Fig 2: Hcmv-miR-UL148D regulates XBP1 splicing and JNK phosphorylation: (A) HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor followed by staurosporine treatment. RNA was isolated and XBP1 mRNA spliced (sXBP1) and unspliced (uXBP1) were measured through qRT-PCR. The uXBP1 and sXBP1 expression level were normalized to that of GAPDH and fold change was calculated and plotted with respect to the untreated control. Results represent the mean ± SEM (n = 3 independent experiments) *, p<0.05, **, p<0.01. (B, C & D) Whole cell lysate was prepared from the hcmv-miR-UL148D mimic or inhibitor transfection and staurosporine treated cells. The expression of p-JNK, uXBP1 and sXBP1 were analyzed in different groups of cells through western blot and ß-actin was used as a loading control. The bands of the blots have been cropped with no further manipulation. The Relative protein expression levels in different groups of cells were analyzed through ImageJ software. Results represent the mean ± SEM (n = 3 independent experiments) **, p<0.01; ***, p< 0.001; ****P<0.0001; ns = non-significant.

Fig 3: Proposed mechanism of TNF-induced neuronal toxicity in the retina: TNF mediates NF-?B activation in astrocytes, which facilitates astroglial survival but also initiates production of neurotoxic pro-inflammatory factors such as TNF in the retina. The lack of NF-?B activity in RGCs in the presence of TNF, as well as sustained JNK activation, both contribute to RGC death, mostly by necrosis (necroptosis). Meanwhile, endogenous factors (called damage-associated molecular patterns or DAMPs) liberated from necrotic RGCs can activate astrocyte pattern recognition receptors (PRRs) and mediate production of neurotoxic pro-inflammatory factors such as TNF. Thus, TNF-mediated RGC necrosis can increase the neurotoxic astrocyte response and can therefore indirectly promote RGC death. t-JNK, transient JNK activation; s-JNK, sustained JNK activation.

Fig 4: Representative (A and C) Western blots and (B and D) densitometric analysis of JNK (total) and phosphorylated JNK (p-JNK) protein levels in the TNF-treated primary RGCs and astrocytes revealed sustained JNK activation in TNF-treated RGCs, while primary astrocytes demonstrated transient JNK activation. Whole-cell extracts (25 µg) were collected from zero to 90 min as well as 2, 3 and 24 h after TNF treatment, and the JNK state was tested by Western blot analysis using an antibody against JNK and p-JNK. The JNK and p-JNK intensities were normalised to GAPDH and the results expressed as percentages of the corresponding values in the cells treated for 0 min (0 m). Values are means ± SEM;n = 3.

Fig 5: Interaction with E. coli O111:B4 lipopolysaccharide and immunomodulatory activity of PN5. (A) CD analysis of the binding structure of AMPs with lipopolysaccharide (LPS); (B) effect of PN5 (2, 4, and 8 µM) on LPS-induced TNF-a production. The concentrations of TNF-a in the culture supernatants were determined by ELISA. (C) Western blot of TNF-a, TLR4, and ß-actin expression. LPS-pretreated RAW 264.7 cells were cultured with PN5 at different concentrations for 24 h. (D) Graphs were generated by quantifying the Western blots using ImageJ software. (E) Immunofluorescence staining showed that PN5 inhibited TNF-a expression induced by E. coli LPS. (F) The Western blot was performed for NF-?B and MAPK signaling activation in RAW264.7 cells. Intracellular NF-?B (p65), I?Ba, p38, ERK, and JNK signals were confirmed. Values represent the mean ± SEM from three individual experiments. **, P < 0.01, and ***, P < 0.001, by unpaired t test.