Fig 2: PTEN and USP18 expression profiles are associated in human lung cancersA. Blocking peptide antagonized PTEN immunostaining in the NIH/3T3 (PTEN positive) cell line. B. Representative staining for human lung cancers with low PTEN and USP18 or high PTEN and USP18 expression profiles. All magnifications were 20X. C. PTEN and USP18 immunostaining are positively correlated in human lung cancer (n = 461) and D. This association was confirmed using a contingency table by grouping negative, low, medium, or high PTEN and USP18 immunostaining profiles in lung cancer cases.

Fig 3: PTEN undergoes ISGylationISG15 conjugates to PTEN in lung cancer cell lines. Immunoprecipitation (IP) with an anti-HA antibody followed by immunoblot (IB) with an anti-PTEN antibody revealed ISG15-PTEN complex formation between HA-tagged ISG15 and A. exogenous and B. endogenous PTEN at ~75 kDa in HOP62 lung cancer cells. C. The ISG15-PTEN conjugation observed by IP was attenuated with introduction of USP18 in HOP62 lung cancer cells. D. Deletions of PTEN at indicated domains were subjected to E. IP by HA-tagged ISG15 using an anti-HA antibody followed by IB with an anti-GFP antibody revealed differential conjugation between PTEN deletions in ED1 lung cancer cells. Arrows displayed ISG15-PTEN conjugation bands.

Fig 4: Modifying USP18 and ISG15 protein levels altered PTEN protein stabilityA. Loss of USP18 expression decreased PTEN protein stability. ED1 murine lung cancer cells stably transfected with a vector control or with one of two individual shRNAs against USP18 were subjected to an 8 hour CHX treatment. PTEN expression was quantified relative to respective actin expression at indicated time points and normalized to the 0 hour (before CHX treatment) time point. B. Gain of USP18 expression increased PTEN levels. ED1 and HOP62 lung cancer cells were independently transfected with a USP18 expression plasmid or a control vector. PTEN expression was normalized to actin expression and compared between vector control and USP18 vector-transfected lung cancer cell lines. C. Engineered gain of ISG15 expression decreased PTEN protein levels. ED1 and HOP62 lung cancer cells were independently transfected with ISG15 or a vector control. PTEN was normalized to actin expression and compared between vector control and ISG15 vector-transfected lung cancer cell lines. D. Decreased PTEN levels conferred by overexpressed ISG15 were rescued by transiently overexpressing GFP-tagged USP18 in HOP62 lung cancer cells. Immunoblots were independently probed with GFP and PTEN recognizing antibodies. PTEN protein levels were normalized to actin expression and compared between vector control and ISG15 vector as well as ISG15 vector/USP18 vector-transfected lung cancer cell lines.

Fig 5: RPPA-based protein profiling revealed USP18 regulated PTEN levelsA. Independent RPPAs are displayed of ED1 and HOP62 lung cancer cell lines stably transfected with a control vector or one of two individual shRNAs against USP18. Signal intensities were normalized and transformed to linear values. RPPA highlighted species showing substantial differences in expression profiles between vector control and USP18 shRNA transfected lung cancer cells are represented by heat-maps for both ED1 and HOP62 lung cancer arrays. Red indicated high and green indicated low protein expression. Cell line clusters are shown as dendrograms. PTEN (red box) was highlighted as one of the proteins showing similar trends in both arrays. B. PTEN expression in the displayed ED1 and HOP62 transfected lung cancer cell lines was quantified using normalized linear values. Cell lines stably transfected with shRNAs against USP18 were compared relative to vector control transfectants. C. Representative immunoblots independently probed with USP18 and PTEN recognizing antibodies confirmed that engineered repression of USP18 decreased PTEN protein levels in ED1 and HOP62 lung cancer cells. PTEN expression was normalized to actin expression and compared between vector control and USP18 shRNA-transfected lung cancer cell lines. D. Decreased PTEN protein levels caused by loss of USP18 expression were rescued by transiently overexpressing GFP-tagged USP18 in ED1 lung cancer cells. Immunoblots were individually probed with GFP and PTEN recognizing antibodies. PTEN was normalized to actin expression and compared between vector control and USP18 shRNA- as well as USP18 shRNA/USP18 vector-transfected lung cancer cell lines.