Implication of CD133 in endocytosis mainly involved the clathrin pathway.

<p>A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133^high (control siRNA) and CD133^low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p<0.05, **p<0.01, ***p<0.001; comparison between control siRNA and CD133 siRNA: Dunnett's test: p<0.05. B) Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells led to an increase in LNC intracellular accumulation. Flow cytometric analysis of intracellular uptake of NR-LNC within CD133^high (Control siRNA) and CD133^low (CD133 siRNA) Caco-2 cells after 1 h of incubation at 37°C/5%CO₂. Results are expressed as percentage of control, thus representing the geomean fluorescence intensity levels obtained for cells treated with vehicle alone. Data represented mean ± s.e.m. obtained from three independent experiments. Dunnett's test: **p<0.01.</p

Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells did not affect short-term Tf exocytosis.

<p>CD133^high (control siRNA) and CD133^low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to Tf-Alexa 488 for 2 h at 37°C/5% CO₂ before the extracellular medium was removed, washed and replaced by fresh medium free from Tf-Alexa 488. Amounts of Tf-Alexa 488 that were not recycled to the extracellular compartment were measured by flow cytometric analysis after further cell incubation at 37°C/5% CO₂ for 1 to 3 h. Data are expressed as a % of Tf-Alexa 488 initially internalized. They represent mean ± s.e.m. obtained from three independent experiments.</p

Recognition of CD133 and modulation of its expression interfere with Tf uptake and TfR accessibility in Caco-2 cells.

<p>A) AC133 antibody treatment inhibited Tf uptake. Constitutively CD133-expressing undifferentiated Caco-2 cells were exposed to Tf-Alexa 488 for 1 h at 37°C/5%CO₂ in the presence of 5 or 10 µg/ml of AC133 or IgG1κ immunoglobulin control. Tf-Alexa 488 that was effectively internalized within cells was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the untreated control. Data represented mean ± s.e.m. from a triplicate obtained from one representative experiment. Dunnett's test: *p<0.05, ***p<0.001. B) Flow cytometric analysis of the expression of TfR (CD71) at the surface of CD133^high (Control siRNA) and CD133^low (CD133 siRNA) Caco-2 cells. Data represented mean ± s.e.m. obtained from three independent experiments. IgG2aκ immunoglobulins were used as negative control immunostaining. Dunnett's test: ***p<0.001. C) Analysis of the expression of AC133, TfR/CD71 and CHC within Caco-2 cells by immunocytochemistry combined with confocal laser scanning microscopy. Note the increase of CD71 expression while AC133 was depleted from the Control siRNA to the CD133 siRNA situation. Bar: 50 µm.</p

Specific siRNA mediated knockdown of CD133 within non-differentiated Caco-2 cells led to an increase in Tf intracellular accumulation but had no impact on Dx and CTB uptake.

<p>A) Immunofluorescence associated flow cytometric analysis of CD133 expression on non-differentiated Caco-2 cells treated with 30 nM of control or CD133-specific siRNA. Results are expressed as a percentage of the control treatment, representing the geomean fluorescence intensity levels obtained after AC133 immunostaining of cells treated with irrelevant siRNA (CD133^high Caco-2 cells); note the effective down regulation of CD133 when CD133-specific siRNA (CD133^low Caco-2 cells) was used. B–D) Flow cytometric analysis of intracellular uptake of Dx-FITC (B), CTB-FITC (C) and Tf-Alexa 488 (D) within CD133^high and CD133^low Caco-2 cells after 1 h of incubation at 37°C/5%CO₂. Results are expressed as percentage of control, thus representing the geomean fluorescence intensity levels obtained for cells treated with vehicle alone. Data represented mean ± s.e.m. obtained from three independent experiments. Dunnett's test: **p<0.01, ***p<0.001</p

Schematic representation of known IREs and nearest related stem loop sequence located in the 3′UTR of the CD133 mRNA (NCBI GenBank accession number NM_001145847.1, nucleotide sequence: from +3544 to +3574).

<p>Note the perfect match between the 5′-A<u>CAGAGU</u>U-3′ loop sequence of the CD133 mRNA and the one present in the TfR1 3′IRE. Note also the high discrepancy between hairpin structures, notably with the presence of secondary loops (dashed line) in the APP 5′IRE. A secondary loop is also present in the CD133 hairpin selected here. CDC14A: dual specificity protein tyrosine phosphatase. DMT1: divalent metal transporter 1. 75 kDa Fe-S: NADH dehydrogenase (ubiquinone) Fe-S protein 1. APP: Alzheimer amyloid precursor protein. Ft-H: ferritin heavy chain.</p

Iron supplementation down regulated AC133/CD133 expression in Caco-2 cells in a dose-dependent fashion.

<p>Combined immunofluorescence and flow cytometry was used to assess the impact of Fe-NTA treatment on AC133/CD133 expression in Caco-2 cells. A) Forward scatter (FSC, approximate cell size) and side scatter (SSC, cell complexity or granularity) profiles of vehicle only treated cells (control) versus Fe-NTA (800 µM) treated cells. B) Cell number distribution versus FITC-fluorescence (arbitrary units) in control and Fe-NTA (800 µM) treatments after IgG1 control or AC133 immunostaining. Dose-response histograms representing AC133/CD133 expression as a function of Fe-NTA (C), FeSO₄ (D), DFO (E) or CoCl₂ (F) treatments at different concentrations as indicated. GFI: geomean fluorescence intensity. Data represent mean ± s.e.m. of a triplicate obtained from one representative experiment. Dunnett's test: *p<0.05, **p<0.01, ***p<0.001.</p

Additional file 1: of The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion

Table S1. Cell populations analyzed in tumor tissues of glioma patients. Supplementary materials and methods. Description of patient characteristics, multiparametric flow cytometry, functional assay, t-SNE analysis and experiments with nanoparticles. (DOCX 22 kb