Article Figures & Data
Figures
- Figure 1.
γ-Glutamylcyclotransferase (GGCT) overexpression promotes mouse embryonic fibroblasts NIH-3T3 proliferation and the hedgehog pathway. (A) GGCT expression levels were compared by western blotting in NIH-3T3 cells cultured in Dulbecco’s modified Eagle’s medium with 2% serum 72 h after inducing GGCT overexpression. Lamin B was used as a loading control. (B) Relative cell viability was compared in NIH-3T3 cells with GGCT overexpression (n=10). ***Significantly different at p<0.001 by two-tailed Student’s t-test. (C) Microarray pathway analysis was performed on genes in NIH-3T3 cells (fold change >2.0) up-regulated by GGCT overexpression. The most significantly affected pathways are shown. GGCT, γ-Glutamylcyclotransferase; OE, overexpression; ACE, angiotensin-converting enzyme; GPCRs, G protein-coupled receptors; MAPK, mitogen-activated protein kinase; ID, inhibitor of differentiation.
- Figure 2.
γ-Glutamylcyclotransferase (GGCT) overexpression promotes NIH-3T3 cell proliferation and has a facilitative effect on the hedgehog pathway. (A) Relative cell viability was compared in NIH-3T3 cells cultured in Dulbecco’s modified Eagle’s medium with 10% serum 72 h after GGCT overexpression (n=12). ***Significantly different at p<0.001 by two-tailed Student’s t-test. (B) The pathway diagram illustrates the key components and interactions within the hedgehog signaling pathway that are affected by GGCT overexpression. In the accompanying heat map, the expression levels of these genes are visually compared between control cells (non-targeting control, NT) on the left and GGCT-OE cells on the right. The heat map uses color coding to represent gene expression levels, with warmer colors (e.g., red) indicating higher expression levels and cooler colors (e.g., blue) indicating lower expression levels. SHH, Sonic hedgehog; IHH, Indian hedgehog; DHH, desert hedgehog; GAS1, growth arrest-specific 1; PTCH, patched; SMO, smoothened; HHIP, hedgehog interacting protein; RAB23, Ras-related protein Rab-23; STK36, serine/threonine kinase 36; IGF2, insulin-like growth factor 2; SUFU, suppressor of fused; CDC2A, cell division cycle 2 kinase A; CCNB1, cyclin B1; SAP18, sin3-associated polypeptide 18; GLI, GLI family zinc finger; SKI, Sloan-Kettering virus; SIN3A, SIN3 transcription regulator family member A; DYRK1A, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A; CREBBP, CREB-binding protein.
- Figure 3.
γ-Glutamylcyclotransferase (GGCT) overexpression in NIH-3T3 cells cultured in Dulbecco’s modified Eagle’s medium with 10% serum affects the hedgehog pathway. Microarray pathway analysis was performed on genes up-regulated (fold change >2.0) in NIH-3T3 cells with GGCT overexpression. Significantly affected pathways are shown. GGCT, γ-Glutamylcyclotransferase; OE, overexpression; SHH, Sonic hedgehog; IHH, Indian hedgehog; DHH, desert hedgehog; GAS1, growth arrest-specific 1; PTCH, patched; SMO, smoothened; HHIP, hedgehog interacting protein; RAB23, Ras-related protein Rab-23; STK36, serine/threonine kinase 36; IGF2, insulin-like growth factor 2; SUFU, suppressor of fused; CDC2A, cell division cycle 2 kinase A; CCNB1, cyclin B1; SAP18, sin3-associated polypeptide 18; GLI, GLI family zinc finger; SKI, Sloan-Kettering virus; SIN3A, SIN3 transcription regulator family member A; DYRK1A, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A; CREBBP, CREB-binding protein.
- Figure 4.
γ-Glutamylcyclotransferase (GGCT) overexpression in mouse embryonic fibroblasts NIH-3T3 facilitates the expression of hedgehog pathway-related genes in NIH-3T3 cells cultured in Dulbecco’s modified Eagle’s medium with 2% serum 72 h after GGCT overexpression. This figure provides an overview of the hedgehog signaling pathway as determined by microarray analysis in NIH-3T3 cells with GGCT overexpression (GGCT-OE). The pathway diagram illustrates the key components and interactions within the hedgehog signaling pathway that are affected by GGCT overexpression. In the accompanying heat map, the expression levels of these genes are visually compared between control cells (non-targeting control, NT) on the left and GGCT-OE cells on the right. The heat map uses color coding to represent gene expression levels, with warmer colors (e.g., red) indicating higher expression levels and cooler colors (e.g., blue) indicating lower expression levels. GPCRs, G protein-coupled receptors; EGFR1, epidermal growth factor receptor 1; EPO receptor, erythropoietin receptor; IL, interleukin; ACE, angiotensin-converting enzyme; EDA signaling, ectodysplasin A signaling; MAPK, mitogen-activated protein kinase; miR-208, microRNA-208; ESC pluripotency, embryonic stem cell pluripotency.
- Figure 5.
γ-Glutamylcyclotransferase (GGCT) knockdown with short hairpin RNA (shRNA) suppresses glioblastoma stem cell proliferation and expression of hedgehog pathway factors. (A) Non targeting (NT) and two GGCT (GGCT-KD1 and GGCT-KD2) shRNAs were transfected into two independent of murine glioblastoma stem cell lines (GSC1 and GSC2) and after 3 days, the expression levels of GGCT, desert hedgehog (DHH) and GLI family zinc finger 1 (GLI1) were compared by western blotting (A) and glioblastoma stem cell proliferation was compared (n=3) (B). Lamin B was used as a loading control. Significantly different at *p<0.05 and ***p<0.001 by one-way analysis of variance with Dunnett test. GSC, Glioblastoma stem cells; GGCT, γ-glutamylcyclotransferase; DHH, desert hedgehog; GLI, GLI family zinc finger; KD, knockdown.
- Figure 6.
Inhibition of cell proliferation by γ-glutamylcyclotransferase (GGCT) knockdown in mouse glioblastoma stem cells is restored by activation of the hedgehog pathway through forced expression of desert hedgehog (DHH). (A) DHH expression levels were compared in glioblastoma stem cells 72 h after DHH overexpression. (B) GGCT knockdown was performed in glioblastoma stem cells with DHH overexpression (DHH-OE), and the levels of GGCT and DHH were compared after another 4 days. Lamin B was used as a loading control. (C) GGCT knockdown was performed in DHH-OE glioblastoma stem cells and after another 4 days the number of cells was compared (n=9). (D) Representative phase-contrast microscopy images of DHH-OE glioblastoma stem cells with GGCT knockdown. Scale bar: 100 μm. (E) Real time quantitative polymerase chain reaction of GLI family zinc finger 1 (GLI1) was performed in DHH-OE glioblastoma stem cells with GGCT knockdown. β-Actin was used as an internal control (n=3). ***Significantly different at p<0.001 by two-way analysis of variance with Bonferroni test. DHH, Desert hedgehog; GGCT, γ-glutamylcyclotransferase; KD, knockdown; OE, overexpression; GLI1, GLI family zinc finger 1.
