Fig 2: A: Quantification of CDKN1B mRNA expression by qPCR. Total RNA was extracted from peripheral blood. White blood cells from peripheral blood were spun down, lysed, and total RNA was purified using the Qiagen RNA easy extraction kit (cat. 52304). RNA was reversed transcribed, and quantitative PCR was carried using the Qiagen one-step SYBR green RT-PCR kit. Q-PCR primers (primer sequences are detailed in Supp. Fig. S4B) spanned the 3' end of exon 1 (forward) of CDKN1B to the 5' end of exon 2 (reverse). The amount of CDKN1B detected was normalized against the ubiquitously expressed ABL1 gene and expressed as a relative value Ratio (ABL1/CDKN1B) = 2CT(ABL1)-CT(CDKN1B). B: Quantification of CDKN1B minimal promoter activity. Region incorporating the 5'UTR of CDKN1B (encompassing the -79 and -73 residues) were amplified from patient DNA and subcloned into the pGL3 basic luciferase reporter vector (Promega cat. E1751). One microgram of pGL3-79C-73G, pGL3-79T-79G, pGL3-79C-73A, or an empty pGL3 vector was cotransfected with 20 ng of pRL-TK (Renilla reporter construct, Promega cat. E2241) into HEK293T cells with the ProFection mammalian transfection system (Promega cat. E1200) according to the manufacturer's protocol. Cells were transfected for 48 hr, followed by a 12 hr period of serum starvation. The dual-luciferase reporter assay system (Promega cat. E1910) was used to assay relative luciferase activity. The data represent the mean of three independent experiments with standard error bars. C: Western blot analysis of CDKN1B from white blood cells. Protein samples were extracted from peripheral blood samples by first isolating white blood cells through centrifugation. These cells were subsequently lysed using RIPA lysis buffer (50 mM Tris HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) and run on a 12% SDS-PAGE. Western blotting was carried out using an anti-p27 antibody (BD Biosciences cat. 610242) and an anti-ß-tubulin antibody (Cell Signaling cat. 2128) (left). Relative intensity of the signal obtained from the western blot was quantified using ImageJ (NIH). Bar chart shows the average of signals (and standard error) quantified from three blots normalized to ß-tubulin level in each independent blood sample (right).

Fig 3: Matador-Glo assay can be coupled with Matador assay. (A) K562 cells stably expressing both Luc146-1H2 and Nluc (K562-Luc146-1H2/Nluc) were treated with digitonin (30 µg/ml) for 90 min. Post-incubation supernatants was carefully transferred to a 384-well plate to measure Luc146-1H2 activity using D-luciferin as a substrate (Matador-Glo assay) and Nluc activity using native coelenterazine as a substrate (Matador assay), respectively. (B) Dual-Luciferase Reporter assay kit from Promega (E1910) was used to measure samples from the same experiment as (A). Luc146-1H2 activity was measured first by adding Luciferase Assay Reagent II (LAR II). After quantifying the Luc146-1H2 activity, the Nluc activity was measured by adding Stop & Glo reagent to the same wells. The Stop & Glo Reagent both quenches the Luc146-1H2 signal and initiates the Nluc luminescence. The values shown are mean ± SE of a representative experiment performed in triplicate for at least two times. Statistically significant differences are shown by asterisks (***) at a level of P < 0.001 and (****) at a level of P < 0.0001.

Fig 4: Identification of interactions between RHDV nonstructural proteins and host factors of RCs. A NCL siRNA inhibited the formation of the RHDV RC. After HA tag affinity purification, the eluted proteins were resolved by SDS-PAGE. The protein bands were visualized with silver staining. PBS acted as a negative control; ß-actin acted as an internal control and was detected by IB with mAb against ß-actin. B Identification of these interactions by M2H assays. Bait and prey plasmids were co-transfected with pG5luc plasmids into subconfluent HEK-293T cells at a molar ratio of 1:1:1 for the pACT:pBIND:pG5luc vector. At 48 ?h post-transfection (hpt), the HEK-293T cells were lysed, and Rluc and Fluc activities were evaluated using the Promega Dual-Luciferase Reporter Assay System. All experimental groups were compared with the negative control group (ACT-Bind). Statistical analysis was performed by Student t-tests. *P ?< ?0.05 and **P ?< ?0.01. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. C These interactions were verified using Co-IP assays. RK-13 ?cells were co-transfected with bait and prey plasmids. Cell lysates were prepared 48 hpt and the proteins were subjected to IP followed by IB analysis. myc fusion proteins acted as bait proteins and Flag fusion proteins acted as prey proteins. RHDV, rabbit hemorrhagic disease virus; RC, replication complex; IP, immunoprecipitation; IB, immunoblotting; mAb, monoclonal antibody; SD, standard deviation.