Journal: Communications Medicine

Article Title: The treatment of primary CoQ deficiency requires the targeting of multiple pathogenic mechanisms

doi: 10.1038/s43856-025-01000-8

Figure Lengend Snippet: Representative image of Western blot of SQOR and the quantitation of the protein bands ( a ), and relative mRNA levels for Sqor ( b ) in the liver. c Representative image of Western blot of Eif2ak2 and the quantitation of the protein bands in the liver. Relative mRNA levels of the genes Cmpk2 ( d ), Mthfd2l ( e ), Psat1 ( f ) and Eif2ak2 ( g ) in liver. Data are expressed as mean ± SD (one-way ANOVA with a Tukey’s post hoc test; n = 5 for each group). Representative images of Western blots of SQOR ( h ), CBS ( i ), PSAT1 ( j ) and EIF2AK2 ( k ), and the quantitation of the protein bands in human skin fibroblast from control and patient 1 (P1). Relative mRNA levels of the genes CMPK2 ( l ), MTHFD2L ( m ), PSAT1 ( n ) and EIF2AK2 ( o ) in human skin fibroblast from control and patient 1 (P1). Representative images of Western blots of SQOR ( p ), CBS ( q ), PSAT1 ( r ) and EIF2AK2 ( s ), and the quantitation of the protein bands in human skin fibroblast from control and patient 2 (P2). Relative mRNA levels of the genes CMPK2 ( t ), MTHFD2L ( u ), PSAT1 ( v ) and EIF2AK2 ( w ) in in human skin fibroblast from control and patient 2 (P2). Data are expressed as mean ± SD (one-way ANOVA with a Tukey’s post hoc test; n = 3 for each group). CBS cystathionine β-synthase, CMPK2 cytidine monophosphate kinase 2, EIF2AK2 eukaryotic translation initiation factor 2 alpha kinase 2, MTHFD2L methylenetetrahydrofolate dehydrogenase (NADP + Dependent) 2-Like, PSAT1 phosphoserine aminotransferase 1, SQOR sulfide:quinone oxidoreductase. Original western-blot membranes corresponding to this figure are provided in Figs. S8 and Fig S9.

Article Snippet: The following primary antibodies were used: anti-COQ4 (Proteintech, 16654-1-AP), anti-COQ5 (Proteintech, 17453-AP), anti-COQ7 (Proteintech, 15083-1-AP), anti-PRODH (Cell Signaling, #22980), anti- SQRDL (Proteintech, 17256-1-AP), anti-CBS (Proteintech, 14787-1-AP), anti-EIF2AK2 (Proteintech, 18244-1-AP), anti-PSAT1 (Proteintech, 10501-1-AP), anti-VDAC1 (Abcam, ab14734) and anti-ACTIN (Invitrogen, #MA5-15739-HRP).

Techniques: Western Blot, Quantitation Assay, Control

Journal: Nature communications

Article Title: Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.

doi: 10.1038/ncomms7239

Figure Lengend Snippet: Figure 7 | GADD34 stabilized by Hippo signalling results in increased Yap phosphorylation and cytoplasmic retention. Western blotting showing the dramatic reduction of GADD34/CReP in the Mst1/2-deficient livers (a) or in the Lats1/2-null mouse embryonic fibroblasts (c). The overexpression of Mst2 (b) or Lats1 (c) resulted in increased GADD34/CReP and decreased phospho-eIF2a levels in HepG2 cells. (d) Western blotting showing that overexpression of Lats1 decreased GADD34 ubiquitination in HepG2 cells. (e) Overexpression of PP1 promoted Yap nuclear translocation. The subcellular localization of transfected PP1 (red) and endogenous Yap (green) was determined by immunofluorescence staining along with DAPI for DNA (blue) in HepG2 cells. Cell transfected with or without PP1 was indicated with arrow or star, respectively. Scale bar, 10 mm. (f) Western blotting showing that GADD34 (WT) but not GADD34 (KARA) downregulated phospho-eIF2a and increased phospho-Yap levels in HepG2 cells. (g) Immunofluorescence staining showing that GADD34 (WT, red) but not GADD34 (KARA, red) promoted endogenous Yap (green) cytoplasmic retention in HepG2 cells treated with 1 mg ml 1 TM for 8 h. Cells transfected with GADD34 (WT or KARA) and control cells were indicated with arrow and star, respectively. Scale bar, 10 mm. (h) The overexpression of Lats1 resulted in the cytoplasmic retention of PP1 in HepG2 cells. The subcellular localization of transfected PP1 (red) was determined in HepG2 cells with or without Lats1 overexpression by immunofluorescence staining along with DAPI for DNA (blue). Cell transfected with or without Lats1 was indicated with arrow or star, respectively. Scale bar, 10 mm. Data are representative of at least three independent experiments.

Article Snippet: Antibodies against CReP (14634-1-AP), GADD34 (10449-1-AP), ATF4 (60035-1-lg), eIF2AK1 (20499-1- AP) and eIF2AK2 (18244-1-AP) were obtained from Proteintech Group Inc. (Wuhan, China).

Techniques: Phospho-proteomics, Western Blot, Over Expression, Ubiquitin Proteomics, Translocation Assay, Transfection, Staining, Control

Journal: Nature communications

Article Title: Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.

doi: 10.1038/ncomms7239

Figure Lengend Snippet: Figure 9 | Elevated PERK-eIF2a-Yap signalling is associated with the development of mouse and human HCC. (a–c) A low dose of TM promotes cell hyperproliferation. Western blot analysis of PERK signalling and Yap levels in HepG2 cells treated with a low dose of TM (10 ng ml 1) for the indicated number of days (a). Low-dose TM-treated HepG2 cells exhibited the formation of significantly more colonies (b) and formed tumours of larger masses in the nude mice (c) compared with the dimethylsulfoxide (DMSO)-treated control cells, n ¼ 8. The black arrow indicates the TM-treated tumour cells; the white arrow denotes the control cells. Data were assessed by Student’s t test and represented as mean±s.d. ***Po0.001. (d) Western blotting with the indicated antibodies in liver tissues isolated from DEN- or DEN-plus-TUDCA-treated mice. (e,f) TUDCA treatment reduced liver masses (e) and the numbers of HCC tumors (f) in DEN-challenged mice, n ¼ 8. Data assessment is same as in c. ***Po0.001. (g) Yapfl/ þ-AblCre mice exhibited significantly reduced numbers of DEN-induced HCC tumours compared with control WT mice. Data assessment is same as in c. ***Po0.001. (h,i) Western blot analysis of Bip, PERK, phospo-eIF2a and Yap in liver cancer tissue (T) and non-tumorous liver tissue (N) isolated from one patient. A total of six representative paired samples are shown (h). See Supplementary Fig. 11 for the remaining 60 paired samples. The intensities of the immunoblot bands were quantified using the Imagine gel software. The ratio of the relative expression of the indicated proteins in the T and N from one patient was plotted and applied with the linear regression t-test (i). (j) A proposed working model for the interplay of the Hippo pathway and the UPR signalling for cell fate determination. Under ER stress conditions, the PERK-mediated eIF2a phosphorylation increases ATF4 expression for the subsequent induction of Yap. Activated Yap increases UPR activity, the size and Ca2 þ storage capacity of ER for cell survival. When ER stress is excessive and homeostasis is not restored, Hippo signalling is activated for stabilizing GADD34/CReP proteins, which assemble with PP1 to form a complex to dephosphorylate eIF2a and reduces the PP1 abundance in the nucleus. In the end, increased Yap phosphorylation inhibits UPR activity and ER membrane expansion and eventually promotes cell death.

Article Snippet: Antibodies against CReP (14634-1-AP), GADD34 (10449-1-AP), ATF4 (60035-1-lg), eIF2AK1 (20499-1- AP) and eIF2AK2 (18244-1-AP) were obtained from Proteintech Group Inc. (Wuhan, China).

Techniques: Western Blot, Control, Isolation, Software, Expressing, Phospho-proteomics, Activity Assay, Membrane