Fig 2: LPIN1 depletion increases gefitinib sensitivity by enhancing apoptosis in TKI-resistant NSCLC cells. (A) H1650 cells stably transduced with a lentiviral vector carrying shLPIN1 (H1650/shLPIN1) or shControl (H1650/pLKO) were treated with gefitinib or DMSO for 120 h. The gefitinib sensitivity of each cell line was determined using varying gefitinib concentrations. (B) H1650 cells were transfected with siRNAs for LPIN1 and then treated with gefitinib or DMSO for 120 h. The gefitinib sensitivity of each cell line was determined using varying gefitinib concentrations. (C) H1650 cells were infected with shLPIN1- or pLKO-harboring lentivirus and treated with gefitinib (10 μM) or DMSO for 10 days. Cell growth was measured by a colony formation assay after 0.5% crystal violet staining. (D) H1650 and HCC827 cells were infected with shLPIN1- or pLKO-harboring lentivirus and treated with gefitinib (10 μM in H1650 or 1 nM in HCC827 cells) or DMSO for 14 days. Cell growth was measured by an anchorage-independent growth assay in soft agar. (E) H1650 cells were transfected with siRNAs for LPIN1; treated with gefitinib (5 μM) or DMSO for 72 h; harvested; stained with Annexin-V, Alexa Fluor® 647 conjugate, and PI; and analyzed via flow cytometry. Etoposide (100 μM) was used as the positive control for inducing apoptosis. (F) Levels of proteins including cleaved PARP and caspase-3 were analyzed via western blotting. β-Actin was used as a loading control. Values are the means ± SD of three independent experiments. Statistical significance was determined by Student’s t-test (*, p < 0.05 and NS, not significant).

Fig 3: Lipin2 silencing interferes with late stages of HCV infection. Huh-7 cells were transduced with lentiviral vectors expressing shRNAs targeting LPIN1 (LPIN1kd) or LPIN2 (LPIN2kd1 and LPIN2kd2). A control cell line was generated in parallel using a lentiviral vector expressing a non-targeting shRNA (control). At day 7 post transduction, the different cell lines were inoculated with HCV (D183v) at MOI 10. Samples of the cells and supernatants were collected 48 h post-transduction to determine HCV RNA levels by RT-qPCR or viral infectivity titers by endpoint dilution. (A) Intracellular HCV RNA levels. (B) Intracellular infectivity. (C) Extracellular infectivity. (D) Extracellular HCV RNA levels. Data are shown as average and SD of six biological replicates from two independent experiments (n = 6). Statistical significance was evaluated using one-way ANOVA and Dunnet´s post hoc test. (** p < 0.01; *** p < 0.001).

Fig 4: Lipin2 silencing interferes with HCV propagation. Huh-7 cells were transduced with lentiviral vectors expressing shRNAs targeting LPIN1 (LPIN1kd) or LPIN2 (LPIN2kd1 and LPIN2kd2). A control cell line was generated in parallel using a lentiviral vector expressing a non-targeting shRNA (control). Seven days post-transduction, cell lysates were subjected to Western-Blot analysis using antibodies against lipin1, lipin2 or tubulin as loading control. (A) Representative images showing lipin1 and lipin2 accumulation in the different cell lines at day 7 post-transduction. (B) Quantitation of lipin1 and lipin2 accumulation in the different cell lines. Data are shown as average and SD of four independent experiments (n = 4). Time 0 displays the limit of detection in this assay. (C) Control and lipin-deficient cells were inoculated with HCV (D183v) at MOI 0.01 and cultured for 5 days. Extracellular infectivity titers were determined in cell supernatants collected at day 3 and 5. Data are shown as average and SD of nine biological replicates from three independent experiments (n = 9). Statistical significance was evaluated using one-way ANOVA and Dunnet´s post hoc test. (** p < 0.01).

Fig 5: Patient-derived EGFR mutant NSCLC cells show LPIN1-dependent gefitinib resistance via PKCδ signaling induction. (A) Gefitinib-resistant lung cancer PDCs (YL05) were transfected with LPIN1- or NC-siRNA and treated with gefitinib (5 μM) or DMSO for (A) 120 h. Cell growth was measured by an MTT assay. (B,C) Gefitinib-resistant lung cancer PDCs were infected with shLPIN1- or pLKO-harboring lentivirus and treated with gefitinib (5 μM) or DMSO for (B) 10 days, and (C) 14 days. Cell growth was measured by (B) a colony formation assay after 0.5% crystal violet staining, or (C) an anchorage-independent growth assay in soft agar. (D,E) YL05 cells were transfected with siLPIN1 or siNC and treated with gefitinib (5 μM) or DMSO (D) for 24 h and harvested to determine the amount of diacylglycerol, (E) for 72 h and harvested to detect PKC activity. (F) YL05/siLPIN1 or YL05/siNC cells transfected with an NF-κB luciferase reporter plasmid were treated with gefitinib (5 μM) or DMSO for 24 h and harvested to determine their NF-κB activity and p-IκBα expression level. (G,H) LPIN1-depleted YL05 cells by (G) siRNA or (H) shRNA were treated with gefitinib (5 μM) or DMSO for 24 h. The cells were subjected to (G) flow cytometry to determine lipid droplet production or (H) Nile red staining to visualize lipid droplets with a fluorescence microscope. Values are the mean ± SD of three independent experiments. Statistical significance was determined by (D–F) Student’s t-test or (G) two-way ANOVA (*, p < 0.05; **, p < 0.01; and NS, not significant).