Additional file 1 of Testosterone upregulates glial cell line-derived neurotrophic factor (GDNF) and promotes neuroinflammation to enhance glioma cell survival and proliferation
Creators: name: Kanwore, Kouminin
name: Kanwore, Konimpo
name: Guo, Xiaoxiao
name: Xia, Ying
name: Zhou, Han
name: Zhang, Lin
name: Adzika, Gabriel Komla
name: Joseph, Adu-Amankwaah
name: Abiola, Ayanlaja Abdulrahman
name: Mu, Peipei
name: Kambey, Piniel Alphayo
name: Noah, Marie Louis N’dzie
name: Gao, DianShuai
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1A. Next-generation sequencing of human astrocytes (U251). The treatment of U251 with testosterone upregulates the cytokines and oncogenes compared with the untreated groups. Next-generation sequencing of human astrocytes (HA). The treatment of HA with testosterone upregulates the cytokines and oncogenes compared with the untreated groups. Figure S1B. Western blot detection of GDNF in human astrocyte (HA), U251, LN229, and U87: The treatment of HA, U251, LN229, and U87 with testosterone significantly increased GDNF protein level compared with the untreated groups. **p&lt;0.01 and ****p&lt;0.0001. Figure S1C. Transwell assay indicating LN229 glioma cell line invasion. The results indicated that the LN229 glioma cell line invasion in the untreated LN229 was slightly lower than in testosterone-treated LN229 in which the invasion ability highly increased. *p&lt;0.05. Figure S1D. Wound-healing assay indicating U251 migration abilities: the results showed that the U251 migration ability is high testosterone-treated U251 compared to the untreated U251 in which the U251 glioma cell line migration was low. **p&lt;0.01 and ***p&lt;0.001. Figure S1E. Western blot detection of Cyclophilin A in U251, LN229, U87, and HA. The results showed that the cyclophilin A protein level in the untreated glioma cell line was lower than in testosterone-treated glioma cell lines in which cyclophilin A protein level significantly increased. ***p&lt;0.001. Figure S1F. LN229 glioma cell line survival test via EDU assay. The staining of the LN229 glioma cell lines with EDU solution showed that the testosterone-treated LN229 glioma cell lines survival significantly increased compared with the untreated LN229 glioma cell lines. *p&lt;0.05. Figure S1G. Brain tissue staining with trypan blue to reveal astrocyte shape. The observation of astrocyte shape showed that the untreated mice brain has a better astrocyte shape followed by testosterone-treated astrocytes. The castrated mice astrocyte axons were completely gone; however, the castrated group treated with testosterone showed a regeneration of axons. ***p&lt;0.001, and ****p&lt;0.0001. Figure S1H. Immunofluorescence detection of pro-inflammatory macrophages in female mice brain. the control group showed few CD86-positive macrophages; however, CD68-positive macrophages were detected. In testosterone-treated female mice, we detected both CD86 and CD68 macrophages significantly. *p&lt;0.05 and **p&lt;0.01. Figure S1I. Co-culture showing interactions of LN229 with Raw264.7 in the untreated and testosterone treated conditions. Figure S1J. Western blot detection of NRF2 in U251, LN229, U87, and HA. The results showed that the NRF2 protein level in the untreated glioma cell line was lower than in testosterone-treated glioma cell lines in which the NRF2 protein level significantly increased. * ***p&lt;0.001. Western blot detection of ERK1/2 and pERK1/2 in U251, LN229, U87, and HA. The results showed that the ERK1/2 and pERK1/2 protein levels in the untreated glioma cell line was lower than in testosterone-treated glioma cell lines in which ERK1/2 and pERK1/2 protein levels significantly increased. the highest difference between the untreated and testosterone-treated pERK1/2 was observed in U251 and HA cells. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, and ****p&lt;0.0001. Figure S1K. Immunofluorescence showing GDNF subcellular localization in human astrocyte (HA). The results showed that GDNF was found in the untreated group HA nucleus, while in testosterone treated group, GDNF is found mainly in the cytoplasm.Immunofluorescence showing GDNF subcellular localization in U251. The results showed that GDNF was found in the untreated group U251 nucleus, while in testosterone treated group, GDNF is found mainly in the cytoplasm. Immunofluorescence showing GDNF subcellular localization in LN229. The results showed that GDNF was found in the untreated group LN229 nucleus, while in testosterone treated group, GDNF is found mainly in the cytoplasm. Figure S1L. Hematoxylin-eosin (H-E) of tumor tissue comparing inflammatory cell between the control (untreated) and testosterone treated groups. The inflammatory cell is higher in testosterone treated tissue than the untreated tissue. ***P&lt;0.001. Figure S1M. LN229 glioma cell line survival test via EDU assay. The staining of the LN229 glioma cell line with EDU solution showed that the Untreated LN229 glioma cell lines survival significantly increased compared with the GDNF-KO LN229 glioma cell lines, which showed a low survival. ****p&lt;0.0001.