Journal: Cell Death & Disease

Article Title: SMAC mimetic drives microglia phenotype and glioblastoma immune microenvironment

doi: 10.1038/s41419-024-07056-z

Figure Lengend Snippet: A Representative experiment of 3 independent experiments of IAP expression levels analyzed by Western blotting after 72 h of vehicle or SMg treatment in C8B4 microglia cell line. B Quantification of GL261-DsRed spheroids area upon vehicle ( n = 10) and SMg treatment ( n = 9) and in the presence or not of the C8B4 cells ( n = 10). Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p -value: *** p < 0.0005. C Concentrations of CD14 and CCL17/TARC quantified in the supernatant of C8B4 and GL261-DsRed co-cultures by ELISA assay after 72 h of vehicle or SMg treatments ( n = 6 independent experiments). Statistical analyses were performed by using Mann–Whitney test; alpha = 0.05, bilateral p-value: ** p < 0.005. D Quantification of GL261-DsRed spheroids area in the presence of the C8B4 cells expressing ML-IAP (siCTRL) or down-expressing ML-IAP (siML-IAP) ( n = 10) after 72 h of treatment vehicle ( n = 10) and SMg treatment ( n = 9). Data from three independent experiments. Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p -value: * p < 0.05 ; ** p < 0.005 ; **** p < 0.0001. E Quantification of GL261-DsRed spheroid area in the presence of C8B4 cells treated with vehicle and SMg for 72 h. C8B4 cells were pre-treated for 24 h with ZVAD (vehicle n = 23, SMg n = 23) and TNFαi (vehicle n = 27, SMg n = 23) or without pre-treatment (vehicle n = 28, SMg n = 25). Statistical analyses were performed by using ANOVA test and ANOVA post-hoc Tukey test; alpha = 0.05, bilateral p -value: *** p < 0.0005. F Representative experiment ( n = 2) of IAP expression levels analyzed by western blotting after 72 h of vehicle or SMg treatment in C8B4 microglia cell line. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. G Representative experiment ( n = 2) of expression levels of p65, phospho-p65, IκBα, phospho IκB, caspase-3, cleaved caspase-3 analyzed by Western blotting in C8B4 microglia cell line after 24 h of vehicle or SMg treatment. H Representative experiment out of 3 experiments of expression levels of CD206 and iNOS analyzed by Western blotting after 24 h of vehicle or SMg treatment. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. Expression level of actin β served as loading control. I iNOS/CD206 ratio in C8B4 microglia cell line after 24 h of vehicle or SMg treatment. C8B4 cells were pre-treated for 24 h with ZVAD and TNFαi. Quantification was performed from 3 independent experiments using ImageJ software and data presented were normalized to actin β expression. iNOS/CD206 ratio fold changes were normalized on vehicle condition. B – E , I Bar graphs represent mean ± s.e.m.

Article Snippet: Supernatants were collected after 72 h of treatment and processed using the proteome profiler human XL cytokine array kit (R&D systems, #ARY022B) or the ELISA kits Quantikine ELISA Mouse CCL17/TARC Immunoassay (R&D systems, MCC170) and Mouse CD14 Immunoassay (R&D systems, MC140) accordingly to the manufacturer protocol (Data represented were normalized to reference spot expression in array membrane).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control, Software

Journal: The Journal of investigative dermatology

Article Title: Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3.

doi: 10.1038/sj.jid.5701102

Figure Lengend Snippet: Figure 3. HDAC inhibition in keratinocytes increases CD14, CYP24A1, and cathelicidin, but not IL-8. (a) NHEKs were stimulated with 1,25D3 (108 M), in the presence of butyrate (BUT; 2 mM) for 24 hours. Transcript abundance of 1,25D3-regulated CD14 and CYP24A1 was determined by qPCR (**Po0.01; Student’s t-test). (b) (Upper panel) Western blot of CD14 protein expression in NHEK stimulated with control (lane 1), 1,25D3 (108M; lane 2), BUT (2 mM; lane 3), or the combination of 1,25D3 and BUT (lane 4) for 24 hours. A band at approximately 50 kDa was detected corresponding to CD14 protein. Membranes were reprobed with an anti-a tubulin antibody and protein abundance quantified by densitometry (right panel). (c) NHEKs stimulated with 1,25D3 (108 M), the TLR2/6 ligand Malp-2 (0.1 mg ml1), or the combination, and cathelicidin or IL-8 transcript abundance determined by qPCR (*Po0.05). (d) HDAC inhibition by BUT did not enhance IL-8 transcript induced by Malp-2.

Article Snippet: After washing in Tris-buffered saline 0.1% Tween-20, membranes were stained with a rabbit polyclonal anti-acetylated histone H4 antibody (Cell Signaling) or a mouse monoclonal anti-CD14 antibody (R&D Systems, Minneapolis, MN), washed again in Tris-buffered saline 0.1% Tween-20 and reprobed with a HRP-coupled goat anti-rabbit antibody (DakoCytomation, Glostrup, Denmark).

Techniques: Inhibition, Western Blot, Expressing, Control, Quantitative Proteomics

Journal: The Journal of investigative dermatology

Article Title: Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3.

doi: 10.1038/sj.jid.5701102

Figure Lengend Snippet: Figure 5. Increased cathelicidin by 1,25D3 is dependent on the VDR and VDR coactivator SRC3 in keratinocytes. (a) Silencing of the VDR blocks cathelicidin induction in keratinocytes. NHEKs were transfected with siRNA oligonucleotides for VDR, DRIP205, SRC3, or control siRNA and stimulated with increasing concentrations of 1,25D3. Expression was normalized to the housekeeping gene L19, which is unaffected by siRNA transfection. (b) Efficiency of siRNA silencing was evaluated by western analysis. (c) siRNA suppression of the VDR coactivator SRC3, but not DRIP205, blocked induction of cathelicidin by 1,25D3. (d) NHEK transfected with siRNA oligonucleotides for SRC3 or DRIP205 and stimulated with 1,25D3 (108 M), butyrate (2 mM), or the combination. Again, silencing of SRC3 blocks induction of cathelicidin and CD14, whereas silencing of DRIP205 has no effect. HDAC inhibition by butyrate is not sufficient to reverse this effect. Data shown are means (7SD) of results from a single representative experiment performed in triplicates. These experiments were repeated using at least two different batches of primary keratinocytes to confirm reproducibility.

Article Snippet: After washing in Tris-buffered saline 0.1% Tween-20, membranes were stained with a rabbit polyclonal anti-acetylated histone H4 antibody (Cell Signaling) or a mouse monoclonal anti-CD14 antibody (R&D Systems, Minneapolis, MN), washed again in Tris-buffered saline 0.1% Tween-20 and reprobed with a HRP-coupled goat anti-rabbit antibody (DakoCytomation, Glostrup, Denmark).

Techniques: Transfection, Control, Expressing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Intestinal Barrier Loss Enables Microbiota-Mediated Purinergic Suppression During Malaria

doi: 10.64898/2026.01.22.701153

Figure Lengend Snippet: ( A ) Resistant and Susceptible mice were treated with 2% DSS for 7 days. Body weight percentage relative to initial weight. ( B ) Representative H&E staining of small intestine sections at day 5 post- P. yoelii infection showing mucosal desquamation (arrow), inflammatory infiltrate in the mucosa (*) and submucosa ($) (scale bar: 200µm) and pathology scores (right). ( C ) Immunofluorescence of the distal small intestine stained for Occludin (purple), ZO-1 (green), and DAPI (blue). White bar 20 µm. Right: Median fluorescence of 40x objective images of intestine or region of interest when the lumen was abundant was measured using Zen Blue Lite. Mean ± SEM. ( D-E ) Longitudinal serum quantification of (D) LBP and (E) sCD14 by ELISA on days 0, 7, 14, and 21 p.i. Statistics: Mann-Whitney (pathology/IFA) and One-way ANOVA/Mixed-effects (longitudinal). comparisons p>0.05: a – Resistant vs. Resistant 2% DSS; b - Resistant 2% DSS vs. Susceptible; c – Susceptible to Susceptible 2% DSS; d – Resistant 2% DSS to Susceptible 2% DSS; e – Resistant to Susceptible 2% DSS. *p<0.05, **p<0.01, ***p<0.001. DSS data are representative of 4 experiments. Pathology scores and sections are accumulated from two independent experiments.

Article Snippet: Serum levels of lipopolysaccharide-binding protein (LBP) (Hycult Biotech, Cat: HK20502), soluble CD14 (R&D Systems, Cat: DY982), angiopoietin-2 (Fisher Scientific, Cat: MANG20), and ICAM-1 (Fisher Scientific, Cat: EMICAM1ALPH) were quantified according to the manufacturers’ instructions.

Techniques: Staining, Infection, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Glia

Article Title: LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14.

doi: 10.1002/glia.22657

Figure Lengend Snippet: FIGURE 8: Role of TLR4 and CD14 in LPS-induced mouse astro- cyte TNFa production: Mouse astrocyte cultures were treated with siRNA specific for TLR4 or CD14, or with non-specific con- trol siRNA (siCtr) and then treated with LPS or not (Ctr), as described in the Materials and Methods section. (A, B) Western blot for TLR4 and CD14 demonstrate the efficiency of RNAi. (C, D) The amount of TNFa protein release in each culture was meas- ured by ELISA and this demonstrated that LPS-induced mouse astrocyte TNFa production was significantly inhibited by TLR4 siRNA. Less robust, but significant inhibition was observed with CD14 siRNA. ***P < 0.001 and *P < 0.05 (vs. siCtr). Results are representative of two independent experiments.

Article Snippet: The mouse CD14 27mer siRNA duplex was purchased from OriGene Technologies, and was transfected using the SiPORT NeoFX Transfection reagent (Life Technologies/Invitrogen) according to the manufacturer’s instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Glia

Article Title: LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14.

doi: 10.1002/glia.22657

Figure Lengend Snippet: FIGURE 7: Differential expression of cell-associated and soluble CD14 in mouse and human astrocytes: (A) Western blot analysis of total cell lysates from mouse astrocyte cultures (Ctr or LPS) demonstrating expression of CD14 protein. Mouse astrocyte CD14 expression is greatly upregulated following LPS stimula- tion (22 h). The blot was incubated with a rat IgG against anti- mouse CD14 (BD Biosciences) showing a single band of 50 kDa protein. Positive control (Std.CD14, first lane “S”) was 4 ng of mouse CD14-Fc fusion protein (85–95 kDa, R&D Systems). In addition, mouse astrocytes also expressed MD2 (26 kDa, pre- dicted size 18 kDa) and TLR4 (95 kDa), the amounts of which did not change after LPS stimulation (right panel). (B) Western blot analyses of human astrocyte cultures (6 LPS) show lack of CD14 expression. The blot was incubated with a rabbit polyclo- nal IgG against anti-human CD14 (Sigma) showing a single band of 50 kDa protein. Positive control was 5 ng of recombinant human CD14 (R&D Systems, first lane “S”), as well as control and LPS-stimulated primary human fetal microglia (last two lanes). MD2 and TLR4 protein expression was also detectable in human astrocytes (right panel). b-actin was used to ensure equal amounts of protein loading. (C) ELISA was performed to deter- mine the amounts of sCD14 in culture supernatants of mouse astrocytes and BV2 cells (left panel) and human astrocytes and microglia (right panel), using two separate commercially available ELISAs (R&D Systems, see Materials and Methods). Data shown are mean 6 SD (n 5 3). Mouse astrocyte sCD14 is barely detecta- ble but shows significant increase after LPS stimulation. BV2 cells show large amounts of basal production of sCD14 which is increased after LPS stimulation (**P < 0.01 vs. Ctr). ELISA for human sCD14 was performed using culture supernatants of human astrocytes and microglial cultures and this shows detecta- ble sCD14 only in microglial cultures. Human microglial sCD14 production is also significantly increased after LPS stimulation (*P < 0.05). Results are representative of three independent experiments.

Article Snippet: The mouse CD14 27mer siRNA duplex was purchased from OriGene Technologies, and was transfected using the SiPORT NeoFX Transfection reagent (Life Technologies/Invitrogen) according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Incubation, Positive Control, Recombinant, Control, Enzyme-linked Immunosorbent Assay

Journal: Glia

Article Title: LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14.

doi: 10.1002/glia.22657

Figure Lengend Snippet: FIGURE 9: Astrocytes in vivo expression CD14 in LPS-injected mouse brains: CD14 immunohistochemistry of the mouse brains that are injected with LPS (left column) or PBS (right column) 24 h before sacrifice. Robust CD14 immunoreactivity is observed in the brains of LPS-injected mice (A) at the site of injection (deep gray matter, thalamus and basal ganglia) whereas PBS injected brains showed no parenchymal CD141 staining (B). Rare vessel-associated CD141 cells are also noted (C). Double labeling with GFAP (blue) shows some of the CD141 cells (brown) are also GFAP1 (D: arrows). Double labeling of PBS-injected mouse brain showing only GFAP stain (E). (F, G) Double label immunofluorescence shows both microglia and astrocytes are CD141 in intracerebral LPS-injected mice: Immunofluores- cence stain for CD14 (red) and cell specific markers, GFAP (astrocytes, green) or Iba-1 (microglia, green), was performed on paraffin- embedded sections of mouse brains injected with LPS, as described in the Materials and Methods section. Shown are examples of astro- cytes (GFAP1) and microglia (Iba-11) that also label for CD14 in these brains. DAPI (blue) was used for nuclear staining. CD14 immuno- reactivity appears cytoplasmic, membrane, as well as granular in these cells. Scale bars represent 20 mm in A, B, D and E, 5 mm in C, 25 mm in G, and 10 mm in F.

Article Snippet: The mouse CD14 27mer siRNA duplex was purchased from OriGene Technologies, and was transfected using the SiPORT NeoFX Transfection reagent (Life Technologies/Invitrogen) according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Injection, Immunohistochemistry, Staining, Labeling, Immunofluorescence, Membrane