Fig 2: In vitro analysis of the effects of CD74-knockdown cells. (A) Knockdown of CD74 attenuated cell proliferation in HT-1376 cells compared with the scramble group. *P<0.05 and **P<0.01 vs. Control shRNA. (B) Flow cytometry indicated that knockdown of CD74 significantly increased the proportion of G1 stage cells, decrease G2 stage and S stage ones, compared with scramble shRNA cells. (C) The cell invasion assay demonstrated that knockdown of CD74 significantly attenuated the invasion ability of HT-1376 cells, (D) and the cells of two groups were counted (mean ± SD) and the data of CD74 shRNA group were presented by a percentage of control shRNA group. (E and F) ELISA test indicated that the secretion of (E) VEGF and MMP-9 (F-a) was significantly reduced in CD74-knockdown-HT-1376 cells compared to the shRNA control, while not significantly reduced in MMP-2 (F-b). CD74, cluster of differentiation; VEGF, vascular endothelial growth factor; MMP, matrix metalloproteinase; sh, short hairpin.

Fig 3: Expression of MIF and CD74 in tissue samples and cells, and protein expression levels in HT-1376 cells following knockdown of CD74. (A) Little or no expression of immunoreactive CD74 was identified in the urothelial layers of normal bladder and NMIBC samples, but sections of MIBC samples exhibited strong immunoreactive signals of CD74. In contrast, MIF demonstrated strong immunoreactions in the normal and UCB samples. (B) Western blotting assay results for MIF and CD74 in the cultured urothelial SV-HUV-1, SW780, 5637, T24, J82 and HT-1376 cell lines. HT-1376 was the only one identified to express CD74, but all cells expressed MIF to a certain extent. (C) Immunofluorescence microscopy (×100, magnification) indicated positive CD74 staining in HT-1376 cells. (D) CD74 shRNA lentiviral particles abrogated the RNA expression of CD74 in the HT-1376 cells compared with untreated and scramble cells. (E) CD74 short hairpin RNA lentiviral particles abrogated the protein expression of CD74 in the HT-1376 cells when compared with the untreated and scramble cells. (F) Knockdown of CD74 modulated the expression levels of not only Cyclin D1, Cyclin E, but also intranuclear NF-κB p65, pAkt, pErk1/2. (G and H) However, no significant changes in Bcl-2, Bad, cleaved PARP and Erk1/2 were observed among all groups. *intranuclear NF-κB. NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; UCB, urothelial cell carcinoma of the bladder; MIF, macrophage migration inhibitory factor; CD74, cluster of differentiation; p65, transcription factor p65; Akt, RAC-alpha serine/threonine protein kinase; p, phosphorylated; Erk1/2, Extracellular regulated protein kinase 1/2; Bcl-2, B-cell lymphoma 2; Bcl-xL, Bcl-extra large; Bax, Bcl-2-associated X protein; PARP, poly(adenosine 5′-diphosphate-ribose) polymerase.