Absence of gD binding to HVEM affects levels of viral RNA but not DNA in infected RS cells.

RS cells were infected with 0.04 pfu/cell of KOS, KOS-Rid1, or KOS-Rid2 viruses. Infected cells were isolated 24 and 48 hr PI and total RNA and DNA from infected cells were isolated. qPCR were performed to detect LAT, gB, and gD DNA, while qRT-PCR were performed to detect LAT, gB, and gD RNA isolated from infected cells. In each experiment, the estimated relative copy number of LAT, gB or gD was calculated using standard curves generated from pGEM-LAT5317, pGem-gB1 or, pGem-gD1, respectively as described in Fig 1 (above). Each point represents the mean ± SEM from 3 experiments.</p

Absence of HVEM affects kinetics of induced reactivation in explanted TG from latently-infected mice.

HVEM-/- mice were ocularly infected with 2X105 pfu/eye of KOS-Rid1, KOS-Rid2, or control KOS after corneal scarification. Twenty-eight days PI, individual TGs were isolated and incubated in tissue culture media as in Fig 1 above. Results are plotted as the number of TG that reactivated daily. Numbers indicate average time that TG from each group first showed CPE ± SEM. For each group 20 TG from ten mice were used.</p

KOS-Rid1 infection affects T cell exhaustion in TG of latently-infected mice.

Total TG RNA from latently-infected mice used to measure LAT expression as described in Fig 1B, were used to measure CD8 and PD-L1 expression by qRT-PCR. CD8 and PD-L1 expression in naive mice were used as a control to estimate relative expression of each transcript in TG of latently-infected mice. GAPDH expression was used to normalize the relative expression of each transcript. Each point represents the mean ± SEM from 20 TG. Panels: A) CD8 transcript; and B) PD-L1 transcript.</p

Predicted glycoprotein D (gD) amino acid sequence of different HSV-1 strains.

gD amino acid sequences for HSV-1 strains 17, McKrae, KOS, ANGPath, and ANG are shown. The putative signal sequence is underlined. Sequence differences between the five HSV-1 strains are shown in “bold” font, and the gD binding site to HVEM is indicated in bold and green font. Sequence differences between AngPath with ANG, strain 17, McKrae and KOS is indicated in yellow font. (PDF)</p

Virus titers and reactivation in KOS-Rid2 infected mice.

A) Virus titers in the eyes of infected mice. WT C57BL/6 mice were infected ocularly with KOS-Rid2 or KOS control virus and the amount of infectious HSV-1 in tear films was determined daily by standard plaque assays as Fig 1. For each time point, the virus titer (Y-axis) represents the average of the titers from 20 eyes ± SEM; and B) Kinetics of induced reactivation in explanted TG from latently-infected mice. On day 28 PI, mice described above were euthanized and TG were harvested for explant reactivation as described in Fig 1D. Results are plotted as the number of TG that reactivated daily. Numbers indicate the average time that the TG from each group first showed CPE ± SEM. For each group 20 TG from ten mice were used.</p

Virus titers and latency-reactivation in KOS-Rid1 infected mice.

A) Virus titers in the eyes of infected mice. Twenty WT C57BL/6 mice were infected ocularly with KOS-Rid1 or KOS virus and the amount of infectious HSV-1 in tear films was determined daily by standard plaque assays as described in Materials and Methods. For each time point, the virus titer (Y-axis) represents the average of the titers from 20 eyes ± SEM; B and C) LAT and gB expression in TG of latently-infected mice. On day 28 PI, mice described above were euthanized and TG were harvested from latently-infected mice. QRT-PCR or qPCR was performed on RNA or DNA from individual mice TG. In each experiment, the estimated relative copy number of gB or LAT was calculated using standard curves generated from pGem-gB1 or pGEM-LAT5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 103 to 1011 copies of gB, or LAT then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of gB DNA and LAT RNA in the TG. Each point represents the mean ± SEM from 20 TG for each virus; and D) Effect of gD binding on kinetics of induced reactivation in explanted TG from latently-infected mice. Twenty WT C57BL/6 mice were ocularly infected with each virus as above and 28 days PI individual TG were harvested from each group. Each TG was incubated in tissue culture media and a 10 μl aliquot was removed from each culture daily and used to infect RS cell monolayers for 10 days as described in Materials and Methods. RS cells were monitored daily for the appearance of CPE for up to 5 days to determine the time of first appearance of reactivated virus from each TG. Results are plotted as the number of TG that reactivated daily. Numbers indicate the average time that the TG from each group first showed CPE ± SEM. For each group 40 TG from 20 mice were used.</p

Effect of ANG and ANGpath viruses on latency-reactivation in infected mice.

C57BL/6 mice were ocularly infected with HSV-1 strains ANG and ANGpath as well as KOS-Rid1, KOS-Rid2, KOS, and RE strains of HSV-1. TG from 20 mice per virus were isolated individually on day 28 PI. qPCR was performed to detect gB DNA (Panel A) and explant reactivation was used to detect reactivation (Panel B) as in Fig 1 above. TG from KOS-Rid2 infected mice was used for measuring gB DNA, while TG from KOS-Rid1 mice was used as a control for reactivation.</p

EPO and gene silencing of PRAS40 reduce caspase-3 activation during OGD.

<p>(<b>A</b>) EPO (10 ng/ml) was applied 1 hour prior to OGD in SH-SY5Y cells. Western blot analysis was performed for the cleaved fragments of caspase 3 with a antibody that identifies both the 19 kDa and 17 kDa fragments of caspase 3 hours following OGD exposure. PRAS40 siRNA transfection significantly reduced the expression of caspase 3 cleaved fragments during OGD exposure and during EPO administration with OGD exposure. (<b>B</b>) Quantitative results of the band density of the western blot analysis for caspase 3 fragments show that PRAS40 siRNA transfection significantly reduced the expression of caspase 3 cleaved fragments during OGD exposure and during EPO administration with OGD exposure (*<i>P</i><0.01 vs. untreated control; ^†<i>P</i><0.01 vs. OGD treated alone). Scrambled siRNA transfection did not alter the expression of the caspase 3 fragments during OGD alone or during EPO with OGD. PRAS40 siRNA transfection did not significantly alter caspase 3 fragment expression in untreated cells when compared to cells receiving no treatments. Each data point represents the mean and SD from 3 experiments.</p

EPO promotes PRAS40 phosphorylation through PI 3-K and Akt during OGD exposure.

<p>(<b>A</b>) Western blot analysis was performed for phosphorylated (p-)-PRAS40 (p-PRAS40, Thr²⁴⁶) in SH-SY5Y cells at 1, 3, or 24 hours (hrs) following OGD exposure. EPO (10 ng/ml) applied to cell cultures 1 hour prior to OGD maintained p-PRAS40 expression at significant levels over 24 hours following OGD (*<i>P</i><0.01 vs. Control; ^†<i>P</i><0.01 vs. OGD at corresponding time points). (<b>B</b>) EPO (10 ng/ml) applied to SH-SY5Y cells significantly increased the expression of phosphorylated (p-) p-PRAS40 3 hours later. EPO (10 ng/ml) applied to SH-SY5Y cultures 1 hour prior OGD significantly increased the expression of p-PRAS40 3 hours following OGD (*<i>P</i><0.01 vs. untreated control; ^†<i>P</i><0.01 vs. OGD treated alone). (<b>C</b>) EPO (10 ng/ml) was incubated with recombinant PRAS40 protein for 30, 60, and 180 min. No significant expression of phosphorylated (p-) PRAS40 was detected. (<b>D</b>) EPO (10 ng/ml) or EPO combined with the P I3-K inhibitors wortmannin (500 nM) or LY294002 (20 μM) were applied to SH-SY5Y cells and western blot analysis for phosphorylated (p-) p-PRAS40 and p-Akt1 (p-Akt1, Ser⁴⁷³) was performed 3 hours later. Wortmannin or LY294002 prevented the expression of p-PRAS40 and p-Akt1 during EPO (10 ng/ml) administration (*<i>P</i><0.01 vs. untreated control; ^†<i>P</i><0.01 vs. EPO treated alone). (<b>E</b>) EPO (10 ng/ml) was applied to SH-SY5Y cells 1 hour prior to OGD and western blot analysis for phosphorylated (p) p-PRAS40 and p-Akt1 was performed 3 hours following OGD. EPO significantly increased the expression of p-PRAS40 and p-Akt1 during OGD exposure. Wortmannin or LY294002 prevented the phosphorylation of PRAS40 and Akt1 during EPO administration following OGD (*<i>P</i><0.01 vs. OGD treated alone; ^†<i>P</i><0.01 vs. EPO/OGD). (<b>F</b>) Transfection of Akt1 siRNA prior to the application of EPO (10 ng/ml) in SH-SY5Y cells significantly limited the expression of Akt1 and significantly reduced the expression of phosphorylated (p-) p-PRAS40 3 hours after administration of EPO. Scrambled siRNA transfection did not alter the expression of Akt1 and p-PRAS40 during EPO application (*<i>P</i><0.01 vs. untreated control; ^†<i>P</i><0.01 vs. EPO treated alone). (<b>G</b>) Akt1 siRNA was transfected into SH-SY5Y cells prior to EPO (10 ng/ml) application and OGD exposure. Western analysis expression of phosphorylated (p-) p-PRAS40 and Akt1 was determined 3 hour following OGD. EPO (10 ng/ml) increased p-PRAS40 expression following OGD. Transfection of Akt1 siRNA significantly limited p-PRAS40 expression during OGD alone and during EPO treatment with OGD (*<i>P</i><0.01 vs. OGD; ^†<i>P</i><0.01 vs. EPO/OGD). In all cases above, each data point represents the mean and SD from 3 experiments.</p

DataSheet1_Comprehensive bioinformatics analysis to identify a novel cuproptosis-related prognostic signature and its ceRNA regulatory axis and candidate traditional Chinese medicine active ingredients in lung adenocarcinoma.docx

Lung adenocarcinoma (LUAD) is the most ordinary histological subtype of lung cancer, and regulatory cell death is an attractive target for cancer therapy. Recent reports suggested that cuproptosis is a novel copper-dependent modulated form of cell death dependent on mitochondrial respiration. However, the role of cuproptosis-related genes (CRGs) in the LUAD process is unclear. In the current study, we found that DLD, LIAS, PDHB, DLAT and LIPA1 in 10 differentially expressed CRGs were central genes. GO and KEGG enrichment results showed that these 10 CRGs were mainly enriched in acetyl-CoA biosynthetic process, mitochondrial matrix, citrate cycle (TCA cycle) and pyruvate metabolism. Furthermore, we constructed a prognostic gene signature model based on the six prognostic CRGs, which demonstrated good predictive potential. Excitedly, we found that these six prognostic CRGs were significantly associated with most immune cell types, with DLD being the most significant (19 types). Significant correlations were noted between some prognostic CRGs and tumor mutation burden and microsatellite instability. Clinical correlation analysis showed that DLD was related to the pathological stage, T stage, and M stage of patients with LUAD. Lastly, we constructed the lncRNA UCA1/miR-1-3p/DLD axis that may play a key role in the progression of LUAD and screened nine active components of traditional Chinese medicine (TCM) that may regulate DLD. Further, in vitro cell experiments and molecular docking were used to verify this. In conclusion, we analyzed the potential value of CRGs in the progression of LUAD, constructed the potential regulatory axis of ceRNA, and obtained the targeted regulatory TCM active ingredients through comprehensive bioinformatics combined with experimental validation strategies. This work not only provides new insights into the treatment of LUAD but also includes a basis for the development of new immunotherapy drugs that target cuproptosis.</p