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