels in alveolar epithelial cells. (H) RT‐qPCR demonstrated the levels of BOK in the peripheral blood of healthy controls and ARDS patients. (I) Pearson correlation analysis signified the expression relation between BOK and miR‐24 in the peripheral blood of ARDS patients. *** p < 0.001.

Article Snippet: Primary human alveolar epithelial cells (Passage 2) were sourced from Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: Quantitative RT-PCR, Binding Assay, Luciferase, Western Blot, Expressing

Journal: Pulmonary Circulation

Article Title: MiR‐24 Attenuates Oxidative Stress and Mitochondrial Apoptosis in Ventilator‐Induced Lung Injury by Targeting Bcl‐2‐related Ovarian Killer

doi: 10.1002/pul2.70171

Figure Lengend Snippet: MiR‐24 protected the alveolar epithelial cells from CS‐induced injury by regulating BOK. (A) RT‐qPCR detected the expression of BOK in CS‐treated alveolar epithelial cells after indicated transfection. (B) CCK‐8 assays revealed the viability of alveolar epithelial cells. (C) RT‐qPCR and (D) western blot analyzes evaluated proinflammatory cytokines levels in CS‐exposed cells. (E) Intracellular ROS levels, (F) MDA and (G) SOD levels in each group. (H) Western blot was used to measure the protein levels of apoptosis‐related proteins in each group. (I) Flow cytometry determined the apoptotic rate of CS‐treated cells in each group. (J) JC‐1 staining of CS‐exposed cells in each group. (K) The ATP levels in CS‐treated cells in each group. *** p < 0.001.

Article Snippet: Primary human alveolar epithelial cells (Passage 2) were sourced from Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Western Blot, Flow Cytometry, Staining