sj-doc-2-inq-10.1177_00469580231155296 – Supplemental material for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies

Supplemental material, sj-doc-2-inq-10.1177_00469580231155296 for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies by Jiayun Lu, Pei Xu, Jinjin Ge, Haiyan Zeng, Weiqun Liu and Peifeng Tang in INQUIRY: The Journal of Health Care Organization, Provision, and Financing</p

sj-doc-1-inq-10.1177_00469580231155296 – Supplemental material for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies

Supplemental material, sj-doc-1-inq-10.1177_00469580231155296 for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies by Jiayun Lu, Pei Xu, Jinjin Ge, Haiyan Zeng, Weiqun Liu and Peifeng Tang in INQUIRY: The Journal of Health Care Organization, Provision, and Financing</p

sj-doc-3-inq-10.1177_00469580231155296 – Supplemental material for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies

Supplemental material, sj-doc-3-inq-10.1177_00469580231155296 for Analysis of Factors Affecting Psychological Resilience of Emergency Room Nurses Under Public Health Emergencies by Jiayun Lu, Pei Xu, Jinjin Ge, Haiyan Zeng, Weiqun Liu and Peifeng Tang in INQUIRY: The Journal of Health Care Organization, Provision, and Financing</p

Interaction of PLIC-1 and Trif.

<p><b>A</b>. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. <b>B</b>. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.</p

Reduction of PLIC-1 level enhanced TLR3-Trif-mediated signaling.

<p><b>A</b>. Knocking down PLIC-1 by shRNA. 0.1 µg of HA-PLIC-1 was transfected with 0.1,1,2,4 µg of indicated shRNA constructs in 293T cells. Western blot was performed to detect HA-PLIC-1. <b>B</b>. shRNA 1602, 733, the scramble construct, and LMP (targeting mouse PLIC-1) were expressed in 293T cells. Knockdown of endogenous PLIC-1 was verified by western blotting using an anti-PLIC-1 antibody. <b>C</b>. Indicated combinations of constructs were transfected into 293T cells. 48 hours following transfection, luciferase activity was determined and normalized against renilla luciferase. <b>D</b>. Reduction of PLIC-1 by shRNA 1602 enhanced the activation of IFN-β promoter activity by poly I∶C stimulation. 293T cells were transfected with indicated plasmids and further stimulated with poly I∶C (40 µg/ml) for 24 hours prior to lysis. Notice that transfection with shRNA construct itself did not activate the IFN-β promoter.</p

Overexpression of PLIC-1 inhibited TLR3 signaling.

<p><b>A</b>. TLR4/MD2 stable HEK cells were transfected with a NF-κB driven luciferase reporter plasmid and increased amount of HA-PLIC-1. 24 hours post-transfection, cells were stimulated with LPS (100 ng/ml) and incubated for additional 24 hours prior to luciferase reporter assay. <b>B</b>. 293T cells were transfected with pISRE-Luc (100 ng), CD4-TLR4 (50 ng), and increased amount of HA-PLIC-1 for luciferase reporter assay. <b>C</b>. 293T cells were transfected with Trif, κB-Luc, and increased amount of HA-PLIC-1 plasmids. A total of 2 µg DNA was transfected in each well in a 12-well plate. 48 hours following transfection, luciferase activity was determined. Data was normalized against renilla luciferase which was transfected as an internal control. Experiments in <b>D</b> were carried out similarly as in <b>C</b> except that p125-Luc, plasmid was used. <b>E</b>. p125-Luc reporter was transfected along with Trif and YFP-PLIC-1 plasmids. <b>F</b>. Experiments were carried out similarly as in <b>D</b> except that 0.3 µg IRF3 expression plasmid was added to stimulate p125-luc reporter. <b>G</b>. HEK cells stably expressing TLR7 were transfected with indicated constructs and stimulated with TLR7 agonist, imiquimod (10 µg/mL) for 24 hours followed by luciferase reporter assay. <b>H</b>. 293T cells were transfected with MAVS and increasing amount of PLIC-1 for reporter assays. <b>I</b>. 2.5×10⁵ J774A.1 cells were transfected with indicated plasmids. Poly I∶C(40 µg/ml) was added to cells for additional period of 24 hours prior to measuring luciferase activity. Data were normalized by using renilla luciferase as an internal control. The error bars in above experiments represent the standard deviation accumulated from at least three experiments.</p

PLIC-1 co-localized with Trif.

<p>The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone (<b>A</b>) or with 0.1 µg Flag-Trif (<b>B</b>) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. <b>C</b>. 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. <b>D</b>. 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.</p

PLIC-1 and Trif co-localized with LC3.

<p>The human hepatoma cell line Huh7.5.1 cells were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 and RFP-LC3 (<b>A</b>), or 0.1 µg YFP-Trif and RFP-LC3 in HEK (<b>B</b>) or Huh7.5.1 (<b>C</b>) cells in a 24-well plate format. 24 hours post-infection, cells were fixed and imaged using a Zeiss Meta LSM510 microscope. In all figures, co-localization was indicated as yellow dots in the merged images.</p

Overexpression of PLIC-1 decreased Trif abundance in a Nocodazole-sensitive manner.

<p><b>A</b>. 293T cells were transfected with indicated plasmids. 48 hours post-transfection, lysates were prepared and subjected to western blot analysis. HCV NS3/4A was known to cleave Trif and was included as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021153#pone.0021153-Li1" target="_blank">[8]</a>. However, it only marginally reduced the level of Trif. PLIC-1 has no effect on the expression of a protein named Flap, which was included as a negative control here. <b>B</b>. 293T cells were transfected with 0.2 µg Flag-Trif and indicated amount of HA-PLIC-1 plasmid for 24 hours. Cells were then treated with 30 µM Nocodazole or MG132 (50 µM) for 6 hours prior to cell lysis. Western blotting was performed to quantitate the level of Flag-Trif, HA-PLIC-1. A non-specific band was indicated as the loading control.</p

PLIC-1 blocked TLR3-induced production of IFN-α during NDV infection.

<p>The parental human lung cancer cell line A549 or the A549 stably expressing shRNA #1602 and 733 (specifically target hPLIC-1) or the negative control shRNA (LMP) were stimulated with poly I∶C (100 µg/ml) for 24 hours and then infected by NDV-GFP (MOI 1). 12 hours post-infection, images were taken to visualize cells that had been productively infected by the green virus. Recombinant IFN-α was added as a positive control to suppress NDV infection. Percentages of positive infection were calculated by averaging the number of green cells over that of blue cells (DAPI staining of nuclei) in 4 fields. The numbers were indicated in the upper right corner of each merged image.</p