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. 2022 Mar 21;13:117. doi: 10.1186/s13287-022-02798-z

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Involvement of TLR2 and TLR4 in the self-renewal of in vitro-expanded spinal cord NPCs. A Representative images of immunofluorescence assay for Sox2 (upper panels, orange) and FoxJ1 (lower panels, green) detection in neurospheres formed using WT, TLR2^−/− and TLR4^−/− NPCs. Inset: double staining images for Sox2 (orange) or FoxJ1 (green) with DAPI (blue); B Quantification of FoxJ1 positive cells by immunoassay expressed as the percentage of total DAPI positive cells. C Upper graph: Quantification of Sox2-positive cells represented as nuclear (full) or cytoplasmic expression (striped). Black stars ( ) vs. TLR4^−/− for cytoplasmic Sox2 and White stars ( ) vs. TLR4^−/− for nuclear Sox2. Lower panel: representative image of typical Sox2 nuclear (*) or cytoplasmic (#) expression co-stained with DAPii (blue); D Gene expression analysis in WT, TLR2^−/−, and TLR4^−/− NPCs of the indicated genes. E Quantification of size (upper graph) and number (lower graph) of neurospheres formed from WT, TLR2^−/−, and TLR4^−/− NPCs 48 h incubation after unicellular disaggregation. * vs. WT; # vs. TLR2^−/−. Representative images of the indicated neurosphere like cultures are shown (right); F (Left) Quantification of BrdU and Ki67 positive cells in WT, TLR2^−/− or TLR4^−/− NPCs represented as percentages of the total cells. * vs. WT; # vs. TLR2^−/−. (Right) Representative images of the double immunofluorescence—BrdU (red) and Ki67 (green). G (Left) Quantification of H2AX positive cells in WT, TLR2^−/− or TLR4^−/− NPCs represent as percentages of the total cells. * vs. WT; # vs. TLR2^−/−. (Right) Representative images of the pH2AX (green) immunofluorescence with DAPI used for nuclei counterstaining (blue) in WT, TLR2^−/− or TLR4^−/− NPCs. H Gene expression by qPCR of cMYC and p21 transcripts in WT, TLR2^−/− and TLR4^−/− NPCs. I Population doubling level analysis in WT, TLR2^−/− and TLR4^−/− NPCs expressed as the mean of three independent experiments. J Quantification of pyknotic nuclei in WT, TLR2^−/− and TLR4^−/− NPCs from DAPI nuclei staining (identified as smaller than normal with hyper condensate chromatin) represented as a percentage of the total cells. Data shown as mean ± SEM. Results were assessed for normality using the Shapiro–Wilk test and one-way ANOVA with Tukey post hoc test; * or ^#p < 0,05; ** or ^## p < 0.01; *** or ^###p < 0.001; ****p < 0.0001

Inline graphic — Involvement of TLR2 and TLR4 in the self-renewal of in vitro-expanded spinal cord NPCs. A Representative images of immunofluorescence assay for Sox2 (upper panels, orange) and FoxJ1 (lower panels, green) detection in neurospheres formed using WT, TLR2^−/− and TLR4^−/− NPCs. Inset: double staining images for Sox2 (orange) or FoxJ1 (green) with DAPI (blue); B Quantification of FoxJ1 positive cells by immunoassay expressed as the percentage of total DAPI positive cells. C Upper graph: Quantification of Sox2-positive cells represented as nuclear (full) or cytoplasmic expression (striped). Black stars ( ) vs. TLR4^−/− for cytoplasmic Sox2 and White stars ( ) vs. TLR4^−/− for nuclear Sox2. Lower panel: representative image of typical Sox2 nuclear (*) or cytoplasmic (#) expression co-stained with DAPii (blue); D Gene expression analysis in WT, TLR2^−/−, and TLR4^−/− NPCs of the indicated genes. E Quantification of size (upper graph) and number (lower graph) of neurospheres formed from WT, TLR2^−/−, and TLR4^−/− NPCs 48 h incubation after unicellular disaggregation. * vs. WT; # vs. TLR2^−/−. Representative images of the indicated neurosphere like cultures are shown (right); F (Left) Quantification of BrdU and Ki67 positive cells in WT, TLR2^−/− or TLR4^−/− NPCs represented as percentages of the total cells. * vs. WT; # vs. TLR2^−/−. (Right) Representative images of the double immunofluorescence—BrdU (red) and Ki67 (green). G (Left) Quantification of H2AX positive cells in WT, TLR2^−/− or TLR4^−/− NPCs represent as percentages of the total cells. * vs. WT; # vs. TLR2^−/−. (Right) Representative images of the pH2AX (green) immunofluorescence with DAPI used for nuclei counterstaining (blue) in WT, TLR2^−/− or TLR4^−/− NPCs. H Gene expression by qPCR of cMYC and p21 transcripts in WT, TLR2^−/− and TLR4^−/− NPCs. I Population doubling level analysis in WT, TLR2^−/− and TLR4^−/− NPCs expressed as the mean of three independent experiments. J Quantification of pyknotic nuclei in WT, TLR2^−/− and TLR4^−/− NPCs from DAPI nuclei staining (identified as smaller than normal with hyper condensate chromatin) represented as a percentage of the total cells. Data shown as mean ± SEM. Results were assessed for normality using the Shapiro–Wilk test and one-way ANOVA with Tukey post hoc test; * or ^#p < 0,05; ** or ^## p < 0.01; *** or ^###p < 0.001; ****p < 0.0001