Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 1. A preprotein bound to OMV under cis and trans condi- tions can be cross-linked to various TOM components. A, a pre- protein can be co-immunoprecipitated with antibodies against Tom40, only when bound to the trans site. Radioactively labeled pSu9-DHFR was bound to OMV for 20 min at 0 or 25 °C in the presence or absence of MTX/NADPH, respectively. The reaction mixtures were chilled on ice and diluted with LSW or HSW buffers (containing 20 or 120 mM KCl, respectively). OMV together with cis or trans site-bound material were reisolated (20 min, 125,000 3 g) and resuspended in SEM buffer with or without MTX/NADPH. Co-immunoprecipitation with antibodies against Tom40 or with antibodies derived from preimmune serum was performed. As a control for the input, an aliquot (Total) representing 25% of the material used for co-immunoprecipitation is shown. B, pSu9- DHFR was bound to OMV under cis and trans conditions and the OMV were washed as described in A. To the indicated samples, 13 mM of F1b

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Immunoprecipitation, Labeling, Derivative Assay, Control

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 2. A presequence polypeptide bound to OMV under cis and trans conditions can be cross-linked to various TOM com- ponents. A, OMV were incubated with radioactively labeled pSu9(17) for 15 min at 0 °C or 25 °C and diluted with LSW or HSW buffers, respectively. After reisolation (20 min, 125,000 3 g), the OMV were resuspended in SEM buffer, and the cross-linking reagents DSG (117 mM) and EDC (1 mM) were added as indicated. After incubation for 40 min at 0 °C, the cross-linkers were quenched. Further analysis was as in Fig. 1B. B, identification of the cross-linking products by immuno- precipitation. OMV were incubated with pSu9(17) for 15 min at 0 °C, diluted with LSW buffer, and reisolated (20 min, 125,000 3 g). The pellets were resuspended in SEM buffer, and DSG (117 mM) was added. Aliquots were removed before (2DSG) and after the cross-linking reac- tion (40 min at 0 °C; 1DSG). Further analysis and immunoprecipita- tion with antibodies directed against Tom20, Tom22, Tom40, and Tom70 or antibodies derived from preimmune serum were as in Fig. 1C. Apparent molecular masses are given on the left.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Incubation, Labeling, Immunoprecipitation, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 3. Cross-linking of preproteins to Tom40 occurs inde- pendently of surface receptors. A, pSu9-DHFR or B, pSu9(17) were incubated with OMV that were pretreated with trypsin (30 mg/ml) or were mock-treated. The binding incubation was for 20 min at 25 °C (pSu9-DHFR) or 0 °C (pSu9(17)). The reaction mixture was chilled on ice and diluted with HSW (pSu9-DHFR) or LSW (pSu9(17)) buffers before reisolation of the OMV. The pellets were resuspended in SEM buffer, and the cross-linker EDC was added as indicated for 40 min at 0 °C. Further analysis was as in Fig. 1. Apparent molecular masses are given on the left. FIG. 4. A purified presequence peptide can be cross-linked to Tom22 and Tom40. The F1b presequence peptide or the control pep- tide CH4 (35 mM) were incubated with OMV that were pretreated with or without trypsin for 20 min at 0 °C as indicated. As a control, an aliquot was removed before the addition of the peptides. The reaction mixture was diluted with LSW and HSW buffers before reisolation of the OMV. The pellets were resuspended in SEM buffer, and 1 mM EDC was added as indicated. After quenching of the cross-linker the proteins were precipitated with trichloroacetic acid, solubilized with sample buffer, and separated by SDS-PAGE. After blotting onto nitrocellulose membranes, immunostaining analysis was performed for A, Tom22 or B, Tom40. In both panels the cross-linking product with a small TOM component is marked with a and the one with the presequence peptide with b.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Incubation, Binding Assay, Purification, Control, SDS Page, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 5. Preproteins accumulated at the outer membrane of intact mitochondria can be cross-linked to the TOM components in a similar fashion as in OMV. A, pSu9-DHFR or B, pSu9(17) were incubated with freshly isolated mitochondria that had been treated with CCCP (38 mM) and apyrase (10 U/ml) to deplete the membrane potential and matrix ATP, respectively. After incubation for 20 min at 0 or 25 °C, the reaction mixture was chilled on ice and diluted with LSW and HSW buffers as indicated. Mitochondria were reisolated (10 min, 12,000 3 g), the pellets were resuspended in SEM buffer containing 38 mM CCCP. DSG or EDC were added as indicated for 40 min at 0 °C. Further analysis was as in Fig. 1B. Apparent molecular masses are given on the left. The position of the cross-linking products of the preproteins with Tom20, Tom22, and Tom40 are marked on the right.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Membrane, Incubation, Isolation

Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 1 HER3 is a candidate driver of the PI3K/mTOR oncogenic signaling circuitry in HNSCC. a Experimental scheme of the kinome siRNA library screen. A “smart pool” of four individual siRNAs targeting each protein kinase gene of the human kinome is distributed in each well of the experimental plates. Cal27 cells were incubated for 72 h and assayed for cell viability, and the Z-score for viability was calculated (Z = (x −µ)/σ) (x stands for each value of cell viability; µ stands for average value; σ stands for standard deviation). b siRNA library targeting human kinases using the kinome siRNA library with Cal27 cells was conducted to search for genes that affect proliferation of HNSCC. Shown are the genes whose knockdown decrease cell viability (Z-score). The blue are the top 20 genes and HER3 is shown in red. (See Supplementary Data 1 for complete list). c Cal27 cells were transfected with the corresponding siRNAs (top 20 hits) for 72 h and cell lysates were analyzed for pS6 by western blotting. Densitometry analysis of western blots was performed using ImageJ. Shown are the top 20 hits of the kinome siRNA screen and their Z-scores, together with changes in pS6 levels after each gene was knocked down, as compared to the non-targeting siRNA group. Similar levels of S6 and GAPDH as loading control were confirmed (Supplementary Fig. 1). d The TCGA (The Cancer Genome Atlas) database was used to determine the relationship between HER3 phosphorylated on tyrosine 1289 (PY1289) and overall survival (OS) (n = 122 HNSCC patients, two sided log-rank test; p = 0.033). e Histogram demonstrating HER3 expression in EpCAM+ E-CAD+ tumor cells (red) and tumor-infiltrating CD4+ and CD8+ T cells (blue) in a fresh surgical specimen from a Stage II T2N0M0 primary tongue squamous cell carcinoma, compared to Cal27 cells (orange) as control. FMO (fluorescence minus one) samples were used to create HER3 + staining gates. f Immunofluorescent staining of CK5, CD8, and HER3 in the same specimen in panel (e), showing HER3 co-expressed with cancer cells (CK5 positive), but not with CD8+

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Incubation, Standard Deviation, Knockdown, Transfection, Western Blot, Control, Expressing, Staining

Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 4 Anti-tumor effect of HER3 kinase inhibition with CDX-3379 antibody in vivo. a WT Cal27 cells, Cal27 cells expressing PIK3CA H1047R or Detroit 562 were transplanted into the flanks of athymic nude mice, and when they reached 150–200 mm3, mice were treated with vehicle diluent or CDX-3379 (10 mg/kg, three times/week) for the indicated days (n = 10 for Cal27; n = 10 for Cal27 PIK3CA H1047R; n = 6 for Detroit 562). Data were reported as mean ± SEM; two-sided Student’s t-test. b Representative H&E stains of mouse tumors from the experiment from panel a. c Representative immunohistochemical analysis of pS6 and BrdU in the short-term treatment (every other day for three times) groups from panel (a) (n = 4 mice per group). Brown chromogen deposition reflects the immunoreactivity; hematoxylin was used as a nuclear counterstain (blue). Scale bars represent 25 μm. Quantification from images using Qupath software and the percentage of positive staining are shown on each image. Data were reported as mean ± SEM, two-sided Student’s t-test, p > 0.05, non-significant or ns; ***p < .001 when compared with the control-treated group. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Inhibition, In Vivo, Expressing, Immunohistochemical staining, Software, Staining, Control

Journal: Nature communications

Article Title: Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer.

doi: 10.1038/s41467-021-22619-w

Figure Lengend Snippet: Fig. 6 Anti-tumor effect of HER3 inhibition in syngeneic HNSCC models and increased durable responses to PD-1 blockade. a C57Bl/6 mice were implanted with 1 × 106 of 4MOSC1 cells into the tongue. After tumors reached ~30 mm3, mice were treated IP with of isotype control, CDX-3379 (20 mg/ kg), anti-PD-1 (10 mg/kg), or a combination of CDX-3379 and PD-1 three times per week for 3 weeks. Individual growth curves of 4MOSC1 tumor-bearing mice are shown (n = 10 per group). b C57Bl/6 mice were implanted with 2 × 106 MOC1 cells into the flanks. After tumors reached approximate 50 mm3, mice were treated same as panel (a). Individual growth curves of MOC1 tumor-bearing mice are shown (n = 8 per group). c A Kaplan–Meier curve showing the survival of mice from panels (a) and (b). The death of animals occurred either naturally, when tumor compromised the animal welfare, when tongue tumor volume (panel a) reached 100 mm3 (n = 10 mice per group), or when flank tumor volume (panel b) reached 500 mm3 (n = 8 mice per group). Two sided log-rank/Mantel–Cox test. d Representative immunohistochemical analysis of pS6 and BrdU in the short-term treatment groups (every other day for three treatments) from panel (a). Brown chromogen deposition reflects the immunoreactivity; hematoxylin was used as a nuclear counterstain (blue). Scale bars represent 25 μm. Quantification from images on the left using Qupath software and the percentage of positive staining are shown on each image. e Immunofluorescent staining of CD8 and CK5 in the short-term treatment from panel (a). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: pS6 (Cell Signaling Technology #2211, 1:200), Brdu (Bio-Rad #OBT0030S, 1:100), and Cleaved Caspase-3 (Cell Signaling Technology # 9661, 1:400).

Techniques: Inhibition, Control, Immunohistochemical staining, Software, Staining

Journal: Experimental & molecular medicine

Article Title: Sestrin2 protects against cholestatic liver injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-mediated pyroptosis.

doi: 10.1038/s12276-022-00737-9

Figure Lengend Snippet: Fig. 5 Sestrin2 regulates AMPK/mTORC1 signaling during cholestasis. A, B Liver tissues were collected from mice 3 days after sham or BDL surgery (n = 6 mice per group) and analyzed by immunoblotting with anti-p-AMPK, anti-AMPK, anti-p-p70S6K, anti-p70S6K, anti-p-S6, and anti-S6 antibodies. Band intensities were quantified and normalized to total protein intensities. C–F HepG2 cells were infected with lentiviruses expressing shRNAs targeting luciferase (sh-Luc) or Sestrin2 (sh-SESN2) and treated with 200 µM CDCA or 750 µM CA for the indicated hours. Cell lysates were immunoblotted with the indicated antibodies. β-Actin served as a loading control. Numbers below the immunoblot bands indicate the fold changes normalized to the control band intensities. G, H Liver tissues were collected from Sesn2+/+ BDL and Sesn2−/−BDL mice (n = 6 mice per group) and analyzed by immunoblotting with the indicated antibodies. Band intensities were quantified and normalized to total protein intensities. The data are representative of two (A, B, G, H) or at least three (C–F) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).

Article Snippet: Immunoblotting was performed using antibodies against human Sestrin2 (Proteintech Group, USA), mouse Sestrin2 (Dr. Jun Hee Lee, University of Michigan); cleaved caspase-3, phospho-eIF2α, PERK, phospho-p70 S6 kinase, p70 S6 kinase, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-AMPK, AMPK, TSC2, NLRP3 (Cell Signaling Technology, USA), ATF4, C/EBP-β, eIF2α, caspase-1, ASC (Santa Cruz Biotechnology, USA); GSDMD (Abcam), GAPDH (Aviva Systems Biology, USA), and β-actin (Developmental Studies Hybridoma Bank, University of Iowa).

Techniques: Western Blot, Infection, Expressing, Luciferase, Control

Journal: Animal nutrition (Zhongguo xu mu shou yi xue hui)

Article Title: Glycine represses endoplasmic reticulum stress-related apoptosis and improves intestinal barrier by activating mammalian target of rapamycin complex 1 signaling.

doi: 10.1016/j.aninu.2021.05.004

Figure Lengend Snippet: Fig. 4. The repressive effect of glycine on the IRE1a-JNK endoplasmic reticulum (ER) stress signaling was dependent on mechanistic target of rapamycin complex 1 (mTORC1). Protein abundances of p-Akt, Akt, p-mTOR, mTOR, p-p70S6K, and p70S6K were determined by Western blot (A) and (B); Cell viability was measured by CCK-8 assay (C). Protein abundances of p-IRE1a, IRE1a, p-JNK, JNK, CHOP, and cleaved-Caspase3 (c-Caspase3), Bcl-2 were determined by Western blot (D). Ultrastructure of rough endoplasmic reticulum under transmission electron microscope (E). Values are means ± SEM of 3 independent experiments. Means without a common letter differ, P < 0.05. Scale bar: 500 nm. p ¼ phosphate; Akt ¼ protein kinase B; mTOR ¼ mechanistic target of rapamycin; p70S6K ¼ p70S6 kinase.

Article Snippet: The antibodies against protein kinase B (Akt) (9272), phosphorylated (p)-Akt (Ser473, 9271), mTORC1 (2972), p-mTORC1 (Ser2448, 5536), p70S6 kinase (p70S6K) (9202), p-p70S6K (Thr389, 9205), p-c-Jun N-terminal kinase (JNK) (Thr183/ Tyr185, 9251), JNK (9252), eukaryotic initiation factor 2a (eIF2a) (2103), p-eIF2a (Ser51, 3398), cleaved-caspase3 (9661), and p53 (2524) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Western Blot, CCK-8 Assay, Transmission Assay, Microscopy