pretrox tight pur  (TaKaRa)

Journal: Cell reports

Article Title: BACH1 inhibits senescence, obesity, and short lifespan by ferroptotic FGF21 secretion.

doi: 10.1016/j.celrep.2024.114403

Figure Lengend Snippet: Figure 1. The secretion from ferroptotic fibroblasts by BACH1 re-expression activates the proliferation and suppresses the expression of SASP in other cells (A–H) The control or Tet-ON BACH1 genes were introduced into Bach1/ iMEFs using retrovirus vectors. 2-ME was removed by culture medium exchange. The culture supernatant from the iMEFs (control: control supernatant, Tet-ON BACH1: ferroptotic supernatant) treated with Dox for 48 h was transferred into Hepa1 cells. The Hepa1 cells were analyzed after treatment with the control or ferroptotic supernatant for 2 (G and H), 3 (F; Figures 2B–2D, 2G, and 2H), or 4 (B–E; Figure 2A) days. (A) Experimental design. (B) GO analysis of the Hepa1 cells. (C) Gene set enrichment analysis of the gene sets related to cell cycle and cytokine expression in the Hepa1 cells. (D) Heatmap displaying the RNA-seq results (counts per million mapped reads) of arbitrarily selected p53 signaling pathway-related genes (KEGG pathway, hsa04115) in the Hepa1 cells. The p values and log2FC (fold change) were calculated using differential expression analysis on edgeR. Cont.: control supernatant, Ferr.: ferroptotic supernatant.

Article Snippet: To overexpress the mouse FGF21 protein using the Tet-ON system, retrovirus particles were prepared by transfecting the retrovirus vector pRetroX-Tight-Pur (Takara Bio), into which the coding region of the mouse Fgf21 gene had been inserted into the multi-cloning site, into Plat-E cells using FuGENE HD (E2311, Promega).

Techniques: Expressing, Control, RNA Sequencing, Quantitative Proteomics

Journal: Cell reports

Article Title: BACH1 inhibits senescence, obesity, and short lifespan by ferroptotic FGF21 secretion.

doi: 10.1016/j.celrep.2024.114403

Figure Lengend Snippet: Figure 2. The secretion from ferroptotic fibroblasts by BACH1 re-expression suppresses senescence in other cells (A–D, G, and H) The control or Tet-ON BACH1 genes were introduced into Bach1/ iMEFs using retrovirus vectors, and Hepa1 cells were treated with the culture supernatant from the iMEFs as in Figure 1A.

Article Snippet: To overexpress the mouse FGF21 protein using the Tet-ON system, retrovirus particles were prepared by transfecting the retrovirus vector pRetroX-Tight-Pur (Takara Bio), into which the coding region of the mouse Fgf21 gene had been inserted into the multi-cloning site, into Plat-E cells using FuGENE HD (E2311, Promega).

Techniques: Expressing, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

doi: 10.1186/1478-811X-11-76

Figure Lengend Snippet: HtrA2/Omi induces monoubiquitination rather than cleavage of its substrate UCH-L1 during TNF-induced necroptosis. A . Wild-type (WT) and HtrA2/Omi-deficient MEF were left untreated or stimulated for 16 h with 20 μM zVAD-fmk and 1 μg/ml CHX in the presence (to induce necroptosis) or absence (for control) of 100 ng/ml TNF before UCH-L1 was detected with a monoclonal antibody that recognizes the full-length 25-kDa form of UCH-L1 (mAB UCH-L1) or, on a parallel blot, with a polyclonal antibody to detect all cleavage fragments of UCH-L1 (pAb UCH-L1). An asterisk marks the 35-kDa band corresponding to the predicted size of monoubiquitinated UCH-L1. B . WT and HtrA2/Omi-deficient MEF were stimulated as in A , and additionally with 0.5 μM staurosporine for 8 h. Lysates were separated on 10–20% w/v Tris-Tricine gels (Biorad) to resolve low molecular weight proteins and immunoblotted with pAb UCH-L1. The blot was deliberately overexposed to visualize faint cleavage fragments. The 10-kDa UCH-L1 cleavage fragment generated by HtrA2/Omi during staurosporine-induced apoptosis is indicated (arrow, red box). C . WT and HtrA2/Omi-deficient MEF were treated with TNF/zVAD/CHX as in A for the indicated times and analyzed for proteins reactive with pAb UCH-L1 by Western blot. An asterisk marks the appearance of the 35-kDa band identical to the predicted size of monoubiquitinated UCH-L1. D . Lysates from WT and HtrA2/Omi-deficient MEF were incubated with 20 μM of an HA-tagged ubiquitin-derived probe (HAUbVME) in 50 mM Tris, 150 mM NaCl, pH 8.0 for 90 min at 37°C and subsequently analyzed by immunoblotting with HA antibody and reanalyzed with pAB UCH-L1. In panels A-D , detection of actin served as a loading control. E . An immunoprecipitation was performed using lysates from necroptotic WT MEF (treated with TNF/zVAD/CHX as in A ) and an antibody for ubiquitin. Subsequently, UCH-L1 was detected by Western blot using pAb UCH-L1.

Article Snippet: Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma).

Techniques: Molecular Weight, Generated, Western Blot, Incubation, Derivative Assay, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

doi: 10.1186/1478-811X-11-76

Figure Lengend Snippet: Inhibition of UCH-L1 protects from TNF-induced necroptosis. A . L929Ts cells were prestimulated for 3 h with 50 μM of the UCH-L1 inhibitor LDN57444 or left unstimulated before addition of 100 ng/ml TNF and 20 μM zVAD-fmk for 5 h. Subsequently, cell death was analyzed by PI staining and flow cytometry. Asterisks indicate statistical significance (t-test), *** p < 0.001. Micrographs additionally show the morphology of untreated L929Ts cells vs. necroptotic cells vs. cells protected by LDN57444. Scale bar: 100 μm. B . L929Ts cells were transfected with an siRNA that does not elicit an RNAi response (negative control, siCtr), with an siRNA specific for murine UCH-L1, and with an siRNA specific for murine RIPK3 (positive control for protection from necroptosis, siRIPK3) as described in “Methods.” 48 h after transfection, cells were treated with 100 ng/ml TNF and 20 μM zVAD-fmk for another 5 h before the decrease of intracellular ATP levels was determined as a marker for cell death. ATP levels are shown relative to controls that were not treated with TNF/zVAD. Asterisks indicate statistical significance (t-test), ** p < 0.01, *** p < 0.001.

Article Snippet: Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma).

Techniques: Inhibition, Staining, Flow Cytometry, Transfection, Negative Control, Positive Control, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

doi: 10.1186/1478-811X-11-76

Figure Lengend Snippet: UCH-L1 as a mediator of caspase-independent, non-apoptotic cell death in diseased kidney podocytes. A . UCH-L1 tet-on podocytes were treated with 20 ng/ml doxycycline for 72 hours (+Dox) or not (-Dox) and 50 μM zVAD-fmk or no inhibitor. Cell death was measured by trypan blue staining. *** p < 0.001. B . UCH-L1 tet-on podocytes were left untreated (-) or treated with doxycycline as in A (+) before PARP-1-reactive bands were detected by immunoblotting. Cell lysates from untreated and apoptotic L929Ts cells (Co, treated with 100 ng/ml TNF and 2 μg/ml CHX for 1 h) are shown as controls. Full-length and cleaved PARP-1 is marked by arrows. Detection of actin served as a loading control. C . Aliquots from the stimulations in B were also analyzed for caspase activity. As negative control, tet- podocytes were treated with doxycycline as in A ; as positive control, lysates from doxycyline-treated tet- podocytes were incubated with cytochrome c and dATP to activate caspases. Subsequently, the activity of caspases-3 and -8 was determined by measuring the cleavage of fluorogenic substrates (zIETD-afc and zDEVD-afc) over 70 minutes. The Western blot below shows that UCH-L1 is indeed upregulated in doxycycline-treated but not untreated UCH-L1 tet-on podocytes and also not in doxycycline-treated negative control tet- podocytes. Treatment with doxycycline was performed as in A . UCH-L1 was detected with mAb UCH-L1, detection of actin served as a loading control. D . Cell death was induced in UCH-L1 tet-on podocytes by treatment with doxycycline as in A in the presence of 50 μM zVAD-fmk (+zVAD-fmk, middle and lower micrographs) or no inhibitor (upper micrographs). Micrographs show the morphology of dying cells within a monolayer of healthy cells (overlay, nuclei are stained blue), and in parallel the nuclear morphology of the same cells after staining with Hoechst dye (Hoechst). Original magnification: x 400.

Article Snippet: Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma).

Techniques: Staining, Western Blot, Activity Assay, Negative Control, Positive Control, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

doi: 10.1186/1478-811X-11-76

Figure Lengend Snippet: Inhibition of UCH-L1 protects podocytes from TNF-induced necroptosis. Podocytes stably transfected with an shRNA construct that causes permanent knockdown of UCH-L1 (shUCH-L1) or with a scrambled negative control shRNA (shCtr) were treated with 100 ng/ml TNF in the presence of 50 μM zVAD-fmk or vehicle for 3 h before loss of membrane integrity as a marker for cell death was measured by trypan blue staining. Asterisks indicate statistical significance (t-test), * p < 0.05. The Western blot below was performed to demonstrate the permanent knockdown of UCH-L1 in shUCH-L1 podocytes, but not in shCtr podocytes. UCH-L1 was detected with mAb UCH-L1, detection of actin served as a loading control. For each stable transfectant, lysates from four independent flasks were analyzed.

Article Snippet: Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma).

Techniques: Inhibition, Stable Transfection, Transfection, shRNA, Construct, Negative Control, Marker, Staining, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

doi: 10.1186/1478-811X-11-76

Figure Lengend Snippet: HtrA2/Omi and UCH-L1 as novel components of TNF-induced necroptosis. The scheme depicts the proposed roles of HtrA2/Omi and UCH-L1 in TNF-induced necroptosis. Binding of TNF to TNF-R1 triggers activation of the necrosomal core complex consisting of RIPK1, RIPK3 and MLKL. Subsequently, the proteins PGAM5L/S and Drp-1 form the mitochondrial attack complex, resulting in the intramitochondrial activation of HtrA2/Omi. Activated HtrA2/Omi then (by cleavage of yet unidentified intramitochondrial substrates) indirectly causes monoubiquitination and activation of UCH-L1, and finally, necroptosis. Accordingly, inhibition of HtrA2/Omi (Ucf-101, knockout) or UCH-L1 (LDN57444, siRNA) protects from necroptosis. Please see Discussion for further details.

Article Snippet: Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma).

Techniques: Binding Assay, Activation Assay, Inhibition, Knock-Out