Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: P. gingivalis strains with fimbriae depleted of all accessory subunits (DAP) poorly activate MDM pro-inflammatory responses. MDMs were treated for A) 4 h or B) 24 h with purified fimbriae (1 μg/ml) from various P. gingivalis strains, including ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD), as well as mutants of accessory fimbrial subunits, ∆ fimE mutant (fimbriae ∆FimE) and ∆ fimC mutant (fimbriae ∆FimC). A) Relative mRNA expression of PGE2 synthesis-related gene COX2 and cytokines IL6 and IL8 ; n = 4–7. B) Secretion of IL-6 and IL-8; n = 5–9. Data are presented as the mean ± SEM compared to WT fimbriae, with significance levels indicated as **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Purification, Mutagenesis, Expressing

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: LPS contamination in fimbriae preparations is negligible and does not influence the pro-inflammatory activation of MDMs. A) MDMs were treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD) strains, as well as LPS-free fimbriae from both strains; n = 3. B) hMDMs were preincubated for 30 min with control (IgG1) or TLR4-blocking antibody (anti-TLR4), then treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and the ∆ ppad mutant (fimbriae ∆PPAD) strains; n = 3. Levels of IL-6 and IL-8 secretion were subsequently measured. Data are presented as mean ± SEM. ‘ns’ indicates not statistically significant. Statistically significant differences are shown in A) between LPS-free fimbriae and fimbriae without the LPS removal step; in B) between IgG1 and anti-TLR4 pretreated samples.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Activation Assay, Purification, Mutagenesis, Control, Blocking Assay

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: PPAD and fimbriae mutants show differences in adhesion and safe entry to host cells. Flow cytometry analysis of MDMs infected for 30 min at MOI = 20 or 100 with P. gingivalis ATCC 33277 WT and PPAD mutant strains—including its isogenic total (∆PPAD) and catalytically inactive (C351A PPAD) strains, as well as fimbriae (∆FimA) mutants—labelled with A) CFSE or B) pHRODO-Red (pHRODO). Phagocytosis and adhesion rates were determined as the percentages of CFSE and pHRODO-Red positive cells, relative to the WT strain; n = 4. Results are shown as mean ± SEM. ** p < 0.01; * p < 0.05; ns, not significant. Comparisons are made to the ATCC WT strain. C) Widefield fluorescence microscopy of MDMs infected for 30 min at MOI 100 with CFSE-labelled P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE ∆FimC, and W83 strains. Nuclei were stained with DAPI, and actin was stained with phalloidin conjugated to Alexa Fluor 647. Scale bars: 30 µm. Images are representative of three independent experiments. White arrows indicate bacteria that are not internalised.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Flow Cytometry, Infection, Mutagenesis, Fluorescence, Microscopy, Staining, Bacteria

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: Phosphorylation of Akt is enhanced by PPAD and accessory fimbriae subunits, with the PI3K/Akt pathway being essential for IL-6 but not for IL-8 secretion. MDMs were infected for A) 10 min, 30 min, or 2 h with P. gingivalis ATCC 33277 WT strain (ATCC WT) and its isogenic PPAD mutant (∆PPAD), or B) for 30 min with ATCC 33277-derived PPAD: total (∆PPAD) and catalytically inactive (C351A PPAD), fimbriae: main FimA subunit (∆ fimA ), accessory subunits FimE (∆ fimE ), and FimC (∆ fimC ) mutants, or the W83 WT strain at MOI 20. Akt phosphorylation levels were determined by western blot analysis, with total Akt as a control and β -actin as the loading control. Representative blots are shown, and densitometry results ( n = 3) are presented as mean ± SEM. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant. In B), data are compared with the ATCC WT strain. C) MDMs were pretreated for 30 min with an Akt inhibitor or left untreated, then infected for 24 h at MOI 20 with P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE, ∆FimC, and W83 strains. Secretion of IL-6 and IL-8 was measured by ELISA; n = 4−7. Results are shown as mean ± SEM; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 compared to ATCC WT without Akt inhibitor; ns, not significant.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Phospho-proteomics, Infection, Mutagenesis, Derivative Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of the oncolytic effects of mutant jin-3 versus R124 reovirus in bladder cancer cell lines in vitro (A) Mean viral S4Q copy number (log fold change S4Q mRNA expression vs. mock treated cells) in bladder cancer cell lines exposed to a range of MOI (0.01–0.1–1–10) of either R124 or jin-3 reoviruses after 24 h. In red, the highest infection with virus is shown. Two-way ANOVA followed by Tukey’s post hoc test. n = 3 (2 replicates). UM-UC-3 cells R124 reovirus MOI1 vs. mock p = 0.0184; ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (B) Viral load (% of viable, single cells expressing Sigma-3 protein) in bladder cancer cell lines exposed to MOI10 of either R124 or jin-3 after 72 h mean (SD) ( N = 3). Two-way ANOVA followed by Tukey’s post hoc test. ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (C) Confocal images of viral protein (Sigma-3, green) and DAPI (blue)-stained bladder cancer cells treated with MOI10 of R124 or jin-3 for 72h. Scale bars, 25 μm. (D) Mean percentage of viable cells after exposure to a range of MOI of either R124 or jin-3 for 6 days. n = 3 (6 replicates). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ); ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (E) Percentage of single, viable, JAM-A protein expressing cells. Mean (SD), n = 3. MOI = multiplicity of infection.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Mutagenesis, In Vitro, Expressing, Infection, Virus, Staining

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of the oncolytic effects of reovirus mutant jin-3 versus wild-type reovirus R124 in 3D cultures in vitro TM00024 bladder PDXOs were exposed to the indicated plaque-forming units (PFUs) of reoviruses for 3 or 7 days. (A) Mean (SD) viral copy number of TM00024 PDXOs treated for 3 days with the indicated PFUs of the reoviruses. Two-way ANOVA followed by Tukey’s post hoc comparison ( n = 3) ∗∗∗ p < .001. (B–F) Viability (mean [SD] was measured using cell titer glo 3D after days 3 and 7 of reovirus exposure. N = 3 (3 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison. ∗∗∗ p < .001. (C) Confocal images of PDXOs (day 7) stained for respectively H&E, panKRT (red); Sigma-3, c-CASP3, or PCNA (green); and DAPI (blue). Scale bars, 20 μm. Cells expressing the respective proteins Sigma-3 (D), c-CASP-3 (E), and nuclear PCNA (F) were counted with ImageJ and divided by the amount of panKRT+_DAPI+ cells. At least five fields were scored per technical replicate. Mean (SD) of N = 3 (3 replicates). ∗∗∗ p < .001, asterisks indicate reovirus infection versus mock same day. Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Mutagenesis, In Vitro, Staining, Expressing, Infection

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue Explanted tissue slices from either RT-112 CDX (A–E) or TM00024 PDX (F–J) were exposed to the indicated PFU of R124 or jin-3 reovirus for 3 days. Tissues were stained for H&E, panKRT (red); Sigma-3, c-CASP3 or PCNA (green), type I collagen (white), and DAPI (blue). Representative confocal images are shown. Scale bars, 20 μm. Cells expressing the respective proteins were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) of N = 2 (4 replicates). The ratio fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E and J). ∗∗∗ p < .001, reovirus infection versus mock. Mean (SD), n = 2 (4 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Infection, Ex Vivo, Cell Culture, Staining, Expressing

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue slices from human bladder cancer patients Explanted tissue slices from patients ( n = 15) diagnosed with bladder cancer were exposed to either mock, R124, or jin-3 reovirus for 3 days and stained for viral protein, c-CASP-3, PCNA (proliferation), and integrity of the cell (nuclear DAPI and panKRT tumor marker). (A–E) Representative confocal images of the mock and 10 7 PFU condition of both viruses from five patients ranging from low-risk NMIBC to MIBC (viral protein [green, Sigma-3], tumor markers [red, panKRT], and nuclei [DAPI, blue]. Scale bars, 20 μm). Cells expressing the respective proteins [(B) Sigma-3, (C) c-CASP3, or (D) nuclear PCNA] were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) (3–4 replicates) with each dot representing one bladder cancer patient. The ratio of fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E). ∗∗∗ p < 0.001, reovirus infection versus mock, one-way ANOVA followed by Tukey’ post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Infection, Ex Vivo, Cell Culture, Staining, Marker, Expressing

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of immune modulation induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines Heat maps of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in bladder cancer cell lines UM-UC-3 (A), T24 (B), HT-1197 (C), RT-112 (D), RT-4 (E), TCCSUP (F), J82 (G), and PDXOs (H) exposed to a range of either R124 or jin-3 reoviruses after respectively 24 h (cell lines) and 3 days (PDXOs). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Expressing, Infection

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of immunogenic cell death induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines (A–C) Cell lines were treated with oncolytic viruses for 48 h at an MOI of 10, and the DAMPs ecto-calreticulin (A) and ecto-HSP90 (B) were measured using flow cytometry. Secreted HMGB1 protein was measured with an ELISA after 48 h of treatment with a dose range of oncolytic viruses (C). ∗∗∗ p < 0.001, $$$ p < 0.001, asterisks indicate reovirus infection versus mock same day, and dollar signs indicate R124 vs. jin-3. Mean (SD), N = 3 (2 replicates). Two-way ANOVA followed by Tukey’s posthoc comparison. (D) Correlation graph of the HMGB1 release ( x axis), percentage of viable cells (size of the dots), and fold change of ecto-CRT ( y axis)-positive cells at MOI 10 of the indicated virus.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Infection, Virus

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Activation of PBMCs in RT-112 co-cultures (A) Brightfield images of RT-112 bladder cancer cells that were cultured in 60% Matrigel and allowed to form 3D structures for approximately 3 days. Subsequently, PBMCs were added at an effector: target ratio of 20:1 in absence or presence of either R124 or jin-3 reovirus at the indicated PFU for an additional 3 days. (B) Fragmented KRT18 was determined as an outcome measure for tumor cell killing 3 days after OV exposure. (C–E) CXCL10 levels and (D) IFN-γ levels were measured 3 days after OV exposure. ( n = 3, 2 replicates). Mean (SD). Two-way ANOVA with Tukey’s post hoc comparison. (E) Heatmap of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in 3D cultured RT-112 cells or RT-112 cells co-cultured with PBMCs exposed to either R124 or jin-3 reoviruses. Mean (SD). p values are depicted (vs. mock; when two depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection and dollar signs R124 versus jin-3. , n = 2 (2 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Activation Assay, Cell Culture, Comparison, Expressing, Infection

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: P. gingivalis strains with fimbriae depleted of all accessory subunits (DAP) poorly activate MDM pro-inflammatory responses. MDMs were treated for A) 4 h or B) 24 h with purified fimbriae (1 μg/ml) from various P. gingivalis strains, including ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD), as well as mutants of accessory fimbrial subunits, ∆ fimE mutant (fimbriae ∆FimE) and ∆ fimC mutant (fimbriae ∆FimC). A) Relative mRNA expression of PGE2 synthesis-related gene COX2 and cytokines IL6 and IL8 ; n = 4–7. B) Secretion of IL-6 and IL-8; n = 5–9. Data are presented as the mean ± SEM compared to WT fimbriae, with significance levels indicated as **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: To test this hypothesis, we treated hMDMs, our study's model, with purified fimbriae from ATCC 33277 wild-type (WT) and its isogenic PPAD-deficient strain (∆PPAD), along with accessory fimbriae subunit mutants (∆ fimC and ∆ fimE ).

Techniques: Purification, Mutagenesis, Expressing

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: LPS contamination in fimbriae preparations is negligible and does not influence the pro-inflammatory activation of MDMs. A) MDMs were treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD) strains, as well as LPS-free fimbriae from both strains; n = 3. B) hMDMs were preincubated for 30 min with control (IgG1) or TLR4-blocking antibody (anti-TLR4), then treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and the ∆ ppad mutant (fimbriae ∆PPAD) strains; n = 3. Levels of IL-6 and IL-8 secretion were subsequently measured. Data are presented as mean ± SEM. ‘ns’ indicates not statistically significant. Statistically significant differences are shown in A) between LPS-free fimbriae and fimbriae without the LPS removal step; in B) between IgG1 and anti-TLR4 pretreated samples.

Article Snippet: To test this hypothesis, we treated hMDMs, our study's model, with purified fimbriae from ATCC 33277 wild-type (WT) and its isogenic PPAD-deficient strain (∆PPAD), along with accessory fimbriae subunit mutants (∆ fimC and ∆ fimE ).

Techniques: Activation Assay, Purification, Mutagenesis, Control, Blocking Assay

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: PPAD and fimbriae mutants show differences in adhesion and safe entry to host cells. Flow cytometry analysis of MDMs infected for 30 min at MOI = 20 or 100 with P. gingivalis ATCC 33277 WT and PPAD mutant strains—including its isogenic total (∆PPAD) and catalytically inactive (C351A PPAD) strains, as well as fimbriae (∆FimA) mutants—labelled with A) CFSE or B) pHRODO-Red (pHRODO). Phagocytosis and adhesion rates were determined as the percentages of CFSE and pHRODO-Red positive cells, relative to the WT strain; n = 4. Results are shown as mean ± SEM. ** p < 0.01; * p < 0.05; ns, not significant. Comparisons are made to the ATCC WT strain. C) Widefield fluorescence microscopy of MDMs infected for 30 min at MOI 100 with CFSE-labelled P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE ∆FimC, and W83 strains. Nuclei were stained with DAPI, and actin was stained with phalloidin conjugated to Alexa Fluor 647. Scale bars: 30 µm. Images are representative of three independent experiments. White arrows indicate bacteria that are not internalised.

Article Snippet: To test this hypothesis, we treated hMDMs, our study's model, with purified fimbriae from ATCC 33277 wild-type (WT) and its isogenic PPAD-deficient strain (∆PPAD), along with accessory fimbriae subunit mutants (∆ fimC and ∆ fimE ).

Techniques: Flow Cytometry, Infection, Mutagenesis, Fluorescence, Microscopy, Staining, Bacteria

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: Phosphorylation of Akt is enhanced by PPAD and accessory fimbriae subunits, with the PI3K/Akt pathway being essential for IL-6 but not for IL-8 secretion. MDMs were infected for A) 10 min, 30 min, or 2 h with P. gingivalis ATCC 33277 WT strain (ATCC WT) and its isogenic PPAD mutant (∆PPAD), or B) for 30 min with ATCC 33277-derived PPAD: total (∆PPAD) and catalytically inactive (C351A PPAD), fimbriae: main FimA subunit (∆ fimA ), accessory subunits FimE (∆ fimE ), and FimC (∆ fimC ) mutants, or the W83 WT strain at MOI 20. Akt phosphorylation levels were determined by western blot analysis, with total Akt as a control and β -actin as the loading control. Representative blots are shown, and densitometry results ( n = 3) are presented as mean ± SEM. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant. In B), data are compared with the ATCC WT strain. C) MDMs were pretreated for 30 min with an Akt inhibitor or left untreated, then infected for 24 h at MOI 20 with P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE, ∆FimC, and W83 strains. Secretion of IL-6 and IL-8 was measured by ELISA; n = 4−7. Results are shown as mean ± SEM; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 compared to ATCC WT without Akt inhibitor; ns, not significant.

Article Snippet: To test this hypothesis, we treated hMDMs, our study's model, with purified fimbriae from ATCC 33277 wild-type (WT) and its isogenic PPAD-deficient strain (∆PPAD), along with accessory fimbriae subunit mutants (∆ fimC and ∆ fimE ).

Techniques: Phospho-proteomics, Infection, Mutagenesis, Derivative Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Transformed HeLa-green fluorescent protein (GFP) cells exhibit anchorage-independent proliferation and colony formation in a 3D culture environment with 0.03 % LA717, unlike surrounding MSCs. Representative whole-well and magnified views at 0, 24, 72, 120, 240, and 360 h. MSCs (20,000 cells/well) were co-cultured with a single HeLa-GFP cell in 200 μL of medium supplemented with 0.03 % LA717 in a low-adhesion 96-well plate. All cells were pre-labeled with CellVue Claret (CVC; pseudo-colored magenta). Bright-field (BF), GFP, CVC, and merged images are shown. Yellow arrows at 0, 24, 72, and 120 h indicate the HeLa-GFP cell/colony in both the GFP and CVC panels; later time points (240 and 360 h) are not arrowed for clarity. Under these conditions, MSCs remained dispersed and did not exhibit anchorage-independent proliferation, whereas the single HeLa-GFP cell proliferated and formed a distinct colony by 240–360 h. A stitched movie of the complete 360-h time-lapse (1-h intervals) is provided as the Supplementary Movie. Images were acquired using a CellVoyager CQ1 (Yokogawa Electric). The inner diameter of each well was 6.4 mm.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transformation Assay, Cell Culture, Labeling

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: The colony-forming efficiency (CFE) of HeLa-GFP cells co-cultured with MSCs in LA717-supplemented medium under various culture conditions. (a) The CFE of HeLa-GFP cells was evaluated under four different concentrations of LA717 (0.03 %, 0.04 %, 0.05 %, and 0.06 %) in 200 μL of culture medium per well. HeLa-GFP cells (0.5 cells/well on average) were co-cultured with 20,000 MSCs per well in a 96-well plate ( n = 240 wells per condition) for 3 weeks without any medium changes or additions. CFE was calculated based on the number of wells containing colonies derived from HeLa-GFP cells, as determined by image analysis. Bar graphs represent the mean and standard deviation (SD) values from three independent experiments, and the colored lines show the individual results from each experiment. (b) The CFE of HeLa-GFP cells was evaluated under varying cell input amounts (λ = 0.125, 0.25, 0.5, and 1.0) in 200 μL of medium containing 0.03 % LA717 and 20,000 MSCs per well. HeLa-GFP cells were seeded into 840, 420, 240, and 120 wells, respectively, and cultured for 3 weeks. The CFE was calculated from the number of wells containing colonies detected by image analysis. Bars represent the mean and SD values of three independent experiments, and colored lines indicate the individual results. No colony formation was observed under control conditions (λ = 0; i.e., MSCs only, 60 wells). Detailed numerical data for each condition in (a) and (b) are provided in , respectively. The equation used to calculate CFE in (a) and (b) is described in , respectively. LA, LA717.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture, Derivative Assay, Standard Deviation, Control

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Fluorescence-based detection of colonies formed by single HeLa cells co-cultured with MSCs in LA717-supplemented medium. Representative images of colonies formed by a single HeLa-GFP or wild-type HeLa cell co-cultured with 20,000 MSCs per well for 3 weeks in 0.03 % LA717-supplemented medium (200 μL/well). Fluorescent staining markedly enhanced the accuracy of colony detection in image analysis. Images were acquired using the CellVoyager CQ1 confocal imaging cytometer (Yokogawa Electric). (a) A colony derived from a single HeLa-GFP cell. Live staining was performed by adding Hoechst 33342 (nucleus), MitoTracker Deep Red (mitochondria), or LysoTracker Red (lysosomes) to the culture medium and incubating the cells for a defined period. The GFP signal confirmed that the colony originated from HeLa-GFP cells, which was also clearly visualized with each dye. (b) A colony derived from a single wild-type HeLa cell. Live staining with Hoechst 33342 and Calcein-AM revealed strong fluorescence signals, indicating colony viability. (c) (Left) A wild-type live HeLa colony stained with Hoechst 33342 and LysoTracker Red. (Right) The same colony was imaged 14 days after fixation with 1 % paraformaldehyde (PFA). Fluorescence signals from Hoechst and LysoTracker staining were retained, indicating that this dye combination is compatible with fixation and long-term storage, allowing flexible scheduling of image acquisition and analysis. The inner diameter of each well was 6.4 mm.

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Fluorescence, Cell Culture, Staining, Imaging, Cytometry, Derivative Assay

Journal: Regenerative Therapy

Article Title: Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

doi: 10.1016/j.reth.2025.101052

Figure Lengend Snippet: Detection sensitivity assessment of the D-LACF assay using MSCs spiked with HeLa-GFP cells. (a) Schematic overview of the sample preparation and culture process. Ten HeLa-GFP cells were isolated using a cell sorter and spiked into a suspension of 10 million MSCs to prepare a test sample containing 0.0001 % HeLa-GFP cells. The mixture was then aliquoted into 300 wells (60 wells × 5 plates) at 20,000 cells per well for D-LACF assay evaluation. (b) Summary of the number of GFP-positive colonies detected per run. Left: spiked condition (MSCs + HeLa-GFP), five independent runs. Right: MSC-only negative controls, three independent runs (all zero). (c) Representative plate maps from Test-1 showing all five 96-well plates; yellow boxes mark wells in which GFP-positive colonies were detected (confirmed by fluorescence and bright-field review). This figure was created using BioRender.com .

Article Snippet: A human cervical cancer cell line, wild-type HeLa (CCL-2, Lot 61647128), was obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Sample Prep, Isolation, Suspension, Fluorescence

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A. Percentage of GFP-positive MEG-01 VP30 cells. PMA-treated or untreated cells were exposed to EBOVΔVP30-GFP at an MOI of 5 and GFP-positive cells were quantified by flow cytometry 2 days after exposure. Data are representative of two independent experiments. B. EBOVΔVP30 titers from MEG-01 VP30 cells. PMA-treated or untreated cells were exposed to EBOVΔVP30-GFP at an MOI of 1. Supernatants were collected daily, and viral titers were determined using Vero VP30 cells. Data are presented as means ± SD of three independent experiments. The dotted lines indicate the lower limit of detection (20 ffu/ml). Statistical significance was assessed by using the multiple unpaired t-test. * p < 0.05, ** p < 0.01.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Flow Cytometry

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A. Detection of EBOV proteins by western blot from PLPs released form EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were collected on day 4 post-exposure from PMA-treated MEG-01 WT or VP30 cells exposed to EBOVΔVP30 at an MOI of 5. The indicated proteins were analyzed by immunoblotting. Data are representative of two independent experiments. B. Relative amount of EBOV gRNA in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). EBOV gRNA was quantified by RT-qPCR using the indicated genome-specific primer pairs and normalized to the gRNA in PLPs from MEG-01 WT cells. Data are presented as means ± SD from two independent experiments performed in triplicate. C. Localization of EBOV GP in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). The platelet marker CD41 (magenta) and EBOV GP (green) were visualized by immunofluorescence microscopy using specific antibodies, along with the corresponding bright-field image. Scale bars, 5 μm. D. Interaction between EBOV NP/VP35 (top panel) and NP/VP40 (bottom panel) in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). NP/VP35 and NP/VP40 complexes (green) were visualized by using a proximity ligation assay (PLA) with specific antibodies and overlaid on the corresponding bright-field image. Scale bars, 5 μm.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Western Blot, Quantitative RT-PCR, Marker, Immunofluorescence, Microscopy, Proximity Ligation Assay

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: Relative expression levels of EBOV mRNA (A) and gRNA (B) in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were collected on day 4 post-exposure from PMA-treated MEG-01 VP30 cells exposed to EBOVΔVP30-GFP at an MOI of 5. EBOV mRNA and gRNA were quantified by RT-qPCR using the indicated specific primer pairs and normalized to the amount in the day 0 samples. Data are presented as means ± SD of three independent experiments performed in triplicate.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A,B. Expression levels of GP and CD41-mCherry in MEG-01 cells stably expressing CD41-mCherry with or without EBOV GP (A), and in PLPs released from these stable cell lines (B). Protein levels were analyzed by immunoblotting using the indicated antibodies. C. Percent of mCherry-positive cells that internalized PLPs containing CD41-mCherry or CD41-mCherry/GP. Huh7 VP30 cells were co-incubated with the indicated PLPs for 1, 3, or 5 h, followed by quantification of mCherry-positive cells by flow cytometry. Data are presented as means ± SD of three independent experiments. Statistical significance was assessed by use of a two-way ANOVA followed by Turkey’s multiple comparisons test. * p < 0.05, **** p < 0.0001.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Expressing, Stable Transfection, Western Blot, Incubation, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: Number of GFP-positive cells following co-incubation with PLPs released from EBOVΔVP30-exposed MEG-01 cells (A). PLPs were collected from PMA-treated MEG-01 WT and VP30 cells exposed to EBOVΔVP30-GFP. Huh7 VP30 cells (2 x 10 5 cells) were co-incubated with the indicated PLPs (2 x 10 6 ) for two days. As a control, cells were cultured in the final wash supernatant (B). GFP-positive cells were quantified by flow cytometry. Data are representative of two independent experiments. C-E. EBOVΔVP30 titers in three different VP30-expressing cell types co-cultured with PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). Huh7 VP30 cells (2 x 10 5 cells, C), HUVEC VP30 cells (2 x 10 5 cells, D), and PMA-differentiated THP-1 VP30 cells (2 x 10 5 cells, E) were co-cultured with the indicated PLPs (2 x 10 6 ). Virus titers on days 2, 4, and 6 were determined using Vero VP30 cells. Data are presented as means ± SD of three independent experiments. The dotted lines indicate the lower limit of detection (1.3 log10 ffu/ml). Statistical significance was assessed by use of a one-way ANOVA followed by Turkey’s multiple comparisons test. *** p < 0.001, **** p < 0.0001.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Incubation, Control, Cell Culture, Flow Cytometry, Expressing, Virus