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Downregulation of P4HB in T98G inhibits chemotaxis and M2 polarization of cocultured microglia. (A) Effect of P4HB knockdown on tumor cell growth in T98G and GL261 cells. (B) Coculture of PMA-treated microglia with differently treated glioblastoma cells. (C) Quantification of microglial chemotaxis toward T98G cells after coculture as shown in (B). (D) Effect of P4HB knockdown on glioblastoma cell growth when cocultured with activated microglia (as in B). (E) Microglia markers identified via RT-qPCR after culturing with P4HB knockdown medium or IL-6 added medium (100pg/ml). (F) Protein expression levels of microglial polarization markers, PD-L1 and shown in (E). * P < 0.05, **** P < 0.0001. Data from at least three independent experiments were analyzed and are presented as mean±SD (A, C, D, and F). Statistical analyses were performed using the nonparametric one-way ANOVA (A, C, D, and F).

Journal: Annals of Medicine and Surgery

Article Title: P4HB regulates tumor-associated macrophage polarization and chemotaxis by enhancing IL-6 cytokine secretion in glioblastoma

doi: 10.1097/MS9.0000000000004049

Figure Lengend Snippet: Downregulation of P4HB in T98G inhibits chemotaxis and M2 polarization of cocultured microglia. (A) Effect of P4HB knockdown on tumor cell growth in T98G and GL261 cells. (B) Coculture of PMA-treated microglia with differently treated glioblastoma cells. (C) Quantification of microglial chemotaxis toward T98G cells after coculture as shown in (B). (D) Effect of P4HB knockdown on glioblastoma cell growth when cocultured with activated microglia (as in B). (E) Microglia markers identified via RT-qPCR after culturing with P4HB knockdown medium or IL-6 added medium (100pg/ml). (F) Protein expression levels of microglial polarization markers, PD-L1 and shown in (E). * P < 0.05, **** P < 0.0001. Data from at least three independent experiments were analyzed and are presented as mean±SD (A, C, D, and F). Statistical analyses were performed using the nonparametric one-way ANOVA (A, C, D, and F).

Article Snippet: The antibodies used for IB were as follows: P4HB at 1:2000 (Cat#PTM-5314, PTM-biolab), CD86 at 1:2000 (Cat#A21198, ABclonal), MHC II at 1:1000 (Cat#bs-8481R, Bioss), CD163 at 1:2000 (Cat#PA5-78961, Invitrogen), CD206 at 1:1000 (Cat#18704-1-AP, Proteintech), p-STAT3(Y705) at 1:2000 (Cat#39595, Proteintech), STAT3 at 1:1000 (Cat#60199-1-Ig, Proteintech), programmed cell death protein ligand 1 (PD-L1) at 1:2000 (Cat#66248-1-Ig, Proteintech), and GAPDH at 1:5000 (Cat#ab8245, Abcam).

Techniques: Chemotaxis Assay, Knockdown, Quantitative RT-PCR, Expressing