Journal: Nature communications

Article Title: Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis.

doi: 10.1038/s41467-017-02406-2

Figure Lengend Snippet: Fig. 2 Exosomal miRNA profile differentiates patients with MS from healthy controls a Heat map of miRNA expression profile in the exosomes from HC and patients with MS. RNA was extracted from exosomes, which were isolated from the plasma of four HC and four patients with MS. A 3D-Gene Human miRNA Oligo chip (TORAY) was used for microarray analysis. MiRNAs with assigned identification number lower than 500 and signal intensity higher than 100 were selected and clustered based on the expression patterns of the eight samples. b Volcano plot of miRNAs in the exosomes. The expression difference of each miRNA between MS-exosome and HC-exosome is plotted on the X axis in log2 scale. p value of the difference by t test is plotted on the Y axis. Blue dots represent miRNAs with signal intensity higher than 100. Green dots represent miRNAs with identification number lower than 500 and signal intensity higher than 100. Four miRNAs pointed by arrows (red dots) were identified as candidate miRNAs upregulated in patients with MS. They were selected from green dots. c PC analysis for the MS and HC samples based on the miRNA expression profiles, using the same data as in b. d,e Quantification of the candidate miRNAs by RT-qPCR. The total amount of RNAs in the exosomes from the same amount of plasma was also examined. Based on the clinical information, patients with MS were divided into those with RRMS or SPMS and into those in remission or relapse phase. An unpaired t test was used for statistical analysis. Error bars represent the mean ± s.d. s.d. standard deviation, n.s. not significant, PC principal component, A.U. arbitrary unit, RT-qPCR reverse transcription quantitative polymerase chain reaction, RRMS relapsing-remitting MS, SPMS secondary-progressive MS

Article Snippet: Using Plasma/Serum Circulating and Exosomal RNA Purification Kit (51000, Norgen Biotek Corp., ON, Canada), total RNA was extracted from the purified exosomes that were obtained from the same amount of the plasma.

Techniques: Expressing, Isolation, Clinical Proteomics, Microarray, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Virulence

Article Title: EGR1 regulates oral epithelial cell responses to Candida albicans via the EGFR- ERK1/2 pathway.

doi: 10.1080/21505594.2024.2435374

Figure Lengend Snippet: Figure 1. EGR1 is expressed following Candida albicans infection of oral epithelial cells. EGR family members whose expression was significantly different after C. albicans SC5314 infection of reconstituted human oral epithelium (ROE) via microarray analysis were studied in TR146 cell monolayers by RT-pcr (a) and Western blot (b). EGR1 overexpression was observed at both RNA and protein levels, whilst only RNA upregulation was detected for EGR3. c) EGR1 protein expression assessed in DOK cell line monolayers after 2 h of infection. d) EGR1 expression in nuclear and cytoplasmic fractions of TR146 monolayers after 2 h of infection. LSD1/KDM1A and Tubulin were used as controls of nuclear and cytoplasmic proteins, respectively. Data shown are the mean of three biological repeats, presented as fold change ± SEM relative to resting cells. Western blot images are representative of three biological repeats. Significance was calculated relative to resting cells, *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Knockdown of gene expression was performed using previously reported siRNAs for EGR1 [22], or commercially available siRNA pools for EGR1 (Santa-Cruz Biotech).

Techniques: Infection, Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Virulence

Article Title: EGR1 regulates oral epithelial cell responses to Candida albicans via the EGFR- ERK1/2 pathway.

doi: 10.1080/21505594.2024.2435374

Figure Lengend Snippet: Figure 2. Production of EGR1 is independent of major fungal virulence factors. EGR1 RNA (a) and protein (b) levels in TR146 epithelial cell monolayers following infection with a parental strain of C. albicans BWP17+cIP30, the yeast-locked mutant efg1Δ/Δ/ cph1Δ/Δ, a candidalysin-deficient ece1Δ/Δ strain, or an hgc1Δ/Δ mutant, which is unable to sustain hyphal growth. (c) Western blot analysis of EGR1 in TR146 cells treated with live wild-type C. albicans, heat-killed yeast, or zymosan (50 μg/mL) for 2 h. (d) EGR1 RNA expression in TR146 cells exposed to SC5314 or uv-killed SC5314 for 2 h. Data shown are the mean of three biological repeats, presented as fold change ± SEM relative to resting cells. Western blot images are representative of three biological repeats. Significance was calculated using one-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, relative to vehicle control; #P < 0.05, ##P < 0.01, ####P < 0.0001, relative to parental strain.

Article Snippet: Knockdown of gene expression was performed using previously reported siRNAs for EGR1 [22], or commercially available siRNA pools for EGR1 (Santa-Cruz Biotech).

Techniques: Infection, Mutagenesis, Western Blot, RNA Expression, Control

Journal: Virulence

Article Title: EGR1 regulates oral epithelial cell responses to Candida albicans via the EGFR- ERK1/2 pathway.

doi: 10.1080/21505594.2024.2435374

Figure Lengend Snippet: Figure 3. NF-κB, ERK1/2, Raf, and EGFR signalling control EGR1 increase upon fungal infection. (a) Western blot analysis of EGR1 in TR146 epithelial cell monolayers. Cells were treated with inhibitors targeting components of signalling pathways, or a vehicle control of DMSO, prior to infection with C. albicans for 2 h. Significance calculated relative to vehicle control (DMSO). (b) EGR1 protein levels in cells stimulated with live or heat-killed (HK) C. albicans or zymosan for 2 h after treatment with an ERK1/2 inhibitor or a vehicle control of DMSO. (c) Dectin-1, EGFR, and EphA2 were inhibited with laminarin, gefitinib, and dasatinib, respectively, and then cells were stimulated with live or heat-killed (HK) C. albicans or zymosan (50 μg/mL) for 2 h. Western blotting was then used to measure EGR1 expression. Significance was calculated relative to uninhibited, SC5314-infected cells. Data shown are the mean of three biological repeats, presented as fold change ± SEM relative to resting cells. Western blot images are representative of three biological repeats. Significance calculated using one-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Knockdown of gene expression was performed using previously reported siRNAs for EGR1 [22], or commercially available siRNA pools for EGR1 (Santa-Cruz Biotech).

Techniques: Control, Infection, Western Blot, Expressing

Journal: Virulence

Article Title: EGR1 regulates oral epithelial cell responses to Candida albicans via the EGFR- ERK1/2 pathway.

doi: 10.1080/21505594.2024.2435374

Figure Lengend Snippet: Figure 4. EGR1 regulates the production of cytokines in oral epithelial cells. TR146 cells were treated with a single siRNA or a pool of siRNAs targeting EGR1 and then infected with C. albicans. (a) Western blot analysis to confirm knockdown of EGR1 in OECs. (b) LDH activity quantification and (c) luminex analysis of culture supernatant at 24 h post-infection. Data shown are the mean of three biological repeats, presented as fold change ± S.E.M relative to resting cells. Western blot images are representative of three biological repeats. Significance relative to non-targeting siRNA control was calculated using one-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Knockdown of gene expression was performed using previously reported siRNAs for EGR1 [22], or commercially available siRNA pools for EGR1 (Santa-Cruz Biotech).

Techniques: Infection, Western Blot, Knockdown, Activity Assay, Luminex, Control