Journal: Journal of Biological Chemistry

Article Title: Lysosomal Two-pore Channel Subtype 2 (TPC2) Regulates Skeletal Muscle Autophagic Signaling

doi: 10.1074/jbc.m114.608471

Figure Lengend Snippet: FIGURE 2. ReducedenduranceandalteredautophagicsignalinginskeletalmuscleofTpcn2/mice.A,mice(male;4–5monthsold)weretestedonatreadmill underdietaryconditionsofregularchowor3-dayfasting.Thedistancestraveled(inmeters)untilexhaustionwererecordedandanalyzed(n6mice/group).Dataare means S.E. (*, p 0.05; **, p 0.01). B, autophagy flux studies under regular diet or extended starvation. Mice (male; 4–5 months old) were fed with regular chow or fasted for 3 days. The TA muscles were then harvested and homogenized for immunoblotting with p-mTOR, total mTOR (t-mTOR), p-AKT, total AKT (t-AKT), p62, LAMP1, and -tubulin. C–F, densitometry quantification of mTOR activity (p-mTOR/total mTOR; C), AKT activity (p-AKT/total AKT; D), normalized (over -tubulin) p62 (E), and LAMP1 (F). Error bars represent S.E. (n 6 mice/group; *, p 0.05; **, p 0.01; NS, not significant; analysis of variance).

Article Snippet: Antibodies for Western blotting were as follows: -tubulin (Sigma), LC3 (clone 5F10, Nanotools), lysosome-associated membrane protein-1 (LAMP1) (Santa Cruz Biotechnology), HA (clone12CA5, Roche Applied Science), dystrophin (Spring Bioscience), p62/ sequestosome-1 (SQSTM1) (Novus Biological), S6K, phosphoS6K (phospho-Thr-389), S6RP (ribosomal protein), phosphoS6RP (phospho-Ser-235/236), AKT and phospho-AKT, and mTOR and phospho-mTOR (phospho-Ser-2448) (all from Cell Signaling Technology).

Techniques: Muscles, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: Primers

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 reduces tumor burden in an oncogene-driven murine model of CCA. A, FGFRs are up-regulated in a YAP-driven murine model of CCA. mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 using qPCR and RNA sequencing of mouse tumors compared with adjacent liver. Thr dashed line represents adjacent liver, which served as the control. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, representative immunostaining images for phospho-FRS2 in vehicle (Veh)- and BGJ398-treated animals. Scale bars: 50 μm. C, liver appearance of mice after intrabiliary injection of myr-Akt and YapS127A Sleeping Beauty transposon-transposase complexes coupled with lobar bile duct ligation and daily intraperitoneal injections of IL-33 (1 μg for 3 days) with (right panel) and without (left panel) BGJ398 treatment (12.5 mg/kg/day) for 2 weeks. D, ratio of tumor weight to liver weight of the ligated lobe expressed as a percentage in vehicle (n = 9)- and BGJ398 (n = 6)-treated animals. *, p < 0.05. E, number of nodules in vehicle (n = 8)- and BGJ398 (n = 6)-treated animals with tumors. *, p < 0.05. F, representative photomicrographs of hematoxylin and eosin-stained tumor sections and adjacent liver are shown in vehicle- and BGJ398-treated animals. Scale bars: 100 μm. G, apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Shown are images (top panel) and the percentage of TUNEL-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. H, immunofluorescence images (top panel) and percentage of Ki67-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, RNA Sequencing Assay, Immunostaining, Injection, Ligation, Staining, TUNEL Assay, Microscopy, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 inhibits YAP activation in a PDX model of CCA. A, representative immunostaining images for nuclear YAP (brown staining) in YAP-positive (PDX1) and YAP-negative (PDX2) tumors. B, mRNA expression of Yap, Ctgf, and Sox4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. C, mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, tumor weight in mg of PDX1 (left panel) and PDX2 (right panel) mice treated for 2 weeks with vehicle (n = 5) or 12.5 mg/kg/day BGJ398 (n = 5). *, p < 0.05. E, representative photomicrographs of hematoxylin and eosin-stained tumors in vehicle- and BGJ398-treated PDX1 (left panel) and PDX2 (right panel) animals. Scale bars: 1 mm. F, immunofluorescence images of CK-19 staining (to outline the biliary epithelium) and YAP in tissue sections obtained from PDX1 mice treated with vehicle or 12.5 mg/kg BGJ398 for 2 weeks. G, mRNA expression of Yap, Ctgf, Sox4, and Mcl-1 in PDX1 animals treated with vehicle or 12.5 mg/kg/day BGJ398 for 2 weeks. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. H, fluorescence images (left panel) and percentage of TUNEL-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. I, immunofluorescence images (left panel) and percentage of Ki67-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Immunostaining, Staining, Expressing, Immunofluorescence, Fluorescence, TUNEL Assay, Microscopy

Journal: bioRxiv

Article Title: The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses

doi: 10.1101/2024.09.09.612163

Figure Lengend Snippet: MGF_360-4L promotes the autophagic degradation of MDA5. (A) HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L and pFlag-MDA5. The cells were then collected and subjected to Western blotting with the indicated antibodies to analyze the effect of MGF_360-4L on MDA5 expression. (B-C). HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L, and the transcript levels of MGF_360-4L (B) and MDA5 (C) were measured via RT‒qPCR. (D) HEK293T cells were cotransfected with pFlag-MDA5 and with or without pHA-MGF_360-4L, followed by treatment with DMSO (negative control), NH 4 Cl (20 mM), MG-132 (10 μM), 3-MA (10 mM), or Z-VAD-FMK (20 μM) for 24 h. Then, the cells were collected, and the protein expression levels of MDA5 and MGF_360-4L were analyzed via Western blotting. (E) HEK293T cells were transfected with pHA-MGF_360-4L or pHA-VEC and infected with VSV at the indicated time points. Cell samples were collected, and the protein expression levels of LC3 and P62 were assessed via Western blotting. (F) HeLa cells were cotransfected with Mcheey-LC3B and EGFP-LC3B with or without pHA-MGF_360-4L. The colocalization of pHA-MGF_360-4L and Mcheey-LC3B was observed via fluorescence microscopy. Scale bars, 10 μm. Fluorescence intensity profiles were measured along the drawn measurement line. (G) HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L, followed by treatment with or without the autophagy inhibitor CQ (50 μM). Protein expression was assessed by Western blotting.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (USA): rabbit anti-DYKDDDK-Tag (CST, 14793S), mouse anti-DYKDDDK-Tag (CST, 8146), rabbit anti-HA (CST, 3724), mouse anti-HA (CST, 2367), rabbit anti-His (CST, 12698), rabbit anti-Myc (CST, 13987), and mouse anti-Myc (CST, 2276); rabbit anti-Rig-I (CST, 3743), rabbit anti-MDA5 (CST, 5321), rabbit anti-MAVS (CST, 24930), rabbit anti-TRAF3 (CST, 33640), rabbit anti-IKKε (CST, 96794), rabbit anti-p-IKKε (CST, 8766), rabbit anti-TBK1 (CST, 3504), rabbit anti-p-TBK1 (CST, 5483), rabbit anti-IRF3 (CST, 4302), rabbit anti-p-IRF3 (CST, 29047), rabbit anti-IRF7 (CST, 72073), rabbit anti-p-IRF7 (CST, 24129), rabbit anti-LC3A/B (CST, 12741), rabbit anti-LC3B (CST, 2775), rabbit anti-SQSTM1/p62 (CST, 23214), rabbit anti-NDP52 (CST, 60732), rabbit anti-NIX (CST, 12396), mouse anti-PARKIN (CST, 4211), rabbit anti-PINK1 (CST, 6946), rabbit anti-BNIP3 (CST, 44060), rabbit anti-ATG5 (CST, 12994), rabbit anti-ATG7 (CST, 8558), rabbit anti-ATG12 (CST, 4180), and rabbit anti-ATG16L1 (CST, 8089) antibodies.

Techniques: Transfection, Western Blot, Expressing, Negative Control, Infection, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses

doi: 10.1101/2024.09.09.612163

Figure Lengend Snippet: MGF_360-4L recruits the selective autophagy receptor p62 to induce mitophagy. (A-B) HEK293T cells were cotransfected with pHA-MGF_360-4L and Myc-OPTN, Myc-NBR1, Myc-TOLLIP, Myc-NDP52, or Myc-p62, and the interactions between proteins were detected by Co-IP. (C) GST-tagged MGF_360-4L protein (10 μg) or GST antigen was added to GST-Sepharose 4B beads, which were incubated for 3 h at room temperature, followed by binding with His-tagged p62 protein (10 μg). The samples were subjected to Western blotting analysis to detect direct interactions between MGF_360-4L-GST and p62-His. (D-E) HEK293T cells were transfected with pFlag-MDA5 with or without pHA-MGF_360-4L, followed by the addition of siRNA targeting p62 or ATG16L1 or a scramble siRNA as a control. The cells were collected, and the protein expression levels were analyzed via Western blotting. (F-H) HeLa cells were transfected with pHA-MGF_360-4L and pMyc-p62, NDP52 or empty vector. The colocalization of MGF_360-4L with p62 or NDP52 was detected via confocal microscopy. Fluorescence intensity profiles were measured along the drawn measurement line.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (USA): rabbit anti-DYKDDDK-Tag (CST, 14793S), mouse anti-DYKDDDK-Tag (CST, 8146), rabbit anti-HA (CST, 3724), mouse anti-HA (CST, 2367), rabbit anti-His (CST, 12698), rabbit anti-Myc (CST, 13987), and mouse anti-Myc (CST, 2276); rabbit anti-Rig-I (CST, 3743), rabbit anti-MDA5 (CST, 5321), rabbit anti-MAVS (CST, 24930), rabbit anti-TRAF3 (CST, 33640), rabbit anti-IKKε (CST, 96794), rabbit anti-p-IKKε (CST, 8766), rabbit anti-TBK1 (CST, 3504), rabbit anti-p-TBK1 (CST, 5483), rabbit anti-IRF3 (CST, 4302), rabbit anti-p-IRF3 (CST, 29047), rabbit anti-IRF7 (CST, 72073), rabbit anti-p-IRF7 (CST, 24129), rabbit anti-LC3A/B (CST, 12741), rabbit anti-LC3B (CST, 2775), rabbit anti-SQSTM1/p62 (CST, 23214), rabbit anti-NDP52 (CST, 60732), rabbit anti-NIX (CST, 12396), mouse anti-PARKIN (CST, 4211), rabbit anti-PINK1 (CST, 6946), rabbit anti-BNIP3 (CST, 44060), rabbit anti-ATG5 (CST, 12994), rabbit anti-ATG7 (CST, 8558), rabbit anti-ATG12 (CST, 4180), and rabbit anti-ATG16L1 (CST, 8089) antibodies.

Techniques: Co-Immunoprecipitation Assay, Incubation, Binding Assay, Western Blot, Transfection, Control, Expressing, Plasmid Preparation, Confocal Microscopy, Fluorescence

Journal: Molecular neurobiology

Article Title: Citalopram reduces aggregation of ATXN3 in a YAC transgenic mouse model of Machado-Joseph disease

doi: 10.1007/s12035-018-1331-2

Figure Lengend Snippet: Assessment of macroautophagy markers shows increased levels of p62 in the spinal cord (B), and of p62 and beclin-1 in the cerebellum (C) of citalopram-treated mice compared to controls. Citalopram treatment did not affect levels of LC3 in any of the three regions (A-C). (A, B, and C) Left panels show immunoblots of indicated proteins in soluble fractions of brainstem, spinal cord and cerebellum. Right panels display quantification of band intensity, with values normalized to Gapdh. Bars represent the average percentage of protein relative to vehicle-treated mice (± SEM). Statistical significance is indicated as *P < 0.05 and **P < 0.01. Mice 1, 2, and 20 were excluded from statistical analysis. Diamond (◆) represents p62 bands.

Article Snippet: Total protein lysates from soluble fractions (100 μg from brainstem, and cerebellum) were resolved on 10% SDS-PAGE gels, and corresponding PVDF membranes were incubated overnight at 4 °C with primary antibodies: mouse anti-HSP70 (1:500; SPA810, Enzo Life Sciences), rabbit anti-HSP90α (1:1000; ab2928, Abcam), mouse anti-HSP90β (1:1000; ADI-SPA842, Enzo Life Sciences), rabbit anti-HSP40 (1:1000; #4868, Cell Signaling), mouse anti-p62/SQSTM1 (1:1000; H00008878-M01, Novus Biologicals), rabbit anti-Beclin1 (1:1000; ab207612, Abcam), rabbit anti-LC3 (1:500; PM036, MBL International Corporation), mouse anti-GAPDH (1:5000; MAB374, Millipore), and rabbit anti-MJD [ 30 ] (1:30000).

Techniques: Western Blot

Journal: International Journal of Nanomedicine

Article Title: Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells

doi: 10.2147/IJN.S115158

Figure Lengend Snippet: Realgar NPs triggered autophagy in K562 cells. Notes: ( A ) The cells were treated with specific concentrations of realgar NPs (0, 5, and 10 μg/mL) for 24 h and then fixed and immunostained with antibodies against LC3 (green), and nuclei were counterstained with Hoechst 33342 (blue); original magnification, ×100. ( B ) K562 cells were treated with specific concentrations of realgar NPs (0, 5, 10, 20, 40, and 80 μg/mL) for 24 h. LC3 protein level was estimated by using Western blot analysis. ( C ) Quantification of LC3-II/LC3-I shown in ( B ). ( D ) The cells were treated with specific concentrations of realgar NPs (0, 5, and 10 μg/mL) for 24 h and then fixed and immunostained with antibodies against P62 (red), and nuclei were counterstained with Hoechst 33342 (blue); original magnification, ×100. ( E ) K562 cells were treated with specific concentrations of realgar NPs (0, 5, 10, 20, 40, and 80 μg/mL) for 24 h. P62 protein level was estimated by using Western blot analysis. ( F ) Quantification of P62 shown in ( E ). n=3; * P <0.05, ** P <0.01, realgar NP treatment groups versus 0 μg/mL group. Abbreviation: NP, nanoparticle.

Article Snippet: Caspase-3 (#9665; Cell Signaling Technology, Danvers, MA, USA), Bcl2 (#2870; Cell Signaling Technology), Bax (#2772; Cell Signaling Technology), P62 (#7695; Cell Signaling Technology), Akt (#9272; Cell Signaling Technology), p-Akt (#4060; Cell Signaling), mTOR (#2983; Cell Signaling Technology), p-mTOR (#5536; Cell Signaling Technology), LC3 (L7543; Sigma-Aldrich, St Louis, MO, USA), Cav-1 (#3267; Cell Signaling Technology), Bcr-Abl (#2862; Cell Signaling Technology), p-Bcr-Abl (#3901; Cell Signaling Technology), CrkL (#3182; Cell Signaling Technology), p-CrkL(Tyr207) (#3181; Cell Signaling Technology), and β-actin (TA-09; ZSGB-BIO, Beijing, China) were used in the present study.

Techniques: Western Blot