Fig 2: TIM knockdown diminishes tumorigenicity of melanoma cells in vivo.Melanoma cells transfected with the non-silencing shRNA (mock sequence) as a negative control or transfected with TIM shRNA were inoculated subcutaneously into male and female mice. (A) Graph showing tumor size measured every 5 days. (B) Representative photographic images of nude mice showing the growth of the xenograft tumors. (C) Graph showing the mean tumor weight of the resected tumor. (D) Images of xenograft tumors dissected from the nude mice 4 weeks after subcutaneous inoculation. (E) Tumor tissues were subjected to immunostaining for determination of TIM, TIPIN, and PCNA expression. Quantitation of histochemical analysis of TIM, TIPIN, and PCNA in tissue samples from tumor xenografts mice.

Fig 3: Knockdown of TIM or TIPIN induces apoptosis of melanoma cells.(A) Cells transfected with TIM- (left panels) or TIPIN- (right panels) shRNA were double-stained with annexin V-FITC and PI and then subjected to flow cytometric analysis. Quantitation of percentage of apoptotic cells expressing knock down of (B) TIM or (C) TIPIN (PI negative and Annexin V positive staining). The values are expressed as means ± S. E. M. of 3 separate experiments. Melanoma cells were transfected with (D) TIM-shRNA or (E) TIPIN-shRNA and cell lysates were prepared after 48 h. The expression levels of TIM, TIPIN, total and cleaved caspase-3, and ?H2AX were detected by Western blotting with GAPDH as the internal control. (F) A375 cells were transfected with the indicated shRNAs and immunofluorescence was conducted. Cells were incubated with an ?H2AX antibody and the nuclei were stained with DAPI. The percentage of cells showing at least five ?H2AX foci (indicated by arrows) were counted and greater H2AX phosphorylation was observed in TIM- or TIPIN-knockdown cells. Quantitative analysis was conducted to measure the intensity of (G) shTIM and (H) shTIPIN-transfected cells relative to mock-control.

Fig 4: Knockdown of TIM/TIPIN inhibits melanoma cell proliferation.Western blotting was performed to analyze the knockdown effectiveness of (A) TIM and (B) TIPIN in melanoma cells. Proliferation of A375 melanoma cells was determined using IncuCyte S3 imaging (Essen BioScience, serial number = IC50471) after transfection with shRNA against (C) TIM or (D) TIPIN relative to mock control. Viability of A375 cells was determined after knockdown of (E) TIPIN or (F) TIM. (G) Representative images photographed at 10× magnification for soft agar colony formation assay are shown. (H) TIM and (I) TIPIN represent means ± standard deviations from triplicate determination for soft agar colony formation.

Fig 5: Reconstitution of the TIM/TIPIN complex visualized by cryo-EM.(A) An SDS-PAGE gel of the purified TIM/TIPIN complex with the protein indicated on the right. (B) Representative gel-filtration chromatography of the TIM-TIPIN complex. The fractions of proteins were collected manually and the absorption was monitored at 280 nm wavelength. (C) The peak fractions were visualized by SDS-PAGE and Coomassie blue staining. The apparent protein sizes correspond well to the expected sizes for TIM (170 kDa including the fused 3xFLAG-tag) and TIPIN (56 kDa). The TIM/TIPIN complex is observed in the peak fraction corresponding to an elution volume of 14.23 ml. Proteins are indicated to the right. Note that only gel filtration peak fractions are shown. (D) 2D class averages show that the TIM/TIPIN complex adopts elongated particles with a well-defined shape. (E) A 3D map of the TIM/TIPIN complex was created from 2D-classes of negative stain by using cisTEM. (F) A raw EM image of the TIM/TIPIN complex. The scale bar corresponds to 28.4 nm. (G) Representative 2D class averages from cryo-EM that are consistent with the particle shapes derived from negative staining. (H) A good 3D map (7.3 Å) of the TIM/TIPIN complex was created from the 2D-class averages from cryo-EM by using cisTEM.