Fig 2: Mouse PHLDA1 can induce caspase-7 (but not caspase-3) activation and detachment of cells. (a) Mouse NIH3T3 cells were transiently transfected with vectors coding for indicated fusion proteins and protein extracts were analyzed by western blot. NT—not transfected cells, TR—transfection reagent only. ACTB and HSPA8 were used as loading controls. Active caspase-3 and caspase-7 were quantitated based on densitometry of replicated blots and results are shown in corresponding charts; asterisks indicate statistically significant (* p < 0.05) differences against the EGFP-transfected cells. (b) An example of the live-cell imaging of NIH3T3 cells transiently transfected with the PMAIP1/EGFP construct showing the apoptotic membrane blebbing in a cell overexpressing PMAIP1, while control cells (transfected with the EGFP vector only, right photo) were not affected. (c) An example of the live-cell imaging of NIH3T3 cells transiently transfected with vectors coding for PHLDA1/EGFP, EGFP/PHLDA1 or EGFP showing detachment of cells overexpressing PHLDA1. Images were recorded in EGFP (green) and brightfield channels for two days. The observation period shown for each vector is given above the panels. The relative time of acquisition is displayed in the upper right corner of each image.

Fig 3: PHLDA1-IHC and p53-IHC positivity rates according to the histological status. The PHLDA1 positivity rate in the neoplastic tissues was higher than that in the non-neoplastic tissues. Notably, an obvious difference was observed in the PHLDA1-IHC positivity rate between the UC-CRC (78.6%) and dysplasia samples (53.8%; P<0.001), and between the UC-CRC and colitis samples (15.6%; P<0.001). Fisher's test was used to compare the ratio differences between the two groups. PHLDA1, pleckstrin homology-like domain, family A, member 1; IHC, immunohistochemistry; UC-CRC, ulcerative colitis-associated colorectal cancer.

Fig 4: Renal interstitial fibrosis is mediated by sXBP1.A Rat renal fibroblasts were treated with TM and the IRE1 endonuclease inhibitor 4µ8c. TM-induced splicing of XBP1, which was prevented by 4µ8c treatment. B Renal fibroblasts were treated with TGF-ß1 (Tß) and ascorbic acid (AA2P; AA) to induce XBP1 splicing. Individually, TGF-ß1 and AA2P increased XBP1 splicing, but the effect was increased with combined treatment. Co-treatment with 4µ8c was able to prevent TGF-ß1-mediated XBP1 splicing. C Renal fibroblasts treated with TGF-ß1 and AA2P show increased collagen deposition, which was inhibited with 4µ8c co-treatment. Bar = 100 µm. *P < 0.05 vs veh; ***P < 0.001 vs veh; #, P < 0.05 vs AA2P + TGF-ß1. D Western blotting demonstrates that expression of TGF-ß1 receptor (TGFß-R1) is significantly reduced in kidneys from WT mice with CKD. Kidneys from TDKO mice with or without CKD do not express TGFß-R1. *P < 0.05 vs sham; #, P < 0.05 vs WT. E Renal fibroblasts were treated with TGF-ß1 and AA2P with or without SB431542 (SB), a TGFß-R1 inhibitor. While TGF-ß1 and AA2P increased mRNA levels of spliced XBP1, co-treatment with SB431542 inhibited XBP1 splicing. *P < 0.05 vs veh; #, P < 0.05 vs Tß + AA. F HK-2 cells were treated with thapsigargin for varying time points (0, 18, 24, 48 h) and subsequently underwent western blotting. Thapsigargin induced TDAG51 expression at 18 and 24 h, but not 48 h. TGFß-R1 was increased after 18 h of TG, but the expression was reduced at 24 and 48 h. *P < 0.05 vs 0 h.

Fig 5: Localization of PHLDA1 in mouse testes. (a) Detection of PHLDA1 by immunofluorescence (using Ab2, red; DNA stained with DAPI, blue) in testes of a control mouse and subjected to heat shock after 2, 4, and 6 h of recovery. Enlargement of the marked areas is shown in the bottom panel; round spermatids and condensing spermatids are marked with arrowhead and arrows, respectively. Representative pachytene spermatocytes (in squares) are further enlarged in the lower corners. (b) Detection of apoptotic DNA breaks (by TUNEL assay, red; DNA stained with DAPI, blue) in seminiferous tubules of untreated mice and after heat shock in vivo and indicated recovery time (2–6 h). (c) Detection of PHLDA1 by immunofluorescence (red; DNA stained with DAPI, blue) in cryptorchid testes 7 and 14 days after surgery (upper panel) and detection of apoptotic DNA breaks (by TUNEL assay, red) in corresponding tissues (bottom panel). Scale bar—50 µm.