Journal: eLife

Article Title: Heat shock factor regulation of antimicrobial peptides expression suggests a conserved defense mechanism induced by febrile temperature in arthropods

doi: 10.7554/eLife.101460

Figure Lengend Snippet: ( A ) Venn diagram showing the downregulated genes by RNA-Seq. RNA-seq was performed 24 hr after WSSV infection (NCBI SRA database accession number PRJNA1110613). ( B ) Heatmap showing the differential expression of downregulated genes encoding potential effector molecules. The color bar indicates the gradient of normalized expression levels. ( C ) mRNA transcription levels of SWD in hemocytes and gills. mRNA transcription levels of SWD in the hemocytes and gills of LvHSF1-silenced shrimp under WSSV challenge. ( D ) Dual-luciferase reporter assays. Dual-luciferase reporter assays were performed to analyze the effects of overexpression of LvHSF1 on the promoter activities of SWD in Drosophila S2 cells in a dose-dependent manner. Protein expression of LvHSF1 was detected with anti-HA Ab, with β-actin used as a protein loading control (panel d). ( E ) Schematic diagram of the SWD promoter regions. Schematic diagram of the SWD promoter regions in the luciferase reporter gene constructs. The HSF1 binding motif sites are shown in red rectangles. ( F ) Dual-luciferase reporter assays with mutated HSF1 binding motifs. Dual-luciferase reporter assays were performed to analyze the effects of overexpression of LvHSF1 on the promoter activities of SWD with mutated HSF1 binding motifs. Protein expression of LvHSF1 was detected with anti-HA Ab, with β-actin used as a protein loading control (panel f). ( G ) Analysis of the SWD promoter. The HSF1 binding site was analyzed using the online JASPAR database. ( H ) EMSA assay. LvHSF1 protein interaction with HSF1 binding sites from the SWD promoter was analyzed in vitro by EMSA assay. Competition assays were performed in the presence of excess unlabeled probes. Statistical significance was calculated using the Student’s t -test (**p<0.01, *p<0.05). All experiments were conducted with three biological replicates, consistently yielding similar results. Figure 5—source data 1. Numerical source data for graphs shown in . Figure 5—source data 2. TIF file with original western blots and boxes indicating the relevant bands shown in . Figure 5—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Drosophila S2 cells (ATCC CRL-1963) were cultured at 28 °C in Schneider’s Insect Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY).

Techniques: RNA Sequencing, Infection, Quantitative Proteomics, Expressing, Luciferase, Over Expression, Control, Construct, Binding Assay, In Vitro, Western Blot

Journal: eLife

Article Title: Heat shock factor regulation of antimicrobial peptides expression suggests a conserved defense mechanism induced by febrile temperature in arthropods

doi: 10.7554/eLife.101460

Figure Lengend Snippet: ( A ) Drosophila S2 cells were seeded in six-well plates and incubated at 27°C or 30°C for 24 hr, then the cells were infected with DCV at a multiplicity of infection (MOI) of 10 at 27°C or 30°C for another 24 hr. The cytopathic signs of S2 cells were observed in an inverted fluorescence microscope. ( B ) The transcriptional expression of DCV in S2 cells with or without DCV infection (black arrow). ( C ) The protein expression of DCV in S2 cells with or without DCV infection. Figure 8—figure supplement 1—source data 1. Numerical source data for graphs shown in . Figure 8—figure supplement 1—source data 2. TIF file with original western blots and boxes indicating the relevant bands shown in . Figure 8—figure supplement 1—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Drosophila S2 cells (ATCC CRL-1963) were cultured at 28 °C in Schneider’s Insect Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY).

Techniques: Incubation, Infection, Fluorescence, Microscopy, Expressing, Western Blot

Journal: eLife

Article Title: Heat shock factor regulation of antimicrobial peptides expression suggests a conserved defense mechanism induced by febrile temperature in arthropods

doi: 10.7554/eLife.101460

Figure Lengend Snippet: Elevated temperature induces a robust expression of LvHSF1, which in turn specifically induces the expression of the antimicrobial peptide (SWD) in shrimp. The SWD directly binds to WSSV envelope proteins and inhibits WSSV replication ( left panel ). Additionally, elevated temperature induces the expression of DmHSF1, which upregulates the expression of Atta, CecA, and Def in Drosophila S2 cells, subsequently restricting the replication of DCV ( right panel ). These findings highlight the roles of HSF1 beyond the classical heat shock response, mediating the thermal regulation of immunity and facilitating the innate immune system’s response against viruses.

Article Snippet: Drosophila S2 cells (ATCC CRL-1963) were cultured at 28 °C in Schneider’s Insect Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY).

Techniques: Expressing