Fig 2: Effects of PRRX1 knockdown on proliferative capacity or drug sensitivity of human osteosarcoma cells.(A) Detection of PRRX1 in human osteosarcoma cell lines by western blot analysis. Total protein was extracted from the human osteosarcoma cell lines SAOS2, MG63, HOS, 143B, and U2OS, and the expression of PRRX1 in each was assessed compared with cell lysates prepared from HEK293T cells overexpressing each PRRX1 isoform as reference. (B) RT-qPCR analysis of PRRX1 in 143B cells after PRRX1 knockdown. The 143B cells were infected with a lentivirus encoding each shPRRX1 clone, and total RNA was extracted to compare the expression level of PRRX1 mRNA. All values were normalized to GAPDH mRNA level (n = 3) (C) Western blot analysis after PRRX1 knockdown. The 143B cells were infected with lentivirus encoding each shPRRX1 clone, and nuclear lysates were extracted to compare the expression level of PRRX1. (D) Comparison of proliferative capacity by WST-1 assay. The 143B/Ctrl or 143B/shPRRX1#1 or #2 cells were seeded in a 96-well plate, and the OD450 was measured at each indicated time point (n = 4, two independent experiments). (E) Comparison of the RNA transcriptome after PRRX1 knockdown. RNA-seq of 143B/Ctrl and 143B/shPRRX1#2 was performed, and data were compared by gene sets enrichment analysis (GSEA). Genes upregulated in 143B/shPRRX1#2 were clustered at the left (n = 2, two independent experiments). (F, G) Comparison of cisplatin (F) or doxorubicin (G) sensitivity after PRRX1 knockdown. Cells were treated with cisplatin or doxorubicin for 72 h, and the ratio of living cells was assessed by WST-1 assay. The ratio of DMSO-treated cells was set to 100% (n = 3, two independent experiments).

Fig 3: Effects of PRRX1 knockdown on tumor growth.(A) Comparison of tumor volume after PRRX1 knockdown. The 143B/Ctrl or 143B/shPRRX1#2 cells were subcutaneously transplanted into nude mice, and the tumor volume at each indicated time point was measured (A). At 21 days after transplantation, mice were sacrificed, and the weights of the developed tumors were measured (B) (n = 5, two independent experiments). (C) Immunohistological analysis of developed xenograft tumors derived from 143B/Ctrl or 143B/shPRRX1#2 cells. Tumor sections were stained with hematoxylin and eosin (H&E), immunostained for PRRX1 or Ki-67, or stained by TUNEL. Representative photomicrographs are shown. (D, E, F) Quantification of Ki-67- or TUNEL-positive cells in tumors developed from each cell line. Two or three fields in each tumor section were assessed, and the intensity of PRRX1 (D) or the numbers of cells positive for Ki-67 (D) or TUNEL (E) were compared (n = 5). (G) Comparison of lung metastasis after PRRX1 knockdown. Lung tissues were harvested 21 days after transplantation, and PRRX1-positive metastatic nodules were quantified. Tissue sections were sampled at 1 mm intervals, and the number of metastatic nodules in each section was added together (n = 5).

Fig 4: Expression of PRRX1 in mouse or human osteosarcoma tissues.(A) Immunostaining of Prrx1 in mouse osteosarcoma tissues. Sections derived from Prrx1-Cre;Rbfl/fl;Trp53fl/fl, Oc-Cre;Rbfl/fl;Trp53fl/fl or Col1a1-Cre;Rbfl/fl;Trp53fl/fl osteosarcomas were stained with Prrx1 and representative photos are shown. (B) Immunostaining of PRRX1 in human osteosarcoma tissues. Sections derived from human osteosarcomas were stained with PRRX1, and representative photos of PRRX1-low or PRRX1-high tumors are shown. (C) Comparison of overall survival between PRRX1-low and PRRX1-high osteosarcoma patients. Kaplan–Meyer survival curve demonstrates significant (log-rank test, p < 0.01) worse overall prognosis for osteosarcoma patients with high expression levels of PRRX1 compared with the PRRX1-low expression group. (D) Correlation of PRRX1 levels with patient clinical and pathological characteristics.

Fig 5: Effects of PRRX1 knockdown on migration or invasion of human osteosarcoma cells.(A, B) Comparison of migration capacity after PRRX1 knockdown by wound healing assay (A) or migration assay (B). For the wound-healing assay, the relative migration of 143B/shPRRX1#1 or #2 cells was lower than that of 143B/Ctrl at 16 h after scratch wound (n = 5, two independent experiments). For the migration assay, transwell plates were used to assess migration. The number of migrated cells was significantly decreased in 143B/shPRRX1#1 or #2 (n = 12, two independent experiments). (C) Comparison of invasion capacity after PRRX1 knockdown by invasion assay. Transwell plates were used to assess the invasion. The number of migrated cells was significantly decreased in 143B/shPRRX1#1 or #2 (n = 12, two independent experiments).