Journal: OncoTargets and Therapy

Article Title:

Mitofusin1 Is a Major Mediator in Glucose-Induced Epithelial-to-Mesenchymal Transition in Lung Adenocarcinoma Cells

doi: 10.2147/ott.s238714

Figure Lengend Snippet: Figure 5 High glucose led to the induction of autophagy in the LAD cell line through MFN1. (A and B) Expression of LC3B-II, BECN-1 and SQSTM1 in A549 cells from the NG+NC, HG+NC, and HG+siMFN1 groups. (C) Cells were transfected with the eGFP-mRFP-LC3 plasmid and exposed to different concentrations of glucose for 24 h. Yellow and red dots refer to autolysosomes and autophagosomes respectively. Scale bar = 50 µm. Data shown are mean ± SEM. *P < 0.05, **P < 0.01 vs NG+NC group. #P < 0.05, ##P < 0.01 vs HG+NC group (n = 6). Abbreviations: LAD, lung adenocarcinoma; NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; siMFN1, small interfering RNA of MFN1; LC3B, microtubule-associated proteins 1A/1B light chain 3B; BECN, beclin-1; SQSTM, sequestosome 1; eGFP, enhanced green fluorescent protein; mRFP, monomer red fluorescent protein.

Article Snippet: Materials Antibodies against SQSTM1 (PB0458, 1:400) was obtained from Boster Biological Technology Co. Ltd. Antibody against MFN1 (ab107129), LC3B (ab48394), Pink (ab23707), Parkin (ab77924) and Snail (ab53519) were purchased from Abcam.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Small Interfering RNA

Journal: Frontiers in immunology

Article Title: Activator-Mediated Pyruvate Kinase M2 Activation Contributes to Endotoxin Tolerance by Promoting Mitochondrial Biogenesis.

doi: 10.3389/fimmu.2020.595316

Figure Lengend Snippet: FIGURE 2 | Mitochondrial biogenesis is induced in PKM2-activated macrophages. (A) Mitochondrial mass was assessed by measuring the uptake of NAO by using flow cytometry (n=3). (B) The autophagy level of RAW264.7 cells after stimulation with TEPP-46 was assessed by measuring the protein expression of p62 and LC3 using Western blotting (n=3). (C) The microstructure of RAW264.7 cells stimulated with TEPP-46 was measured using transmission electron microscopy (TEM) (n=3). (D) mtDNA copy number was determined by measuring MTND1 relative to B2M using RT-PCR in RAW264.7 cells transducted with a control siRNA (con siRNA) or siPKM2 (n=4). (E) Western blot analysis of mtTFA in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). (F) The mitochondrial OCR was measured in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 by using a Seahorse XF96e Extracellular Flux analyzer. Dashed vertical lines indicate the addition of 1 mM oligomycin (Oligo), 0.5 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 mM rotenone plus 1 mM antimycin A (Rot/Ant) (n=3). (G–I) Quantitative analysis of basal respiration, ATP-linked respiration and maximal respiration in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). *p < 0.05.

Article Snippet: We used antibodies against PKM2 (Cell Signaling, #4053, 1:1,000), PKM1 (Cell Signaling, #7067, 1:1,000), PGC-1a (Cell Signaling, #2178, 1:1,000), PGC-1b (Abcam, ab176328, 1:1,000), p62 (Cell Signaling, #16177, 1:1,000), LC3 (Abcam, ab192890, 1:1,000), mtTFA (Abcam, ab252432, 1:1,000), NRF1 (Abcam, ab221792, 1:1,000), NRF2 (Abcam, ab137550, 1:1,000), p-AMPK (Cell Signaling, #4186, 1:500), AMPK (Cell Signaling, #4150, 1:1,000), SIRT1 (Abcam, ab189494, 1:1,000), p-Akt (Abcam, ab38449, 1:500), Akt (Abcam, ab8805, 1:500), p-PI3K (Cell Signaling, #17366, 1:1,000), PI3K (Cell Signaling, #4255, 1:1,000), and GAPDH (Santa Cruz, sc365062, 1:1,000).

Techniques: Cytometry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 3. Cdk inhibition down-regulates SQSTM1/p62 but fails to affect LC3 processing during

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques: Inhibition

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 4. SQSTM1/p62 down-regulation results in cargo loading failure and inefficient autophagy.

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 5. Expression of SQSTM1/p62 diminishes the increased lethality of BH3-mimetics in p62-

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 9. A mechanistic model of SQSTM1/p62 and NBK/Bik acting as novel molecular switches

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

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: 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: Journal of nanobiotechnology

Article Title: Extracellular vesicles from adipose-derived stromal/stem cells reprogram dendritic cells to alleviate rat TMJOA by transferring mitochondria.

doi: 10.1186/s12951-025-03478-9

Figure Lengend Snippet: Fig. 6 Transferred functional mitochondria activate the ERK1/2/FoxO1/autophagy pathway. (A) GO analysis of variably expressed messenger RNA associ ated signaling pathways in EVs vs. LPS groups. (B) Top 20 common pathways from RNA-seq and Metabolomics analyses between EVs vs. LPS groups. (C) Representative immunofluorescence images of LC-3 in the DC, LPS, EVs and R-EVs groups. (D) Representative immunofluorescence images of mitoSOX in the DC, LPS, EVs and R-EVs groups. (E) Quantification of LC-3 and mitoSOX in the DC, LPS, EVs and R-EVs groups. (F-G) Illustrative Western Blots (F) and quantification (G) for JNK, P-JNK, P38, P-P38, ERK1/2, P-ERK1/2, FoxO1, P-FoxO1, LC-3, p62 and Actin in LPS, EVs and R-EVs groups. (H-I) Illustrative Western Blots (H) and quantification (I) for FoxO1, P-FoxO1, LC-3, p62 and Actin in LPS, EVs, AS and DMSO groups. (J) Representative immunofluorescence images of CD11c and LC-3 expression in different groups. (K) Quantification of colocalization between CD11c and LC-3. (L) Schematic diagram illustrating the autophagy activation pathway

Article Snippet: The membrane was incubated at a temperature of 4 °C for the duration of the night with primary antibody: anti-FoxO1 (2880, Cell Signaling Technology), anti-P-FoxO1 (9461, Cell Signaling Technology), anti-LC-3 (83506, Cell Signaling Technology), anti-p62 (39749, Cell Signaling Technology), anti-ERK1/2 (sc514302, Santa Cruz Biotechnology), anti-P-ERK1/2 (sc81492, Santa Cruz Biotechnology), anti-JNK (9252, Cell Signaling Technology), anti-P-JNK (9251, Cell Signaling Technology), anti-P38 (8690, Cell Signaling Technology), anti-P-P38 (28796-1-AP, Proteintech), or Actin (4970, Cell Signaling Technology).

Techniques: Functional Assay, Protein-Protein interactions, RNA Sequencing, Immunofluorescence, Western Blot, Expressing, Activation Assay

Journal: BMC cancer

Article Title: Anti-proliferative activity of RIHMS-Qi-23 against MCF-7 breast cancer cell line is through inhibition of cell proliferation and senescence but not inhibition of targeted kinases.

doi: 10.1186/s12885-023-11547-1

Figure Lengend Snippet: Fig. 4 RIMHS-Qi-23 acts through affecting autophagy and necrosis pathways: a-d Immunoblotting of MCF-7 cell lines homogenates following treatment with serial concentration, incubated with cyclophlin A, p62, LC3 and β-actin as loading control e-g Quantification of immunoblots band values relative to β-actin were normalized to non-treated cells and represent mean ± SEM. Capital letters represent p values from One-Way ANOVA followed by a post-hoc test (similar letters = a statistically non-significant difference, while different letters = a statistically significant difference). Bold values denote significant p values (≤ 0.05)

Article Snippet: Primary antibodies against LC3B (GeneTex catalog no. GTX127375, dilution 1:1000), p62 (SantaCruz Biotechnology catalog no. sc-48389, dilution 1:500), cyclophilin A (Cell signaling catalog no. 2175, dilution 1:1000) and β-actin (Abcam ab227387, dilution 1:5000) were incubated overnight at 4 °C.

Techniques: Western Blot, Concentration Assay, Incubation, Control

Journal: Blood cancer journal

Article Title: The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy.

doi: 10.1038/s41408-022-00747-w

Figure Lengend Snippet: Fig. 3 THZ-P1-2 induces markers of apoptosis, DNA damage, and blockage of autophagic flow. A Western blot analysis for PARP1, γH2AX, p62/SQSTM1, and LC3B in total extracts from MV4-11, OCI-AML3, Jurkat, and NALM6 cells treated with vehicle or with increasing doses of THZ- P1-2 (1.6, 3.2, and 6.4 µM) for 24 h. Membranes were reincubated with α-tubulin antibody and developed with the SuperSignal™West Dura Extended Duration Substrate system and GBox. Band intensities of cleaved-PARP1, γH2AX, p62/SQSTM1, and LC3B were corrected by α- tubulin expression and normalized by vehicle-treated cells. B Heatmap depicting the gene expression profile of leukemic cell lines treated with vehicle or THZ-P1-2 (6.4 μM) for 24 h. The blue color in the heatmap indicates decreased mRNA levels, while red indicates induced mRNA levels, which were normalized by vehicle-treated cells (n = 4). Leukemia cells that showed the lowest rates of THZ-P1-2-induced apoptosis were considered resistant. C Network for THZ-P1-2 modulated genes constructed using the GeneMANIA database (https://genemania.org/). A total of seven genes (BBC3, ATG5, MAP1LC3B, MCL1, CDKN1B, BAX, and BCL211) were significantly modulated in all cell lines tested and are illustrated as crosshatched circles; the interacting genes included by modeling the software are indicated by circles without crosshatched. The main biological interactions and associated functions are described in the literature.

Article Snippet: Antibodies directed against PARP1 (#9542), γH2AX (#9718), SQSTM1/p62 (#88588), LC3B (#2775), and α-tubulin (#2144) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Gene Expression, Construct, Software