Fig 2: The expression of ankyrin repeat domain containing 26 is upregulated by the overexpression of GATA binding protein 2. A: Agarose gels after polymerase chain reaction (PCR) to assess the expression of GATA binding protein 2 (GATA2) in HEL, K562, human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and human induced pluripotent stem cells derived from urothelium (hiPSC-U); B: Quantitative real-time PCR (RT-qPCR) analyses showing the promoting effect of GATA2 on ankyrin repeat domain containing 26 (ANKRD26) mRNA expression. K562 cells were transfected with GATA2 plasmids and control plasmids; C: Western blots showing the effect of GATA2 on promoting ANKRD26 protein expression in K562 cells; D: RT-qPCR analyses showing the effect of GATA2 on promoting ANKRD26 mRNA expression. HEL cells were transfected with GATA2 plasmids and control plasmids; E: Western blots showing the effect of GATA2 on promoting ANKRD26 expression at the protein level in HEL cells. The error bars represent the means ± SD of triplicate samples. bP < 0.01, calculated by Student’s t test. GATA2: GATA binding protein 2; ANKRD26: Ankyrin repeat domain containing 26; RUNX1: Runt related transcription factor 1; FLI1: Friend leukemia integration 1; hiPSC-BM: Human induced pluripotent stem cells derived from bone marrow; hiPSC-U: Human induced pluripotent stem cells derived from urothelium.

Fig 3: The regulation of ankyrin repeat domain containing 26 by GATA binding protein 2 overexpression in bone marrow- and urothelium-derived cell lines. A-D: Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analyses showing that GATA binding protein 2 (GATA2) promoted ankyrin repeat domain containing 26 (ANKRD26) expression in two bone marrow-derived cell lines (THP-1 and MOLM-13); E-H: RT-qPCR and western blot analyses showing that GATA2 promoted ANKRD26 expression in two urothelium-derived cell lines (LNCaP and DU145). The error bars represent the means ± SD of triplicate samples. aP < 0.05; bP < 0.01, calculated by Student’s t test. GATA2: GATA binding protein 2; ANKRD26: Ankyrin repeat domain containing 26.

Fig 4: The transcription factor GATA binding protein 2 is potentially involved in regulating ankyrin repeat domain containing 26 expression. A: A heatmap showing the expression patterns of transcription factor (TF) genes in human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and human induced pluripotent stem cells derived from urothelium (hiPSC-U); B: A volcano plot showing the differentially expressed TF genes in hiPSC-BM and hiPSC-U. Red dots represent TF genes with significant differential expression; C: Venn diagram showing overlaps of ankyrin repeat domain containing 26 (ANKRD26) related transcription factors from the Cistrome DB, animal TFDB, ENCODE and transcriptome (differentially expressed transcription factors)[41]. DETFs: Differentially expressed transcription factors; GATA2: GATA binding protein 2; hiPSC-BM: Human induced pluripotent stem cells derived from bone marrow; hiPSC-U: Human induced pluripotent stem cells derived from urothelium.

Fig 5: Inhibition of invasion- and migration-related transcripts in T. gondii-infected HTR-8/SVneo cells is SMAD2- and GATA2-independent. (A, B) Cells were serum-starved and pretreated with 10 μM SB431542 or an equivalent volume of vehicle (i.e., DMSO) for 1 h then stimulated with 5 ng/mL recombinant human TGF-β1 for 2 h, infected with Tg RH for 0.5–2 h (A) or 2 h (B), or left uninfected (Control) for 2 h. (A) Cells were lysed, and samples were processed for western blot analyses to monitor the phosphorylation status (S465/S467) and total levels of SMAD2. β-actin was used as a loading control, and an antibody raised against T. gondii profilin-like protein was used to assess infection of HTR-8/SVneo cultures. Total protein extracts from extracellular tachyzoites (“Tg only”) were used to control for any cross-reactivity of the antibodies against T. gondii proteins. (B) Cultures were fixed and processed for confocal immunofluorescence microscopy. Samples were stained with DAPI (shown in blue), used as a nuclear marker, and for phosphorylated SMAD2 at S465/S467 (shown in white). Original magnification (left panels), 4 times-enlarged insets (right panels). Parasitophorous vacuoles (PVs) are outlined with dashed lines to indicate the presence of parasites within infected cells. (C) Serum-starved HTR-8/SVneo cultures were infected with T. gondii RH strain (Tg RH) or left uninfected for the indicated times. Cells were lysed, and samples were processed for western blot analyses to monitor the expression levels of GATA2. (A–C) Images are representative of three independent experiments.