Journal: bioRxiv

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of HU-mCherry labelled (green) wild-type cells at 0, 8, and 16 minutes after CIP exposure, showing DNA distribution. Scale bar: 5 µm. ( B ) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 34-141 cells from a single representative biological replicate (see Materials and methods for detailed explanation). ( C and D ) Cells were immobilized on agar pads and imaged using spinning disk microscopy. Results shown are from images captured 12, 14, 16, and 18 minutes after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. ( C ) Average HU-mCherry fluorescence intensity along the cells’ long axis. ( D ) Kymograph heat map of relative HU-mCherry intensity distribution over time. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin exposure in Escherichia coli

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of ΔrecN cells at 0, 8, and 16 minutes after CIP exposure, showing HU-mCherry fluorescence (green) to represent DNA distribution. Scale bar: 5 µm. ( B ) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 17-61 cells from a single representative biological replicate (see Materials and methods for detailed explanation). ( C and D ) Cells immobilized on agar pads and imaged using spinning disk microscopy. Results are shown from images captured 12, 14, 16, and 18 minutes after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. ( C ) Average HU-mCherry fluorescence intensity along the cells’ long axis. ( D ) Kymograph heat map of relative HU-mCherry intensity distribution over time. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Tolerogenic lung allograft microenvironment suppresses pathogenic tissue remodeling following respiratory virus infection in mice

doi: 10.1016/j.ajt.2025.08.021

Figure Lengend Snippet: Acute Sendai virus infection affects both transplanted and native lungs in an allogeneic lung transplant model. (A) Schematic depicting the experimental model. Balb/c left lungs were transplanted into B6 mice and ≥30 days later, recipient mice were inoculated with either phosphate-buffered saline (mock) or Sendai virus strain 52 (SeV-52). Lungs were analyzed on 7 and 49 dpi. (B) Disease progression was monitored by measuring weight loss throughout the experiment. The area under the curve (AUC) was calculated, and significance of data was compared using 2-tailed unpaired t test. **** P < .0001. n = 5–6 mice per group. (C) Lungs were recovered on 7 dpi and stained for SeV nucleoprotein (NP) (white). Cellular nuclei were stained with Hoechst (blue). Widefield, ×10 magnification, scale bars: 500 μm. Images are representative of 2 independent experiments, 5 mice per group. (D) Quantification of SeV NP-stained airways as percent of total airways per lung section. ns = not significant. n = 5 mice per group. (E) Lungs were recovered on 7 dpi and stained with hematoxylin and eosin (H&E). Brightfield, ×10 magnification, digital zoomed insets are indicated at the right side of the corresponding section. Scale bars, 200 μm. (F) H&E sections of mock-infected and SeV-infected left (transplanted) and right (native) lungs were blindly scored for histopathological changes. Area affected, percentage of airway structures affected, and intensity of alveolitis and peribronchitis were determined for each individual section. Individual weighted scores ± standard error of the mean (SEM) values are indicated. (G) International Society for Heart and Lung Transplantation (ISHLT) A grade rejection scores for lung allografts. Mock, n = 3; SeV-52, n = 4. (H) Quantification of bronchus-associated lymphoid tissue (BALT)-like area (as percentage of total lung area) in lung allografts. Mock, n = 3; SeV-52, n = 4. Significance of data was compared using 2-tailed unpaired t test. * P < .05; ** P < .01; ns, not significant.

Article Snippet: Images were acquired with Zeiss Axio observer widefield fluorescence microscope (5× and 20×) and analyses performed with ZEN blue v.3.3 software.

Techniques: Virus, Infection, Saline, Biomarker Discovery, Staining, Transplantation Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Tolerogenic lung allograft microenvironment suppresses pathogenic tissue remodeling following respiratory virus infection in mice

doi: 10.1016/j.ajt.2025.08.021

Figure Lengend Snippet: Pathogenic remodeling following long-term Sendai virus (SeV) infection occurs in native lungs but not in tolerant allografts. (A) SeV-infected lungs were stained for the tissue remodeling and chronic lung lesion markers keratin (Krt)5 (green) and Krt8 (magenta). Nuclear staining is displayed in blue (Hoechst). Widefield, ×5 magnification tiled whole lung section images. Scale bars, 500 μm. (B) Quantification of Krt5 + and Krt8 + stained area related to total lung area. n = 7 per group. (C) Lung homogenates were analyzed for expression of Krt5 , Krt8 , and p63 by quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). n = 6 per group. (D) SeV-infected lungs were stained for Krt5 (turquoise), amphiregulin (Areg) (magenta), and Foxp3 (yellow) with immunofluorescence. Nuclear staining is displayed in blue. White arrowheads indicate Areg + Foxp3 + double-positive cells. Widefield, 20x magnification. Scale bars, 200 μm. (E) Quantification of Areg + , Foxp3 + , and Areg + Foxp3 + cells per lung section. Eight power fields per sample were randomly acquired using an automated slide scanner, 3 slides per condition. (F) Correlation between Areg + Foxp3 + cell counts per lung section analyzed, and Krt5-stained (left plot) and Krt8-stained (right plot) areas. Simple linear regression was performed to obtain the best fit curve and the 95% confidence interval (light gray area). Pearson r coefficient values and P values are displayed. Each dot represents a lung section from a single animal, 4 animals per condition. All remaining data significance were analyzed using 2-tailed unpaired t tests. * P < .05; ** P < .005; *** P < .001; **** P < .0001.

Article Snippet: Images were acquired with Zeiss Axio observer widefield fluorescence microscope (5× and 20×) and analyses performed with ZEN blue v.3.3 software.

Techniques: Virus, Infection, Staining, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Tolerogenic lung allograft microenvironment suppresses pathogenic tissue remodeling following respiratory virus infection in mice

doi: 10.1016/j.ajt.2025.08.021

Figure Lengend Snippet: Similar progression of postacute Sendai lung disease in syngeneic grafts and native lungs. (A) Schematic depicting experimental model. B6 left lungs were transplanted into B6 mice and ≥30 days later, recipient mice were inoculated with either phosphate-buffered saline (PBS) (mock) or SeV-52. Lungs were analyzed on day 49 post infection. (B) Disease progression was monitored by measuring weight loss throughout the experiment. The AUC was calculated, and significance of data was compared using 2-tailed unpaired t test. **** P < .0001. n = 5 mice per group. Lungs were recovered at 49 dpi and stained with hematoxylin and eosin (H&E) (C) to assess histopathological changes. (D) H&E sections of mock- and SeV-infected left (transplanted) and right (native) lungs were blindly scored for histopathological changes. Mean and individual weighted score values per section ± standard error of the mean (SEM) are indicated. (E) International Society for Heart and Lung Transplantation (ISHLT) A grade rejection scores in mock-infected and SeV-infected syngeneic grafts; 3 mice per condition. (F) Lung sections were analyzed by immunofluorescence staining for keratin (Krt)5 (green) and Krt8 (magenta). Brightfield (C) and widefield (F) microscopy, ×10 magnification. Scale bar, 500 μm. (G) Quantification of Krt5-stained and Krt8-stained areas. Three mice per condition. Significance of data was compared using Mann–Whitney test; ns, not significant.

Article Snippet: Images were acquired with Zeiss Axio observer widefield fluorescence microscope (5× and 20×) and analyses performed with ZEN blue v.3.3 software.

Techniques: Saline, Infection, Biomarker Discovery, Staining, Transplantation Assay, Immunofluorescence, Microscopy, MANN-WHITNEY

Journal: Cell reports

Article Title: Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration

doi: 10.1016/j.celrep.2022.110473

Figure Lengend Snippet: (A) Co-immunostaining for F4/80 (green) and Na+ K+ ATPase (red) on the paraffin sections of mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. Fold change in the intensity of F4/80 normalized to nuclei and plotted in comparison to control kidneys at day 14 post-AA treatment (n=4). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (B) FACS sorting strategy for macrophages from control and Stat3 KO mouse kidneys at 14-days post-AA treatment. TaqMan base quantitative RT-PCR for (C) inflammatory mediators/M1 markers and (D) M2 markers from macrophages isolated from mouse kidneys at 14-days post-AA treatment. Fold changes were plotted in comparison to control mice at 14-days post-AA treatment (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E) Co-immunostaining of F4/80 (green) and IL-10 (red) and IL-10 (red) alone on the paraffin sections of mouse kidneys 14-days post-AA treatment. Scale bar=10μm. Fold change in the intensity of IL-10 normalized to nuclei and plotted in comparison to control kidneys at day-14 post-AA treatment (n=4). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (F) Representative immunostaining for pH2AX (green) in the control and Stat3 KO mouse paraffin kidney sections at 14-days post-AA injection. Scale bar=10μm. Images were captured on a widefield immunofluorescence microscope using 60X objective. Quantitation of interstitial and tubular pH2AX positive cells (n=4). Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotted in comparison to control mice at 14-days post-AA treatment. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G) Co-immunostaining for PDGFRβ (green) and pH2AX (red) on the paraffin sections of control mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. PDGFRβ and pH2AX double-positive cells are shown by white arrows. Number of PDGFRβ and pH2AX double-positive cells were counted and plotted (n=4). (H) Immunostaining for Ki67 (cyan) in the mouse kidneys 14-days post-AA injection. Images were captured on a widefield immunofluorescence microscope using 60X objective. Quantitation of interstitial and tubular Ki67 positive cells (n=4). Scale bar=10μm. Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotted in comparison to control mice at 14-days post-AA treatment. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (I) Co-immunostaining for PDGFRβ (green) and Ki67 (red) on the paraffin sections of control mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. PDGFRβ and Ki67 double-positive cells are shown by white arrows. Number of PDGFRβ and Ki67 double-positive cells were counted and plotted (n=4).

Article Snippet: Three-to-five images were captured using 60X objective either on a Nikon Confocal Imaging system (Nikon C1 Eclipse, Nikon, Melville, NY) or Axio Imager A2m widefield microscope (Carl Zeiss, White Plains, NY).

Techniques: Immunostaining, Comparison, Control, Two Tailed Test, Quantitative RT-PCR, Isolation, Injection, Immunofluorescence, Microscopy, Quantitation Assay