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Correction to: Metabolic Brain Disease (2022)
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Supplementary Information for
Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway
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Extended Materials and Method
Supplementary Fig. 1–4
Supplementary Table 1–2
Extended Materials and Method
Isolation and culture of primary mouse microglia
Primary mouse microglia were isolated from 6 to 8-week-old male C57BL/6 J mice (Beijing SPF Biotechnology) as previously described with modifications (Cardona et al. 2006). Briefly, after perfusion, brains were diced into 2 mm pieces and then further homogenized using a 15-ml dounce homogenizer containing 3 ml of digestion cocktail (0.25% trypsin with 0.025 U/ml DNase I). Homogenized tissue was filtered through a 70 μm cell strainer to obtain a single cell suspension, centrifuged at 300×g for 5 min at 18 °C and resuspended in 37% Percoll (17–0891-02, GE Healthcare). Transfer the cell suspension (37% layer) to 15 ml conical tubes containing 4 ml of 70% Percoll at the bottom. Then on top of the 37% layer slowly pipette 4 ml of 30% Percoll, followed by 2 ml of PBS. Centrifuge the gradient 40 min at 300×g with no brake and ultimately the microglia was harvested from 70 to 37% interphase. Isolated cells were further cultured in six-well plates coated with poly-d-lysine. The DMEM/F12 culture medium was supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 ng/mL murine recombinant macrophage colony stimulating factor (M-CSF, 51112-MNAH, Sino Biological). The culture medium was changed twice a week.
Supplementary Fig. 1. Castor1 overexpression inhibited M1 related genes expression. BV2 cells were transfected with pcDNA3.1-vector or pcDNA3.1-Castor1 plasmid for 24 h without any other stimulations and then the M1 or M2-related genes were examined. The results demonstrated that Castor1 overexpression inhibited the expression of some M1 related markers, including iNOS (P < 0.05), IL-β (P < 0.05), TNF-α (P < 0.05) and IL-6 (P < 0.001). However, there were no change in the expression of M2-related genes. Data are expressed as SEM, *P < 0.05, **P < 0.01 compared to the pcDNA3.1-vector group.
Supplementary Fig. 2. Castor1 knockdown promotes the transcription of M1-related genes. Castor1 knockdown was performed using Castor1-targeted shRNA cloned into the plasmid. qPCR analysis revealed that Castor1 expression was decreased 73% by shCastor1–1 (P = 0.056), 54% by shCastor1–2 (P < 0.001), 74% by shCastor1–3 (P < 0.05), and 53% by shCastor1–4 (P < 0.001) separately compared to the shNC. To further investigate the effect of Castor1 knockdown on the M1 polarization, BV2 cells were transfected with shNC or shCastor1–4 for 12 h and then treated with LPS/IFN-γ for 12 h. Compared with shNC groups, there was a significant increase in the expression of of M1-related genes such as iNOS (P < 0.01), IL-β (P < 0.01), TNF-α (P < 0.001) and IL-6 (P < 0.01). Data are expressed as SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to the shNC group.
Supplementary Fig. 3. The Expression of Castor1 in IL-4 treated BV2 cells. BV2 cells were treated with 20 ng/mL IL-4 for 12 h. The expression of M2-related markers and Castor1 was detected. The data shown that Castor1 expression was significantly upregulated in M2 polarization conditions. Data are expressed as SEM, *P < 0.05, **P < 0.01compared to the CON group.
Supplementary Fig. 4. The Expression of Castor1 in LPS/IFN-γ or IL-4 treated primary cultured microglia cells. Primary microglia cells were treated with 100 ng/mL LPS concurrent with 10 ng/mL IFN-γ or 20 ng/mL IL-4 for 12 h. The expression of M1- or M2-related markers and Castor1 was detected. The results shown that Castor1 expression was significantly decreased in M1 polarization conditions and upregulated in M2 polarization conditions. Data are expressed as SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to the CON group.
Supplementary Table 1. The sequence used for shCastor1
sequence (5′–3′) | |
---|---|
shNC | CTTACGCTGAGTACTTCGA |
shCastor1–1 | GCTGTTGACATCTCTGCTTAC |
shCastor1–2 | GCCCGTTACTGGAGACGATTC |
shCastor1–3 | GCTGGTGATGAACGTATCTCA |
shCastor1–4 | GGATGAGGAGGGCTTTAAAGA |
Supplementary Table 2. Primer sequence used for qPCR analysis
Gene | Primer sequence (5′–3 | |
---|---|---|
CD86 | Forward: | ACGATGGACCCCAGATGCACCA |
Reverse: | GCGTCTCCACGGAAACAGCA | |
iNOS | Forward: | GGCAGCCTGTGAGACCTTTG |
Reverse: | TGCATTGGAAGTGAAGCGTTT | |
IL-β | Forward: | CCTGCAGCTGGAGAGTGTGGAT |
Reverse: | TGTGCTCTGCTTGTGAGGTGCT | |
TNF-α | Forward: | AGCCCACGTCGTAGCAAACCAC |
Reverse: | AGGTACAACCCATCGGCTGGCA | |
IL-6 | Forward: | AGGAGACTTCACAGAGGATACC |
Reverse: | GAATTGCCATTGCACAACTCTT | |
MCP-1 | Forward: | GCATCCACGTGTTGGCTCA |
Reverse: | CTCCAGCCTACTCATTGGGATCA | |
Arg1 | Forward: | TTAGGCCAAGGTGCTTGCTGCC |
Reverse: | TACCATGGCCCTGAGGAGGTTC | |
CD206 | Forward: | TCAGCTATTGGACGCGAGGCA |
Reverse: | TCCGGGTTGCAAGTTGCCGT | |
IL-10 | Forward: | GGCAGAGAACCATGGCCCAGAA |
Reverse: | AATCGATGACAGCGCCTCAGCC | |
β-Actin | Forward: | CTCTGGCTCCTAGCACCATGAAGA |
Reverse: | GTAAAACGCAGCTCAGTAACAGTCCG |
Reference
Cardona AE, Huang D, Sasse ME, Ransohoff RM (2006) Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 1(4):1947–1951. https://doi.org/10.1038/nprot.2006.327
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Hu, H., Lu, X., Huang, L. et al. Correction to: Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway. Metab Brain Dis 38, 1437–1440 (2023). https://doi.org/10.1007/s11011-023-01163-0
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DOI: https://doi.org/10.1007/s11011-023-01163-0