Fig 2: Downregulating USP44 inhibited the tumorigenesis in vivo(A) The photograph presented all nude mice model, the white arrow points to the tumor originated in U87-USP44-NC cells and the black arrow points to the tumor originated in U87-USP44-KD2 cells. (B) 30 days after subcutaneous injection, all nude mice were sacrificed and tumor tissues were sampled. (C) There was a statistically significant difference between the two group data of tumor volume (**p<0.01). (D) Excised tumor tissues were stained with HE and Ki-67. Representative photos were taken in 40 × magnification. Scale bar, 200μm.

Fig 3: Knockdown of USP44 induces apoptosis in glioma cells(A) U251-USP44-KD2 cell and NC cell stained with PE and 7-AAD were analyzed by flow cytometry, the difference percentage of early and late apoptosis was displayed in the right histogram (**p<0.01). (B) A172-USP44-KD2 cell and NC cells were analyzed as mentioned above; the data was shown in histogram on the right(**p<0.01). (C/D) Apoptosis-related proteins (Bcl-2, BAX) were detected by western blot with specific antibodies and ß-actin was served as the control of sample loading. The rate of BAX/BCL-2 was presented under the blotting.

Fig 4: Antibody identification and intracellular localization of USP44(A) A sketch map shows the fundamental structure of USP44 protein, the antibody-binding site and the potential nucleolus localization signal (red). (B) Flag-tagged USP44protein was expression in 293T cells, the IF signals of USP44 (green) and Flag (red) existed in same localization of nuclear. DAPI (blue) was used for nuclei staining. Scale bar, 50µm. (C) The co-localization of USP44 (green) and B23 (red) in nucleolus (blue) in U251MG cell was presented by immunofluorescence. DAPI was used for nuclei staining. Scale bar, 50µm/10µm. (D) IF assay analyzed expression of endogenous USP44 in U251-NC cell and U251-USP44-KD2 with an Alex Fluor 594 labeled secondary antibody (red), DAPI was used for nuclei staining, Scale bar, 50µm. (E/F) Expression of USP44 in glioma cell lines and the knockdown efficiency of two shRNA were tested by immunoblotting with antibody pAb21808.

Fig 5: Knockdown of USP44 inhibit malignant biological behavior of glioma cells in vitro(A) Changes in cell proliferation were assessed by WST-1 after silencing USP44 in both U251MG (left) and A172 (right) cells (*p<0.05). (B) Clone formation assay was performed with U251-USP44-KD2, A172-USP44-KD2 cells and the corresponding NC cells. The statistical significance was presented in column chart on the right (*p<0.05). (C/D) Migration and invasion were tested withU251-USP44-KD2, A172-USP44-KD2 and corresponding NC cells by transwell assay, results were presented in histogram on the rightKnockdown of USP44 decreased the cell numbers in lower surface significantly (*p<0.05). Scale bar, 50µm. (E) Expression of MMP2 and MMP9 were probed in U251-USP44-KD1/2, A172-USP44-KD1/2 and corresponding NC cells by western blot.