Journal: Animal Cells and Systems

Article Title: Autophagy-driven MHC-I downregulation enables NK cell-mediated clearance of intracellular uropathogenic E. coli in urinary tract infection

doi: 10.1080/19768354.2025.2588910

Figure Lengend Snippet: Infection of bladder epithelial cells by UPEC induces a reduction in MHC-I. (A) Reduction in MHC-I expression in BECs following UPEC infection. Cells were stained for MHC-I (green), UPEC (red), and DAPI (gray). The left panel shows saline-treated cells, while the right panel shows infected cells. MHC-I expression was assessed by mean fluorescence intensity. (B) To quantify MHC-I reduction, BECs were infected with UPEC at different MOIs (100, 200, 500, or 1000) and harvested at 6 and 24 h post-infection for Western blot analysis. GAPDH was used as a loading control. Densitometry analysis of MHC-I was performed using three independently conducted Western blot images. (C) Quantification of transcription levels post-infection. BECs were infected with UPEC at an MOI of 500. After 4 h of post-infection, qPCR was performed for HLA-A , HLA-B , or HLA-C . (D) Vesicle association of MHC-I after UPEC infection. BECs were infected with magnetic bead-labeled UPEC and harvested at 6 or 24 h post-infection. Samples were probed for various proteins (FimH, LAMP1, SLC7A11, RAB27b, or MHC-I) at different time points. The cell lysate fraction was also analyzed as the loading control. Data are shown as the mean ± SEM. Quantitative data from two to three independent experiments were analyzed by unpaired two-tailed Student’s t-test or ordinary one-way ANOVA. * p < 0.05; *** p < 0.001; **** p < 0.0001; n.s, not significant.

Article Snippet: Thunderbird next SYBR qPCR mix and CFX384 Touch Real-Time PCR Detection System (Bristol Myers Squibb) were used to conduct qRT-PCR.

Techniques: Infection, Expressing, Staining, Saline, Fluorescence, Western Blot, Control, Labeling, Two Tailed Test

Journal: Animal Cells and Systems

Article Title: Autophagy-driven MHC-I downregulation enables NK cell-mediated clearance of intracellular uropathogenic E. coli in urinary tract infection

doi: 10.1080/19768354.2025.2588910

Figure Lengend Snippet: Autophagy-mediated degradation of MHC-I in UPEC-infected BECs. (A) Human BECs were treated with bafilomycin A1 (50 nM) or MG132 (0.1 µM or 1 µM) overnight. The following day, the cells were infected with UPEC at MOI 500 and subsequently probed for MHC-I expression via Western blot. The cells were harvested 24 h later and then probed for MHC-I via Western blot. GAPDH antibody was used as a control. Densitometry analysis of MHC-I was performed using three independently conducted Western blot images. (B) Confocal microscopy was performed to visualize the colocalization of MHC-I along with the intracellular vesicle components. The infected BECs with UPEC (MOI 200) were stained for LC3B (blue), UPEC (red), MHC-I (green), or the nucleus (gray). To visualize the colocalization of MHC-I with lysosomal vesicles, UPEC-infected cells were stained for LAMP1 (blue), UPEC (red), MHC-I (green), and nucleus (gray) after 20 h of infection. The regions outlined with white dashed lines in the panels of the first column are enlarged and shown in the remaining panels. Optical fields (100 μm × 100 μm) were randomly chosen from two to three independent experiments. Colocalization between the channels was measured in terms of Pearson’s coefficient. (C, D, E) First, the siRNA knockdown efficacy of NBR1 , BECLIN1 , or ATG5 was examined using qPCR. Then, BECs were again transfected with NBR1 , BECLIN1 , or ATG5 siRNAs, followed by UPEC infection (MOI, 500). MHC-I levels were analyzed 24 h post-infection via Western blot. Densitometry analysis of MHC-I was performed using three independently conducted Western blot images. Data are shown as the mean ± SEM. Quantitative data from two to three independent experiments were analyzed by unpaired two-tailed Student’s t-test or ordinary one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s, not significant.

Article Snippet: Thunderbird next SYBR qPCR mix and CFX384 Touch Real-Time PCR Detection System (Bristol Myers Squibb) were used to conduct qRT-PCR.

Techniques: Infection, Expressing, Western Blot, Control, Confocal Microscopy, Staining, Knockdown, Transfection, Two Tailed Test

Journal: Animal Cells and Systems

Article Title: Autophagy-driven MHC-I downregulation enables NK cell-mediated clearance of intracellular uropathogenic E. coli in urinary tract infection

doi: 10.1080/19768354.2025.2588910

Figure Lengend Snippet: CD48 upregulation in UPEC-infected bladder epithelial cells promotes NK cell activation and the elimination of target cells. (A) Activation ligand expression in infected BECs. The harvested cells were analyzed for elevated expression levels of known activation ligands (CD48, B7-H6, MICA, BAG6, CD112, CD155, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6) using qPCR. (B) The CD48 protein level was measured 24 h after UPEC infection using Western blot analysis. Densitometry analysis of CD48 was performed using three independently conducted Western blot images. (C) First, the CD48 siRNA (0.5 μg) knockdown efficacy was examined using qPCR. Then, before UPEC infection, BECs were transfected with si CD48 , followed by infection with UPEC (MOI 500). After 24 h, NK cells were added and co-incubated overnight. The next day, the bacterial burden was assessed. Data are shown as the mean ± SEM. Quantitative data from two to three independent experiments were analyzed by unpaired two-tailed Student’s t-test or ordinary one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s, not significant.

Article Snippet: Thunderbird next SYBR qPCR mix and CFX384 Touch Real-Time PCR Detection System (Bristol Myers Squibb) were used to conduct qRT-PCR.

Techniques: Infection, Activation Assay, Expressing, Western Blot, Knockdown, Transfection, Incubation, Two Tailed Test

Journal: Animal Cells and Systems

Article Title: Autophagy-driven MHC-I downregulation enables NK cell-mediated clearance of intracellular uropathogenic E. coli in urinary tract infection

doi: 10.1080/19768354.2025.2588910

Figure Lengend Snippet: CXCL10 and CXCL12 mediate NK cell recruitment to UPEC-infected bladder epithelial cells. (A) NK cell migration through a Transwell plate. Cells were fixed and stained for granzyme B (red), nucleus (gray), and tubulin (blue). The number of transmigrated NK cells was counted and compared. Optical fields (100 μm × 100 μm) were randomly chosen from two to three independent experiments. (B) Enhanced expression of chemokine transcription for NK cell recruitment. Transcription levels of CXCL10 , CXCL12 , CXCL5 , CXCL11 , CXCL1 , CCL5 , or CCL19 were measured via qPCR after BECs were infected with UPEC for 24 h. (C) CXCL10 and CXCL12 are required to recruit NK cells in the Transwell. CXCL9 -/- , CXCL10 -/- , or CXCL12 -/- BECs were generated by CRISPR–Cas9. These knockout cells or mock control BECs were infected with UPEC (MOI 200) and plated on the basal well of the Transwell insert. Transmigrated NK cells were compared by counting cells in the basolateral side of the Transwell system. Data are shown as the mean ± SEM. Quantitative data from two to three independent experiments were analyzed by unpaired two-tailed Student’s t-test or ordinary one-way ANOVA. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Thunderbird next SYBR qPCR mix and CFX384 Touch Real-Time PCR Detection System (Bristol Myers Squibb) were used to conduct qRT-PCR.

Techniques: Infection, Migration, Staining, Expressing, Generated, CRISPR, Knock-Out, Control, Two Tailed Test