Fig 2: RNA sequencing analysis upon PHF14 depleted cells. a 137 upregulated and 120 downregulated genes (Log2 (Fold Change) = - 1/= 1) were differentially expressed upon PHF14 knockdown with 5% False Discovery Rate (FDR). b Validation of the RNA sequencing analysis was performed by quantitative real time PCR. Pearson correlation of RNA Seq and qPCR is 0.68. c, d Gene ontology (GO) and KEGG pathway analyses results summarized that functional categories of genes significantly enriched upon PHF14 knockdown

Fig 3: PHF14 silencing in glioma cell lines decelerates proliferation and clonogenic growth. a, b The knockdown efficacy of PHF14 examined in U251, U87MG and A172 cells by qRT-PCR (a) and Western blot (b). c, d Cellular DNA replication is mitigated after PHF14 knocked down. Ten thousand U87MG, U251 and A172 cells per well were seeded in 96-well plates overnight. EdU and Hoechest co-staining was performed on the second day to assess cellular DNA replication activities (c) (Scale bar = 100 µm). Values are counted as EdU + cells/total cells (d). e–g PHF14 silenced cells showed weaker proliferation and colony formation potential. 500 U251 and A172 cells per well were cultured in 6-well plates in Complete growth medium for 10 days and then stained by 1% crystal violet for colony formation assay (e) and single colony morphology (f) (Scale bar = 200 µm). The number of colonies containing over 50 cells were counted up for analysis (g). h Cell colonies were stained by Phalloidin-cy3 after 10 days of culture for assessment of actin filaments containment and cellular protrusions (Scale bar = 50 µm) (*P < 0.05; **P = 0.01; ***P < 0.001; ****P < 0.0001)

Fig 4: PHF14 is overexpressed in glioblastoma multiforme (GBM) and lower PHF14 expression correlates with better outcome in GBM and LGG. a The cellular location of PHF14 confirmed by immunofluorescence (Scale bar = 50 µm). b Representative stainings of PHF14 in normal brain, grade II, grade III and grade IV glioma (Scale bar = 50 µm). c Correlation of the staining intensity of PHF14 with glioma grade. d–f Correlation of PHF14 mRNA expression with increasing WHO glioma grade according to REMBRANDT, TCGA and FRENCH data. g, h The relationship between PHF14 mRNA expression and molecular pathology of glioma. i, j Upregulation of PHF14 is a dismal prognostic factor in LGG by TCGA database. k Upregulation of PHF14 is a dismal prognostic factor in GBM by REMBRANDT database (*P < 0.05; **P = 0.01; ***P < 0.001; ****P < 0.0001)

Fig 5: PHF14 silencing attenuates growth of glioblastomas and delays the onset of deaths in vivo. a 3 × 105 U87MG scrambled or PHF14 silenced cells were implanted into 8-week-old nude mice (6 mice for each group). Once neurological disorders appeared, the animal would get sacrificed. The median overall survival of tumor bearing mice was prolonged from 32 days (n = 6) to 44.5 days (n = 4) after PHF14 was knocked down (P < 0.05). Two nude mice in PHF14 silencing group were excluded because no tumor was found after euthanized. b Ki-67 immunostainings of sliced brains of executed animals. PHF14 silenced cells represented weaker intensity, suggesting its lower proliferation rate (b, c) (Scale bar = 100 µm)