Journal: mBio

Article Title: Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus

doi: 10.1128/mBio.01206-15

Figure Lengend Snippet: EmaA is an adhesin and involved in autoaggregation. (A) Mean adherence to human epithelial cells by A. aphrophilus with the wild-type emaA gene, an interrupted emaA gene, and the interrupted emaA gene reverted to the wild type. (B) Mean autoaggregation of A. aphrophilus with the wild-type emaA gene, an interrupted emaA gene, and the interrupted emaA gene reverted to the wild type. Each graph shows data from biological replicates performed in triplicate. Error bars represent standard errors. An asterisk denotes a P value of <0.05 obtained from a paired t test.

Article Snippet: Quantitative adherence assays were performed as previously described using Chang epithelial cells (ATCC CCL 20.2) ( ).

Techniques:

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: Infection and Immunity

Article Title: Regulators Encoded in the Escherichia coli Type III Secretion System 2 Gene Cluster Influence Expression of Genes within the Locus for Enterocyte Effacement in Enterohemorrhagic E. coli O157:H7

doi: 10.1128/iai.72.12.7282-7293.2004

Figure Lengend Snippet: FIG. 6. Adhesion to human epithelial cells. (A to C) Int407 cells infected with the parent strain (A), etrA mutant LZ2 (B), or eivF mutant LZ3 (C) and subjected to Hemacolor staining. Magnification, 400. (D) Semiquantitative analysis of adherence. The number of microcolonies (a microcolony consisted of 10 adherent bacteria) per cell was calculated from 20 fields for each experiment, and the means were determined from three independent experiments. An average of 140 cells were examined in each field.

Article Snippet: Adhesion of E. coli strains to Int407 cells (human embryonic intestine; ATCC CCL 6) was quantified essentially as described previously (38).

Techniques: Infection, Mutagenesis, Staining, Bacteria

Journal: eLife

Article Title: SIRT-1 is required for release of enveloped enteroviruses

doi: 10.7554/elife.87993

Figure Lengend Snippet: Figure 1. Enterovirus D68 (EV-D68) infection changes SIRT-1’s subcellular localization. H1HeLa cells were transfected with either scramble control or SIRT-1 siRNAs for 48 hr. The cells were then infected with EV-D68 (multiplicity of infection [MOI = 0.1]) for 5 hr. The intracellular (A) and extracellular (B) particles were collected for plaque assay. (C) Cells were transfected with the indicated plasmids for 24 hr before being infected with EV-D68 (MOI = 0.1) for 5 hr. The extracellular particles were collected and analyzed by a plaque assay. (D) H1HeLa cells were infected for the indicated time points for

Article Snippet: Cell culture, plasmids, and viruses H1HeLa cells were purchased from ATCC (CRL- 1958) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine saline, 1× penicillin/streptomycin, and 1× sodium pyruvate.

Techniques: Infection, Transfection, Control, Plaque Assay

Journal: eLife

Article Title: SIRT-1 is required for release of enveloped enteroviruses

doi: 10.7554/elife.87993

Figure Lengend Snippet: Figure 2. SIRT-1 promotes autophagy but decreases enterovirus D68 (EV-D68) extracellular titers in autophagy-deficient ATG-7 KO cells. (A) H1HeLa cells were transfected with scramble or SIRT-1 siRNA for 48 hr. The cells were subsequently starved or treated with CCCP (10 µM) for 4 hr. Lysates were harvested and analyzed by western blot. (B) Cells were transfected with the indicated siRNAs for 48 hr before being fixed and subjected to immunofluorescence analysis (IFA) against endogenous LC3. (C) H1HeLa and ATG-7 KO cells were infected with 0.1 MOI of EV-D68 for 5 hr. The

Article Snippet: Cell culture, plasmids, and viruses H1HeLa cells were purchased from ATCC (CRL- 1958) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine saline, 1× penicillin/streptomycin, and 1× sodium pyruvate.

Techniques: Transfection, Western Blot, Immunofluorescence, Infection

Journal: eLife

Article Title: SIRT-1 is required for release of enveloped enteroviruses

doi: 10.7554/elife.87993

Figure Lengend Snippet: Figure 3. The proviral activity of SIRT-1 is mediated through endoplasmic reticulum stress (ER stress). (A) H1HeLa cells were infected with enterovirus D68 (EV-D68) (MOI = 0.1) for 30 min. The cells were washed and replenished with complete media with or without 2 µM thapsigargin (TG) for 5 hr. Viral titers were determined by plaque assay. (B) Cells were transfected with either scramble control siRNA or SIRT-1 siRNA for 48 hr. The cells were infected and treated as in A, and viral extracellular titers were similarly measured by plaque assay. Cells were transfected with the indicated siRNAs for 48 hr

Article Snippet: Cell culture, plasmids, and viruses H1HeLa cells were purchased from ATCC (CRL- 1958) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine saline, 1× penicillin/streptomycin, and 1× sodium pyruvate.

Techniques: Activity Assay, Infection, Plaque Assay, Transfection, Control

Journal: eLife

Article Title: SIRT-1 is required for release of enveloped enteroviruses

doi: 10.7554/elife.87993

Figure Lengend Snippet: Figure 5. SIRT-1 is critical for poliovirus (PV), but not coxsackievirus B3 (CVB3), release from cells. (A) H1HeLa cells were mock-infected or infected with PV or CVB3 for 4 hr. The cells were fixed, and immunofluorescence analysis (IFA) was done against SIRT-1 MOI = 30. (B, C) Cells were transfected with scramble or SIRT-1 siRNAs for 48 hr before being infected with PV (MOI = 0.1) for 5 hr. Viral titers were determined by a plaque assay. (D, E) Cells were

Article Snippet: Cell culture, plasmids, and viruses H1HeLa cells were purchased from ATCC (CRL- 1958) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine saline, 1× penicillin/streptomycin, and 1× sodium pyruvate.

Techniques: Infection, Immunofluorescence, Transfection, Plaque Assay

Journal: eLife

Article Title: SIRT-1 is required for release of enveloped enteroviruses

doi: 10.7554/elife.87993

Figure Lengend Snippet: Figure 7. SIRT-1 KD reduces extracellular vesicle-mediated release of infectious enterovirus D68 (EV-D68) viral particles. H1HeLa cells were transfected with SIRT-1 and Scramble siRNAs for 48 hr. (A) Cells were infected with EV-D68 (MOI = 0.1) for 5 hr, and EVs were isolated for viral titer measurement by plaque assay. (B) Cells were transfected as in A, and the whole-cell lysates (WCL) were collected and prepared for western blot against CD63. (C) H1HeLa cells were transfected with the indicated siRNAs for 48 hr. The cells were either left uninfected or infected with MOI 30 of EV-D68 for 4 hr. The

Article Snippet: Cell culture, plasmids, and viruses H1HeLa cells were purchased from ATCC (CRL- 1958) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine saline, 1× penicillin/streptomycin, and 1× sodium pyruvate.

Techniques: Transfection, Infection, Isolation, Plaque Assay, Western Blot