Journal: Cellular and Molecular Life Sciences: CMLS
Article Title: The immune sensitivity caused by DUSP11, an RNA 5′-end maturation phosphatase, is adjusted by a human non-coding RNA, nc886
doi: 10.1007/s00018-025-05607-x
Figure Lengend Snippet: The increased nc886 by DUSP11 KD suppresses innate immune responses. siRNAs were transfected at 0 h, PAMP (or vehicle control) was treated at 36 h, and cells were harvested at 48 h. A qRT-PCR of IFN-β. y-axis: 2 −ΔΔCt value with the leftmost bar set to 1. –RT reactions were performed in parallel and subtracted from + RT values. The p-value in red letters represents the statistical significance of the siDUSP11/siControl values between the two PAMP-treated cell lines. More specifically, three siDUSP11/siControl values of 293 T-vector (the ratio of bar 4 to bar 2) and those of 293 T-U6:nc886 (the ratio of bar 8 to bar 6) were calculated and were subjected to Student's t-test. B A dot plot showing the relative TPM values of 42 ISGs that were upregulated by more than twofold in siDUSP11 with PAMP compared to siControl without PAMP in 293 T-vector cells. The TPM values of these ISGs were normalized to siControl in 293 T-vector without PAMP as shown in Fig. 3A. The p-value in red letters represents the statistical significance of the difference between the two cell lines and was calculated as follows: siDUSP11/siControl values for these 42 ISGs were calculated and the 42 ratio values of 293 T-vector were analyzed against 42 values of 293 T-U6:nc886 using Student's t-test. C A heat map depicting TF activity from MSigDB analysis. Z-scores for each of four samples indicate the change of a TF activity upon PAMP-treatment. Among 1137 TFs, those of Z-score > + 4 in 293 T-vector cells with siDUSP11 (= column 2) were selected and displayed. D A heat map of Z-scores for KEGG pathways (selected from total 186). All other descriptions are the same as panel C , except for the Z-score cut-off (> + 3 here) and the color scale (see scale bar below). E Northern hybridization and Western blot of indicated genes. All descriptions are the same as Fig. C, except that EtBr staining is shown for equal loading. F A graph showing the expression of PAMP-triggered IFN-β and ISGs calculated from 2 −ΔΔCt values of qRT-PCR. The fold-induction values of a siDUSP11 sample relative to the corresponding siControl sample were calculated (y-axis). siRNAs were transfected at 0 h, PAMP (or vehicle control) was treated at 40 h, and cells were harvested at 48 h. G Western blot of DUSP11 (top panel), quantification (bottom panel), and qRT-PCR of nc886 (bottom panel), after KSHV infection. At 24 h post-infection, > 90% of the cells were infected. In the lower graph, nc886 expression is 2 −ΔΔCt values (left y-axis), with the value from Huh7 cells with mock infection at 6 h set to 1. DUSP11 and β-actin bands in the top Western blot were quantified using Image J, to calculate DUSP11/ β-actin, which is a normalized DUSP11 expression value. The ratio of normalized values between KSHV and mock infection (right y-axis) in Huh-7 cells was shown at each time point, with the ratio at 6 h set to 1. H nc886 expression, as measured by qRT-PCR, at day 1, 3, and 5 after infection of rKSHV.219 at the indicated multiplicity of infection (MOI). LEC are KSHV-permissive (upper panel); BEC are non-permissive and were included as a negative control (lower panel). I KSHV infectivity at each indicated MOI (x-axis) at 24 h post-infection, as measured by GFP expression using flow cytometry. J A dot plot showing the expression of 81 ISGs (that were upregulated in DUSP11-low patients) between nc886-low and nc886-high HNSCC patients. Each dot indicates an average of TPM values (in three nc886-low patients or in seven nc886-high patients) that were calculated from RNA-seq data
Article Snippet: KSHV was infected to primary LEC and BEC, which were procured from Lonza (Walkersville, MD) and were cultured in the EGMTM−2 MV medium kit (Lonza), as previously described [ ].
Techniques: Transfection, Control, Quantitative RT-PCR, Plasmid Preparation, Activity Assay, Northern Blot, Hybridization, Western Blot, Staining, Expressing, Infection, Negative Control, Flow Cytometry, RNA Sequencing
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