Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: The bimodal role of SIRT6 in melanoma growth at different stages in vivo. (A) Primary and metastatic melanoma cells stably transfected with SIRT6 overexpression vectors or SIRT6 shRNA vectors were subcutaneously injected into NOD/SCID nude mice (n = 5 per group) for the generation of subcutaneous xenograft tumors. Tumor volumes including tumor length (L) and width (W) were measured using vernier calipers every 3 d from d 6 after injection and then calculated according to the formula (L × W2)/2. Data are presented as means ± SD (from 5 individual mice). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (B) Tumor weights were analyzed at the terminal time point. At the end of 33 d, tumors were excised and weighed. The data are shown as means ± SD (from 5 mice per group). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (C and D) Tumors from sacrificed mice were dissected 33 d after subcutaneous injection and are shown in the indicated row representatively (n = 4 per group). (E and F) Representative immunoblotting analysis showing the expressions of IGF-AKT signaling-related proteins and autophagy markers (LC3 and SQSTM1) in tumors from sacrificed mice as indicated (n = 3 per group). WT1 and WT2, SIRT6-WT1 and SIRT6-WT2; FLAG, FLAG-tagged control vector; shRNA1 and shRNA2, SIRT6-shRNA1 and SIRT6-shRNA2; Ctr-shRNA, control shRNA. N, FLAG or Ctr-shRNA; W and T, SIRT6-WT1 and SIRT6-WT2; S and H, SIRT6-shRNA1 and SIRT6-shRNA2.

Article Snippet: Retroviral vectors synthesizing ATG5 shRNA or control shRNA were purchased from Ori Gene (TR314610).

Techniques: In Vivo, Stable Transfection, Transfection, Over Expression, shRNA, Injection, Western Blot, Plasmid Preparation

Journal: Cancer Research

Article Title: Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

doi: 10.1158/0008-5472.can-11-1094

Figure Lengend Snippet: Figure 2. Targeting autophagy restores IGR-Heu tumor cell susceptibility to CTL-mediated lysis. A, expression of Atg5 and Beclin1 by immunoblot in normoxic (pO2 21%) and hypoxic (pO2 1%) cells transfected with Atg5, Beclin1 (BECN1), or luciferase (Luc) siRNA. B, CTL-mediated cytotoxicity of IGR-Heu tumor cells at different E:T ratios. Data represent 3 independent experiments with SD. Statistically significant differences (indicated by asterisks) are shown (*, P < 0.05; **, P < 0.005; and ***, P < 0.0005).

Article Snippet: Atg5 short hairpin RNA (shRNA) expressing cells were generated by infection with commercial human APG5 shRNA lentiviral particles (Santa Cruz Biotechnology) according to manufacturer's protocol.

Techniques: Lysis, Expressing, Western Blot, Transfection, Luciferase

Journal: Cancer Research

Article Title: Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

doi: 10.1158/0008-5472.can-11-1094

Figure Lengend Snippet: Figure 3. Targeting autophagy inhibits hypoxia-induced pSTAT3. A, expression of pSTAT3 by immunoblot in IGR-Heu cells transfected with siRNA targeting autophagy genes and cultured under normoxia (pO2 21%) and hypoxia (pO2 1%). B, expression of pSTAT3 in IGR-Heu cells untreated () or treated (þ) with autophagy inhibitors HCQ (60 mmol/L) or 3 MA (1 mmol/L) and cultured under normoxia (N) or hypoxia for 16 hours. Hypoxia was generated as described in Fig. 1C. C, accumulation of hypoxia- dependent autophagosomes shown by confocal microscopy in hypoxic GFP-LC3 expressing IGR-Heu cells treated with HCQ (60 mmol/L). Nuclei were stained with 40,6-diamidino-2- phenylindole. Bar, 10 mm.

Article Snippet: Atg5 short hairpin RNA (shRNA) expressing cells were generated by infection with commercial human APG5 shRNA lentiviral particles (Santa Cruz Biotechnology) according to manufacturer's protocol.

Techniques: Expressing, Western Blot, Transfection, Cell Culture, Generated, Confocal Microscopy, Staining

Journal: Cancer Research

Article Title: Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

doi: 10.1158/0008-5472.can-11-1094

Figure Lengend Snippet: Figure 4. Autophagy and ubiquitin proteasome system cooperate to regulate pSTAT3 in hypoxic IGR- Heu cells. A, expression of pSrc by immunoblot in normoxic (N) and hypoxic cells transfected with Luciferase (Luc), Beclin1 (BECN1) or Atg5 siRNA. B, expression of pSrc and pSTAT3 by immunoblot in normoxic (N) and hypoxic cells untreated () or treated with Src kinase inhibitor (PP2, 10 mmol/L). C, immunoblot analysis of ubiquitinated protein in hypoxic cells untreated () or treated (þ) with 3MA or Bz. D, expression of pSrc and pSTAT3 in normoxic (N) and hypoxic cells untreated () or treated (þ) with Bz. Hypoxic cells were transfected with Luc, BECN1, or Atg5 siRNA. E, immunoblot analysis of pSrc and pSTAT3 polyubiquitination. Left, immunoblot analysis of the expression of HA-ubiquitin in control (C) untransfected and transfected cells cultured under normoxia (N) or hypoxia (H). Middle and right panels represent immunoprecipitation experiments using anti-HA antibody followed by immunoblot using anti-pSrc (middle) or anti-pSTAT3 (right).

Article Snippet: Atg5 short hairpin RNA (shRNA) expressing cells were generated by infection with commercial human APG5 shRNA lentiviral particles (Santa Cruz Biotechnology) according to manufacturer's protocol.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Transfection, Luciferase, Control, Cell Culture, Immunoprecipitation

Journal: Cancer Research

Article Title: Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

doi: 10.1158/0008-5472.can-11-1094

Figure Lengend Snippet: Figure 5. Involvement of p62 in autophagy-dependent degradation of hypoxia-induced pSTAT3. A, expression of pSTAT3 and pSrc by immunoblot in normoxic (N) and hypoxic cells untransfected () or transfected with Atg5 siRNA and Beclin1 and/or p62 siRNAs. Luc siRNA was used as control. B, immunoprecipitation (IP) of pSTAT3 and p62 in normoxic (N) or hypoxic (H) cells using p62 antibody on whole-cell lysate (1 mg). C, nuclear and cytoplasmic (cyto) expression of pSTAT3, pSrc, and p62 by immunoblot in cells under normoxia (N) and different duration of hypoxia (H).

Article Snippet: Atg5 short hairpin RNA (shRNA) expressing cells were generated by infection with commercial human APG5 shRNA lentiviral particles (Santa Cruz Biotechnology) according to manufacturer's protocol.

Techniques: Expressing, Western Blot, Transfection, Control, Immunoprecipitation

Journal: Cancer Research

Article Title: Blocking Hypoxia-Induced Autophagy in Tumors Restores Cytotoxic T-Cell Activity and Promotes Regression

doi: 10.1158/0008-5472.can-11-1094

Figure Lengend Snippet: Figure 6. (Continued) B, growth curve of control (B16-F10) or Beclin1-deficient (B16-F10 shRNA-Beclin1) engrafted tumor in C57BL/6 mice (n ¼ 10). Data (mean SEM) represent 3 independent experiments. Statistically significant differences (indicated by asterisks) are shown. (*, P < 0.05; **, P < 0.005; and ***, P < 0.0005). C, TUNEL staining of apoptotic cell death in tumors from mice shown in B. (a) and (b) show the apoptotic foci, and (c) and (d) show TUNEL-positive nuclei in indicated tumors. Boxes represent an enlarged region of apoptotic cells. Images represent 2 independent experiments using samples isolated from 3 individual mice. Statistically significant differences (indicated by asterisks) are shown (*, P < 0.05; **, P < 0.005; and ***, P < 0.0005).

Article Snippet: Atg5 short hairpin RNA (shRNA) expressing cells were generated by infection with commercial human APG5 shRNA lentiviral particles (Santa Cruz Biotechnology) according to manufacturer's protocol.

Techniques: Control, shRNA, TUNEL Assay, Staining, Isolation

Journal: Autophagy

Article Title: Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy

doi: 10.1080/15548627.2017.1360466

Figure Lengend Snippet: KISS1 regulates autophagy in the brain metastatic cells. (A) Immunofluorescence staining of paraffin-embedded sections of BrCa and brain metastatic BrCa tissues using anti-KISS1 (red, cytoplasmic and nuclear staining), anti-BCL2 (green, cytoplasmic staining) fluorescently-labeled antibodies and DAPI (blue, DNA staining) to visualize nuclei. Numerous cells with robust KISS1 expression identified by bright red signal (white arrows) or yellow and orange signal (in cells with visible colocalization of KISS1 and BCL2 signals), are present in representative tissue specimens. In contrast, brain metastatic BrCa tissue contains almost no KISS1-positive cells, but large number of cells expressing BCL2 suggesting a correlation between downregulation of KISS1 in metastatic cells and their resistance to apoptosis. Image magnification is 400X, scale bar: 20 µm. (B) Relative expression of BCL2+- and KISS1+-positive cells detected in 4 BrCa tissue specimens and 4 brain metastatic lesions of BrCa. The data are presented as amount of BCL2- and KISS1-positive cells detected in the patient samples ± SD. All changes are statistically significant (P = 0.0172 calculated by the Student t test). (C, D) Expression of human KISS1 in Ad-KISS1 vector-transduced MDA-MB-231Br cells results in suppression of autophagy. The cells (2 × 105) were infected with Ad or Ad-KISS1 vectors at a multiplicity of infection of 1 and the levels of autophagy-related proteins SQSTM1, LC3-II, ATG5 and ATG7 were analyzed by western blot (C), followed by densitometry-based semi-quantification, which is presented as a relative protein expression normalized by GAPDH as a loading control (D); black bars, control adenovirus (Ad)-infected cells; red bars, cells infected with human KISS1-encoding adenovirus (Ad-KISS1). The experiment was performed 3 times and average data are presented. (E to H) Knockdown of KISS1 by shRNA-mediated silencing induces autophagy in MDA-MB-231Br and 4T1Br cells. The cells were transduced with lentiviral vector or siRNA duplexes to generate stable or transient cell lines with silenced KISS1/Kiss1 expression. Stable cell lines with silenced KISS1 expression (KISS1-KD61 and KISS1-KD79), based on MDA-MB-231Br (E, (F)top panels), or transient 4T1Br-based cell lines Kiss1-KDA, Kiss1-KDB and Kiss1-KDC with siRNA-silenced Kiss1 expression (G, H), were used to analyze the autophagy response by assessing expression levels of ATG5, ATG7, SQSTM1 as well as LC3 form I to form II conversion rate by western blot (E, G). Cells expressing shScrambled or noncoding siRNA were used as a nontargeting control. The relative protein expression was quantified by semi-quantitative densitometry (F, H). The experiment was performed 3 times and representative data are shown.

Article Snippet: shRNA plasmids targeting the human ATG5 gene (Origene, TG314610; sh ATG5 versions A, B, C and D) and “scrambled” negative control (nontargeting shRNA cassette in pGFP-V-RS vector plasmid TR30013, Origene) were transfected into MDA-MB-231Br using DharmaFECT1 transfection reagent (GE Dharmacon, T-2001–01).

Techniques: Immunofluorescence, Staining, Labeling, Expressing, Plasmid Preparation, Infection, Western Blot, shRNA, Transduction, Stable Transfection

Journal: Cell Death and Differentiation

Article Title: Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

doi: 10.1038/cdd.2010.117

Figure Lengend Snippet: Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot

Article Snippet: Four HuSH 29mer shRNA constructs against human ATG5 (Locus ID=9474), with sequences listed in , were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Inhibition, Infection, shRNA, Staining, Microscopy, Western Blot

Journal: Cell Death and Differentiation

Article Title: Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

doi: 10.1038/cdd.2010.117

Figure Lengend Snippet: ATG5 shRNA constructs sequence

Article Snippet: Four HuSH 29mer shRNA constructs against human ATG5 (Locus ID=9474), with sequences listed in , were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: shRNA, Construct, Sequencing

Journal: Nano Convergence

Article Title: Ultrasmall iron oxide nanoparticles induced ferroptosis via Beclin1/ATG5-dependent autophagy pathway

doi: 10.1186/s40580-021-00260-z

Figure Lengend Snippet: Reversal effect on the USIONPs-induced ferroptosis by the interference of Beclin 1/ATG5 for 48 h. A - a Inhibitory rates of U251 cells after silencing of Beclin1 gene; A - b Concentrations of iron in the U251 cells after silencing Beclin1 gene; A - c Relative levels of lipid ROS in the U251 cells after silencing Beclin1 gene; A - d Relative levels of ROS in the U251 cells after silencing Beclin1 gene; A - e Representative images of expression of autophagy and ferroptosis related proteins in the U251 cells after silencing Beclin1 gene; A - f Relative expression of autophagy and ferroptosis related proteins in the U251 cells after silencing Beclin1 gene; B - a Inhibitory rates of U251 cells after silencing ATG5 gene; B - b Concentrations of iron in the U251 cells after silencing ATG5 gene; B - c Relative levels of lipid ROS in the U251 cells after silencing ATG5 gene; B - d Relative levels of ROS in the U251 cells after silencing ATG5 gene; B - e Representative images of expression of autophagy and ferroptosis related proteins in the U251 cells after silencing ATG5 gene; B - f Relative expression of autophagy and ferroptosis related proteins in the U251 cells after silencing ATG5 gene. * P < 0.05, statistically significant compared with control group. # P < 0.05, statistically significant compared with 200 μg/mL USIONPs

Article Snippet: The lentivirus(LV)expressing Beclin 1 shRNA (Sense: 5ˊ-CCCGTGGAATGGAATGAGATT- 3ˊ; Antisense: 5ˊ-AATCTCATTCCATTCCACGGG-3ˊ) and ATG5 shRNA (Sense:5ˊ-CCTGAACAGAATCATCCTTAA-3ˊ; Antisense: 5ˊ-TTAAGGATGATTCTGTTCAGG-3ˊ) were generated and produced by Keygen Inc.Co.

Techniques: Expressing

Journal: Nano Convergence

Article Title: Ultrasmall iron oxide nanoparticles induced ferroptosis via Beclin1/ATG5-dependent autophagy pathway

doi: 10.1186/s40580-021-00260-z

Figure Lengend Snippet: Stimulation of USIONPs-induced ferroptosis by the overexpression of Beclin 1/ATG5. A - a Inhibitory rates of U251 cells after overexpression of Beclin1 gene for 48 h; A - b Concentrations of iron in the U251 cells after overexpression of Beclin1 gene for 48 h; A - c Relative levels of lipid ROS in the U251 cells after overexpression of Beclin1 gene for 48 h; A - d Relative levels of ROS in the U251 cells after overexpression of Beclin1 gene for 48 h; A - e Representative images of expression of autophagy and ferroptosis related proteins in the U251 cells after overexpression of Beclin1 gene for 48 h; A - f Relative expression of autophagy and ferroptosis proteins in the U251 cells after overexpression of Beclin1 gene for 48 h; B - a Inhibitory rates of U251 cells after overexpression of ATG5 gene for 48 h; B - b Concentrations of iron in the U251 cells after overexpression of ATG5 gene for 48 h; B - c Relative levels of lipid ROS in the U251 cells after overexpression of ATG5 gene for 48 h; B - d Relative levels of ROS in the U251 cells after overexpression of ATG5 gene for 48 h; B - e Representative images of expression of autophagy and ferroptosis proteins in the U251 cells after overexpression of ATG5 gene for 48 h; B - f Relative expression of autophagy and ferroptosis related proteins in the U251 cells after overexpression of ATG5 gene for 48 h. * P < 0.05, statistically significant compared with control group. # P < 0.05, statistically significant compared with 200 μg/mL USIONPs

Article Snippet: The lentivirus(LV)expressing Beclin 1 shRNA (Sense: 5ˊ-CCCGTGGAATGGAATGAGATT- 3ˊ; Antisense: 5ˊ-AATCTCATTCCATTCCACGGG-3ˊ) and ATG5 shRNA (Sense:5ˊ-CCTGAACAGAATCATCCTTAA-3ˊ; Antisense: 5ˊ-TTAAGGATGATTCTGTTCAGG-3ˊ) were generated and produced by Keygen Inc.Co.

Techniques: Over Expression, Expressing