Fig 2: AHNAK and NFATC1 increases cell migration, invasion and proliferation in BLCA. (A, B) Transwell migration and invasion assays of T24 and UMUC3 cells transfected with siRNAs against AHNAK and NFATC1 (100× magnification). (C, D) MTT assay of T24 and UMUC3 cells transfected with siRNAs against AHNAK and NFATC1. **p<0.01, ***p<0.001, ****p<0.0001.

Fig 3: QKI deficiency amplified the activation of the NF-?B and MAPK signaling pathways during osteoclastogenesis.a–e Western blot analysis of RANKL-induced p65, I?Ba, p38, ERK, JNK in BMMs from KO and control mice, and quantitative analysis of the western blot data, respectively. n = 4, *P < 0.05, **P < 0.01. f Western blot analysis of NFATc1 expression after blocking I?Ba, p38, ERK, and JNK by inhibitors. g Quantitative analysis of the western blot data evaluating NFATc1 expression after blocking signaling pathways. n = 4, **P < 0.01, ***P < 0.001.

Fig 4: DV activates osteoclasts and upregulates osteolytic activity via CLEC5A. a Human osteoclasts (1 × 105/well) were preincubated with anti-CLEC5A mAb or isotype control, followed by DV infection. Cells were fixed at 24 h postinfection for immunofluorescence staining and were examined under a confocal microscope (Olympus FV1000). Scale bars, 10 µm. Green color: NFATc1; red color: Hoechst 33342; yellow color: merge of NFATc1 (green) and Hoechst 33342 (red). b Human osteoclasts (5 × 106) were preincubated with anti-CLEC5A mAb or isotype control, followed by DV infection. Cells were harvested at 12 h postinfection for WB analysis. c Determination of osteolytic activity by “pit formation” assay. Mature human osteoclasts were seeded on the bone slices and incubated with DV for 2 h in the presence of isotype control or CLEC5A mAb. The resorbed areas (pits) were examined under a microscope (Nikon) at day 6 after DV infection. Five images per each bone slice were randomly photographed, and the resorbed areas were counted and represented as mean ± SEM (under each picture) from three independent experiments. d Supernatants from bone slices incubated with human osteoclasts and DV were harvested at day 6 after DV infection to determine C-telopeptide of type I collagen (CTX-1) level by ELISA (CrossLaps for Culture). Data were collected and expressed as mean ± SEM ANOVA tests were performed and all the infections were performed at M.O.I. = 5

Fig 5: Lack of NFATc1 activity arrests pro-B cell differentiation to pre-B cells. a Cellularity in the BM of WT and Vav-CreNfatc1fl/fl mice. b B220+ B cell distribution both as a percentage of the population and as the absolute numbers in the BM of WT and Vav-CreNfatc1fl/fl mice. c Flow cytometry profiles showing the distribution of BM cells based on B220 and CD43 expression in WT and Vav-CreNfatc1fl/fl mice. d Quantification of the percentage of distribution and absolute numbers of B220+CD43+ pro-B cells in the BM of WT and Vav-CreNfatc1fl/fl mice. e Quantification of the percentage of distribution and absolute numbers of B220+CD43- pre-B cells in the BM of WT and Vav-CreNfatc1fl/fl mice. f, g B220+CD43+CD24-BP1- pre-pro-B, B220+CD43+CD24+BP1- pro-B, and B220+CD43+CD24+BP1+ large pre-B cell distributions in the BM (f) and the quantification of their percentage of distribution (g) from Vav-CreNfatc1fl/fl mice compared with WT controls. h, i B220+CD43-IgM-IgD- small pre-B, B220+CD43-IgM+IgD- immature B, and B220+CD43-IgM+IgD+ mature B cell distributions in BM (h) and the quantification of their percentage of distribution (i) from WT and Vav-CreNfatc1fl/fl mice. j Distribution of B cells gated on the B220+CD43- population in BM from Il7-/- and Il7r-/- mice based on IgM and IgD expression compared with littermate WT mice. Numbers inside each dot plot represent the percentage of the respective population. Data represent one of three independent experiments (n = 3 per group) and are shown as the mean ± s.d., unpaired t test