Fig 2: PLAC8 expression level in lung cancer patients and lung cancer cell lines. (a) PLAC8 expression level in lung cancer tissues and nontumor tissues (left: immunohistochemistry; right: statistical analysis). (b) PLAC8 expression level in serum of lung cancer patients and healthy people. (c) Expression of PLAC8 in serum of patients with lung cancer before and after operation. (d) PLAC8 mRNA expression level in lung cancer cell lines. (e, f) PLAC8 protein expression in lung cancer cell lines, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. All these cell biological function assays were triplicate.

Fig 3: Downregulation of PLAC8 inhibits the proliferation of lung cancer in vitro and in vivo. (a) Protein levels confirm downregulation of PLAC8. (b) Downregulation of PLAC8 expression inhibits lung cancer cell clone proliferation. (c) PLAC8 downregulates inhibition of lung cancer proliferation in lung cancer mouse model. (d) Immunohistochemical detection of lung cancer in mice, ∗∗p < 0.01; ∗∗∗p < 0.001. All these cell biological function assays were triplicate.

Fig 4: Cell growth changes in the presence of upregulated PLAC8 in vitro and in vivo. (A) Growth of SW480 cells in accordance with PLAC8 expression. All growth curves were relative the cell numbers at initial culture (0 h). SW480 (green line), parental SW480 cells; RFP-SW480 (blue line), SW480 cells infected with pLAS3w.RFP-C.Ppuro which expressed RFP as control infection; overPLAC8-SW480 (red line), SW480 cells infected with PLAC8-containing pLAS3w.Ppuro which overexpressed PLAC8. The statistical significance was calculated with Student’s t test (*p < 0.05). (B) Representative images of migrated SW480 cells in accordance with PLAC8 expression. All migrated cells were evaluated using a polyethylene terephthalate hanging cell culture insert with 0.4 μm pores. The filter membrane was removed, fixed with methanol, and stained with crystal violet. Migrated cells were detected after culturing cells for 30 and 60 min. Scale bar, 100 μm. (C) Relative migrated cell numbers of SW480 cells in accordance with PLAC8 expression. RFP, cells without overexpressed PLAC8 (blue line) or overexpressed PLAC8 (overPLAC8, red line). Migrated cells were counted after culturing cells for 30 and 60 min in three-to-four random fields under a light scope. The statistical significance was calculated with Student’s t test (#p < 0.1). (D) Representative images of migrated SW620 cells in accordance with PLAC8 expression. shLUC, targeting luciferase and normally express PLAC8; shPLAC8, PLAC8 knockdown. All migrated cells were evaluated and stained as SW480 cells with the exception of the detecting time (20 and 40 min). Scale bar, 100 μm. (E) Relative migrated cell numbers of SW620 cells in accordance with PLAC8 expression. shPLAC8, blue line; shLUC, red line. Migrated cells were counted after culturing cells for 20 and 40 min in three-to-four random fields under a light scope. The statistical significance was calculated with Student’s t test (*p < 0.05). (F) Relative mRNA levels of genes in shPLAC8-SW620 cells. All mRNA levels were relative to the level of glyceraldehyde-3-phosphate dehydrogenase and were compared to the relative level of shLUC-SW620 cells. Blue bar, shLUC620 cells; red bar, shPLAC8 cells. PLAC8, Placenta Specific 8; PCNA, Proliferating Cell Nuclear Antigen; MK167, Marker of Proliferation Ki-67. EPHB2, EPH Receptor B2; LGR5, Leucine Rich Repeat Containing G Protein-Coupled Receptor 5. The statistical significance was calculated with Student’s t test (*p < 0.05; **p < 0.01). Data are mean ± s.d. (G) Growth of xenograft tumor in accordance with PLAC8 expression. Four-week-old SCID mice were inoculated subcutaneously with 1 × 106 shLUC-SW620 cells or shPLAC8-SW620 cells on the right side of the back. Representative IVIS images demonstrated the tumor growth at the day post injection as indicated. IVIS, Non Invasion In Vivo Imaging System. (H) Size of xenograft tumor in accordance with PLAC8 expression. The dimensions of injection sites were measured roughly at the indicated time points while study. Tumor volumes (mm3) were calculated as described in Methods. shLUC-SW620 cells, red line; shPLAC8-SW620 cells, blue line. (I) Representative nanoPET/CT images in accordance with PLAC8 expression. A 10-minute PET scan was acquired after injection and one-hour distribution of F-18-FDG using a nanoScan PET/CT. The nanoPET and nanoCT images were fused. The left panel shows higher tracer uptake in the lung, heart, and bladder, but the right panel shows higher tracer uptake only in the heart and bladder. PET, positron emission tomography; CT, computed tomography. L, left side view; R, right side view. (J) Lung tissues of SCID mice in accordance with PLAC8 expression. Mice with xenograft tumors were euthanized and the lung organs were removed. The black arrowheads indicated the sites of metastatic tumors. Error bars showed standard deviation obtained from 2 to 3 independent experiments or from 3 to 5 mice for size of xenograft tumor. The statistical significance was calculated with Student’s t test (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 5: PLAC8 regulates lung cancer cell growth by regulating Wnt/β-Catenin signaling pathway. (a) Expression of p-Catenin in lung cancer tissues and nontumor tissues. (b) Lung cancer cell were treated by 100 ng/mL PLAC8 for the indicated times. (c) Effects of overexpression and downregulation of PLAC8 on Wnt/β-Catenin signaling pathway (Western blot). (d) Dual luciferase reporter gene analysis reveals that PLAC8 regulates Wnt/β-Catenin signaling pathway activity in A549 and H838 cells, ∗∗p < 0.01; ∗p < 0.001. All these cell biological function assays were triplicate.