Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Inhibition of branched actin leads to increased endosome size. (A-J) . HeLa cells were ( A-C ) untreated, ( D-E ) treated with the formin inhibitor SMIFH2 (25 µM), ( F-G ) treated with the inactive control CK-689 (300 µM), or ( H-J ) the ARP2/3 branched actin inhibitor CK-666 (300 µM) for 50 min. Cells were fixed and stained with EEA1 and cortactin. Merged images (panels C and J ) show decreased cortactin at endosomes upon CK-666 treatment. (K) . Quantification of the effects of the inhibitors on endosome size as depicted in A-J. Imaris software was used to render EEA1-decorated endosomes as surfaces, and endosome size for each treatment (µm 2 ) was normalized to the average endosome size of the untreated group. Quantification represents 30 images from three independent experiments, including ∼50,000 endosomes per treatment. A two-tailed Mann-Whitney nonparametric test was used to determine p -values between treatment groups.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Control, Staining, Software, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin recycling is impaired upon ARP2/3 inhibition. (A-D) . HeLa cells on coverslips were incubated with fluorophore-labeled transferrin (Tf-488) for 10 minutes of uptake. Cells were ( A ) untreated, ( B ) treated with 25 µM SMIFH2, ( C ) treated with 300 µM CK-689, or ( D ) treated with 300 µM CK-666 during the transferrin uptake. Images are representative of a treated coverslip after uptake. (E-H) . Cells were chased in ( E ) complete media, ( F ) complete media containing 25 µM SMIFH2, ( G ) complete media containing 300 µM CK-689, or ( H ) complete media containing 300 µM CK-666 for 40 minutes to allow recycling. Images are representative of cells on an treated coverslip after chase. (I) . The arithmetic mean intensity of each image was analyzed using Zeiss Zen Blue software after uptake and chase. For each treatment condition, the internalized mean for the uptake was set at 100%, and the arithmetic mean intensity for each recycling image was expressed as a percentage of the normalized uptake for that condition. Quantification represents 30 images from three independent experiments. Statistical significance was determined using a two-tailed unpaired t -test.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Labeling, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin and EGF are delayed at early endosomes upon ARP2/3 inhibition. (A-D) . HeLa cells were incubated with Tf-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( A-B ) complete media or ( C-D ) in media containing 300 µM CK-666. Representative images are from the 30 min chase time point. (E). The percentage of Tf-488 fluorescence in EEA1 endosomes was calculated by measuring the area of Tf-EEA1 overlap as a percentage of total Tf fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 30 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 15 min time point. (F). A bar graph showing the individual data points from the 30 min time point in ( E ). (G-J) . HeLa cells were serum starved for 1 h, then incubated with EGF-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( G-H ) complete media or ( I-J ) in complete media containing 300 µM CK-666. Representative images are from the 45 min time point of media chase. (K). The percentage of EGF-488 fluorescence in EEA1-marked endosomes was calculated by measuring the area of EGF-EEA1 overlap as a percentage of total EGF fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 15 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 30 min time point. (L). A bar graph showing the individual data points from the 45 min time point in ( K ).

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Branched actin inhibition, but not formin inhibition, decreases actin at RAB5 QL endosomes. (A-P) . HeLa cells were transfected with mCherry-RAB5 Q79L and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin is bounded by cortactin at endosomes. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. (M-P) . Representative fluorescence intensity profiles of endosomes from ( M ) untreated, ( N ) SMIFH2-treated cells, ( O ) CK-689-treated cells, or ( P ) CK-666-treated cells. The intensity profile of Tf is in green, and the intensity profile of cortactin is in magenta. Tf vertices above 130% that occur within 20 degrees of a cortactin value above 130% are represented with a red circle; these peaks are “bounded”. Tf vertices above 130% that are not within 20 degrees of a cortactin value above 130% are represented with a grey circle and are “not bounded”. (Q). The percentage of “bounded” Tf peaks was quantified from fluorescence intensity profiles (as represented in ( M-P )). Quantification is from three independent experiments, and from 37 endosomes for the untreated group, 43 endosomes for SMIFH2-treated, 38 endosomes for CK-689-treated, and 37 endosomes from the CK-666-treated group. Statistical significance was determined using a two-tailed unpaired t -test (ns: p > 0.05). (R, S) . Representative models for the quantification and results of the experiment. A circle was drawn around the endosome membrane (red) that intersects with the regions of Tf (green) and cortactin (magenta), and fluorescence intensity at each degree around the circle was measured. ( R ) Untreated cells have Tf in confined regions on the endosome and are adjacent to regions of cortactin ∼60% of the time. ( S ) CK-666-treated cells have regions of Tf that are broader and “not bounded” by cortactin.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Transfection, Incubation, Fluorescence, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Tf occupies less discrete regions on the endosome when branched actin is inhibited. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show Tf localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of Tf on the endosome (blue arrows). (M). Model for quantification. A circle (blue) was drawn around the endosome membrane (red) that intersects with the regions of Tf, and the fluorescence intensity at each degree around the circle was measured. (N-Q) . Representative fluorescence intensity profiles of Tf on the endosome from ( N ) untreated, ( O ) SMIFH2-treated, ( P ) CK-689-treated, and ( Q ) CK-666-treated cells. The profiles depicted are from the endosomes indicated with a magenta-colored star ( A-L ). (R). Tf-containing regions from the fluorescence intensity profiles ( N-Q ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of Tf-containing regions are Tf “peaks”. The graph shows the average profile of 72 Tf peaks from 37 endosomes for the control group, 70 peaks from 43 endosomes from SMIFH2-treated cells, 73 peaks from 38 endosomes from CK-689-treated cells, and 70 peaks from 37 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of Tf peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV3 for statistical information.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Transfection, Incubation, Membrane, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: EGF segregation on the endosome is affected by branched actin inhibition. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) SMIFH2, ( G-I ) CK-689, or ( J-L ) CK-666 for 4 min. Following the pre-treatment, cells were incubated with EGF-488 (and the respective inhibitor) for 17 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show EGF localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of EGF on the endosome (blue arrows). (M-P) . Representative fluorescence intensity profiles of EGF on the endosome from the ( M ) untreated, ( N ) SMIFH2-treated, ( O ) CK-689-treated, and ( P ) CK-666 treated cells. The profiles depicted are from the endosomes indicated with a magenta star in ( A-L ). (Q) . EGF-containing regions from the fluorescence intensity profiles ( M-P ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of EGF-containing regions are EGF “peaks”. The graph shows the average profile of 84 EGF peaks from 47 endosomes that were analyzed for the control group, 86 peaks from 51 endosomes from SMIFH2-treated cells, 72 peaks from 39 endosomes from CK-689-treated cells, and 86 peaks from 49 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of EGF peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV4 for statistical information.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Incubation, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: The degradative and retrieval subdomains on endosomes coalesce upon branched actin inhibition. (A-F) . HeLa cells were transfected with mCherry-RAB5 Q79L and were co-incubated with EGF-488 and anti-CD59 antibody for 20 min. During the last 5 min of uptake, either ( A-C ) no inhibitor or ( D-F ) 300 µM CK-666 was added to the media. Pearson’s and Manders’ correlation coefficients for each representative image are listed. (G). ImageJ was used to calculate Pearson’s correlation coefficient for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test. (H, I) . ImageJ was used to calculate Manders’ correlation coefficients (M1 and M2) for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Incubation, Two Tailed Test

Journal: Vaccine

Article Title: Fluorescence-barcoded cell lines stably expressing membrane-anchored influenza neuraminidases.

doi: 10.1016/j.vaccine.2025.127157

Figure Lengend Snippet: Fig. 1. Standard NA mAbs brightly and specifically label K530-NA cell lines. Shown are flow cytometry histograms depicting the binding of recombinant IgG versions of standard NA mAbs to K530 cell lines expressing membrane-anchored NAs. Each row corresponds to a monoclonal cell line stably expressing a single type of NA. Each column corresponds to the binding profile for a single mAb. MAbs were incubated with pooled cell lines comprising Option 1 or Option 2 (Table 1), and the resultant data were concatenated into a single figure.

Article Snippet: PBMCs in RPMI-1640 medium plus 10 % FBS were incubated with irrelevant mouse IgG1 (MG1K; Rockland) to block nonspecific binding and then labeled with fluorochrome-conjugated mAbs.

Techniques: Flow Cytometry, Binding Assay, Recombinant, Expressing, Membrane, Stable Transfection, Incubation

Journal: Vaccine

Article Title: Fluorescence-barcoded cell lines stably expressing membrane-anchored influenza neuraminidases.

doi: 10.1016/j.vaccine.2025.127157

Figure Lengend Snippet: Fig. 3. Use of K530-NA cells to determine the binding breadth of newly identified NA mAbs. Shown are flow cytometry histograms depicting the binding of re combinant IgG versions of NA mAbs from donors T1, T2, or T3 to K530 cell lines expressing membrane-anchored NAs, as in Fig. 1.

Article Snippet: PBMCs in RPMI-1640 medium plus 10 % FBS were incubated with irrelevant mouse IgG1 (MG1K; Rockland) to block nonspecific binding and then labeled with fluorochrome-conjugated mAbs.

Techniques: Binding Assay, Flow Cytometry, Expressing, Membrane

Journal: Vaccine

Article Title: Fluorescence-barcoded cell lines stably expressing membrane-anchored influenza neuraminidases.

doi: 10.1016/j.vaccine.2025.127157

Figure Lengend Snippet: Fig. 4. Analysis of serum IgGs elicited by infection of rhesus macaques with H3N2 influenza virus. A) Pre-immune (day 0) or immune (day 27 or 28) blood plasma from three rhesus macaques (6451, T651, T771) infected with A/Aichi/2/1968 (H3N2) influenza virus was incubated with K530-NA cell lines. The degree of IgG labeling of each cell line was determined by flow cytometry, as in Fig. 1. Vertical, black lines denote the threshold for specific labeling of cell-surface NA, determined according to the labeling intensity observed for K530 cells expressing no NA (top row). B) Fold-change in the fluorescence intensity of plasma IgG labeling of selected K530-N2 cell lines, after either infection with H3N2 virus (Fig. 4A) or immunization with recombinant H3 HA (Supplementary Fig. 3B). Fold-change was calculated as the ratio of the geometric mean fluorescence intensity (geoMFI) resulting from labeling with the immune plasma IgG, divided by the geoMFI resulting from labeling with pre-immune plasma IgG. Each symbol represents a single animal. The difference in group means was analyzed by two-tailed t-test with Welch’s correction, as described in Materials and Methods.

Article Snippet: PBMCs in RPMI-1640 medium plus 10 % FBS were incubated with irrelevant mouse IgG1 (MG1K; Rockland) to block nonspecific binding and then labeled with fluorochrome-conjugated mAbs.

Techniques: Infection, Virus, Clinical Proteomics, Incubation, Labeling, Flow Cytometry, Expressing, Fluorescence, Recombinant, Two Tailed Test

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: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection induces expression of a subset of YAP target genes. (A) Volcano plot of gene expression in bulk RNA-sequencing of End1/E6E7 immortalized epithelial cells (End1s) during infection with Chlamydia trachomatis ( Ct ) serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (B) Table of selected transcription factors identified as potential targets of infection-associated modulation via cross-referencing of differentially expressed genes identified in (A) with the ChIP Enrichment Analysis (ChEA) database of transcription factor target genes. See also Supplementary Data S2. (C) Volcano plot of gene expression in bulk RNA-sequencing of primary human endocervical epithelial cells (HCECs) during infection with Ct serovar L2 compared to mock infection, at 24 hours post-infection (hpi). n = 3, with a minimum of 3x10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes whose expression differed most significantly (lowest FDRP) from the mock infection. (D) Venn diagram of differentially expressed genes identified in (A, C) cross-referenced with the ChEA database of YAP target genes. (E) Scatter plot of gene expression of YAP-responsive (ChEA), differentially expressed genes in either Ct serovar L2-infected End1s (x-axis) or HCECs (y-axis). All fold changes are relative to each cell type’s respective mock-infected control; blue line: linear regression model of correlation; grey shading: 95% confidence interval. R 2 and p-values calculated using Pearson’s correlation. (F) Heatmap of YAP target gene expression in Ct serovar L2-infected End1s (left columns) and HCECs (right columns). All fold changes are relative to each cell type’s respective mock-infected control; only genes differentially expressed (FDRP ≤ 0.05) in both cell types are shown. (G) Dot plot of GO biological process term enrichment in the set of YAP target genes differentially expressed in Ct serovar L2 infection of End1s or HCECs (top 25 most significantly enriched terms shown). Dot size: number of term-associated genes found in set, dot color: adjusted p-value.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Expressing, Gene Expression, RNA Sequencing, Control, Targeted Gene Expression

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

doi: 10.3389/fcimb.2023.1098420

Figure Lengend Snippet: Chlamydia infection promotes YAP nuclear translocation. (A) Expression of CTGF and CYR61 at 24 hpi in mock- and Ct L2-infected End1 cells, as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; n.s.: not significant (p-values > 0.05), asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (B) Expression of CTGF at 24 hpi in mock- and Ct L2-infected End1 cells transfected with non-targeting (NT) or YAP1-targeting siRNA (10 nM for 24 h prior to infection), as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (C) Expression of CTGF, INHBA, and BMP2 at 24 hpi in mock- and Ct L2-infected End1 cells treated with the YAP-TEAD inhibitor verteporfin (Vpf, 5 μM for 16 h starting at 8 hpi), as measured by RT-qPCR. n = 5 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons. (D) Representative micrographs of YAP (green) translocation into the nuclei (blue) of confluent mock- and Ct L2-infected End1 cells at 24 hpi. Asterisks: chlamydial inclusions, scale bar: 20 μm. (E) Quantification of YAP nuclear translocation in (D) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (F) Representative micrographs of YAP nuclear translocation of confluent mock- and Ct L2-infected primary human cervical epithelial cells at 24 hpi. Asterisks, chlamydial inclusions, scale bar: 20 μm. (G) Quantification of YAP nuclear translocation in (F) as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (H) Quantification of YAP nuclear translocation at 2, 4, 8, 12, 18, and 24 hpi in confluent mock- and Ct L2-infected End1 cells as a ratio of nuclear to cytosolic YAP fluorescence. n = 5 biological replicates, 50 cells measured per sample. Blue dots: mock-infected cells, red dots: Ct L2-infected cells, black bars: group means, asterisks: p-value ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons. (I) Representative micrographs of YAP nuclear translocation at 18 hpi in confluent mock- and Ct L2-infected End1 cells treated with chloramphenicol (Cm, 50 μg/mL for 1 h at 17 hpi) or DMSO. Asterisks: chlamydial inclusions; scale bar: 20 μm. (J) Quantification of YAP nuclear translocation in (I) . n = 3 biological replicates, 50 cells measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank-sum tests and Bonferroni’s correction for multiple comparisons.

Article Snippet: Primary human cervical epithelial cells (HCECs, ATCC PCS-0480-011, Lot 80306190) were cultured at 37° C with 5% atmospheric CO 2 in Cervical Epithelial Cell Basal Medium (CECBM, ATCC PCS-480-032) supplemented with all contents of a Cervical Epithelial Growth Kit (ATCC PCS-080-042).

Techniques: Infection, Translocation Assay, Expressing, Quantitative RT-PCR, Control, Transfection, Fluorescence