Fig 1: WSSV infection activates the pentose phosphate pathway (PPP) in shrimp.(A–F) Temporal expression profiles of viral and PPP-related genes. Hemocytes and intestinal tissues were collected from shrimp at 0, 6, 12, 24, 36, and 48 h post-injection (hpi) with WSSV or PBS. The mRNA levels of the viral gene IE1 (A, B) and the host genes G6PD (C, D) and TKTL2 (E, F) were determined by qPCR. (G, H) Western blot detection of VP28, TKTL2, and G6PD proteins in hemocytes (G) and intestinal tissues (H) at the indicated time points. (I, J) Enzymatic activity of TKT and G6PD in hemocytes collected at 24 hpi. WSSV- or PBS-injected lysates were used to measure the activity of G6PD (I) and TKT (J). (K, L) Quantification of PPP metabolites. Hemocytes collected at 24 hpi were analyzed for (K) NADPH levels using a detection kit and (L) R5P levels by LC-MS. Data are expressed as mean ± SD from three biological replicates. Asterisks indicate statistically significant differences compared with PBS control. *p< 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.
Fig 2: IE1 enhances PPP flux through interaction with TKTL2.(A-F) Effects of IE1 overexpression on PPP metabolites in High Five cells transfected with empty vector pIZ-V5‑His (EV), pIZ-V5-IE1, pIZ-FLAG-TKTL2, or both pIZ-V5-IE1 and pIZ-FLAG-TKTL2. (A) Western blot detection; (B) NADPH levels and (C) R5P levels; (D-F) ROS levels analyzed by microplate reader (D) and flow cytometry (E, F). (G-L) Effects of IE1 knockdown (G) in shrimp on (H) NADPH, (I) R5P, and (J-L) ROS levels. (M-S) Effects of IE1 overexpression on PPP flux under TKT and G6PD inhibition. High Five cells transfected with EV or pIZ-V5-IE1 were treated with 25 μM OT (TKTL2 inhibitor), 25 µM 6AN (G6PD inhibitor), or DMSO control. (M) Western blot detection; (N) Inhibitor cytotoxicity; (O) NADPH, (P) R5P, and (Q-S) ROS measurements. Data are presented as mean ± SD from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
Fig 3: IE1 enhances the enzymatic activity of TKTL2.(A, B) Effect of IE1 overexpression on TKT activity in High Five cells transfected with empty vector pIZ‑V5‑His (EV), pIZ‑V5‑IE1, pIZ‑FLAG‑TKTL2, or both pIZ‑V5‑IE1 and pIZ‑FLAG‑TKTL2 constructs. (A) Western blot detection of protein expression; (B) TKT enzymatic activity measured using a commercial kit. (C–E) Effect of IE1 knockdown on TKT activity in shrimp. Shrimp injected with dsIE1 or dsEGFP were challenged with WSSV and hemocytes were collected at 24 and 36 hpi to assess (C, D) IE1 knockdown efficiency and (E) TKT enzymatic activity. (F) In vitro TKT assay showing direct enhancement of TKTL2 activity by IE1 using purified recombinant proteins. Data represent mean ± SD from three biological replicates. *p< 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
Supplier Page from Abcam for Transketolase Activity Assay Kit (Fluorometric)