Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Knockdown of MYH10 suppresses neurodegeneration caused by FTD-associated mutant CHMP2B in iPSC-derived cortical neurons. (A) Schematic of the experimental timeline of the differentiation of cortical neurons from iPSCs and the lentiviral transduction strategy. (B) Viability of iPSC-derived cortical neurons transfected with CHMP2B WT , CHMP2B intron5 , and control or MYH10 shRNAs. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey post-hoc test for multiple comparisons. (C) Western blot analysis of the lysates of iPSC-derived cortical neurons with anti-MYH10, anti-SQSTM1, anti-CHMP2B, anti-ACTB/β-actin, or anti-LC3 antibodies. (D-F) Quantification of MYH10 knockdown (D) and autophagic flux based on the ratio of LC3-II (E) or SQSTM1 (F) relative to ACTB in iPSC-derived cortical neurons. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by one-way ANOVA with Tukey post-hoc test for multiple comparisons.

Article Snippet: GFP-SQSTM1 , Sino Biological , HG17112-ANG.

Techniques: Mutagenesis, Derivative Assay, Transduction, Transfection, Western Blot

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 is essential for autophagosome formation. (A) Autophagic flux assay. Lysates of wild-type (WT) or MYH10 knockout (KO) U2OS cells were subjected to amino acid starvation in the absence or presence of bafilomycin A 1 (BafA1, 100 nM) for 2 h and analyzed by western blot with antibodies against MYH10, SQSTM1, ACTB, or LC3. (B-C) Quantification of autophagic flux based on the difference of LC3-II (B) or SQSTM1 (C) relative to ACTB in WT or MYH10 KO U2OS cells. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. (D) Representative immunofluorescence images of LC3- and SQSTM1-positive autophagosomes in wild-type (WT) or MYH10 knockout (KO) U2OS cells after amino acid starvation in the absence or presence of bafilomycin A 1 (BafA1, 100 nM) for 2 h. Scale bar: 20 μm. (E-F) Number of LC3- (E) or SQSTM1-positive (F) puncta per cell. Cells were counted with the ITCN plugin using FIJI. Values are mean ± SEM of n > 15 randomly selected cells. ****P < 0.0001, ns, not significant by two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.

Article Snippet: GFP-SQSTM1 , Sino Biological , HG17112-ANG.

Techniques: Flux Assay, Knock-Out, Western Blot, Immunofluorescence

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 recruits autophagic receptor proteins to autophagosomes. (A) Co-immunoprecipitation analysis of MYH10 and autophagic receptors. HEK293T cells were transfected with vectors expressing proteins tagged with Flag-MYH10 and green fluorescent proteins (GFP, GFP-OPTN, GFP-CALCOCO2, or GFP-SQSTM1), and were subjected to co-immunoprecipitation with anti-Flag agarose beads in the absence or presence of amino acid starvation for 2 h. Western blotting was done with anti-Flag antibody or anti-GFP antibody. (B) Cellular localization of GFP-MYH10 with monomeric red fluorescent protein (mRFP)-autophagic receptors (OPTN, CALCOCO2, and SQSTM1). U2OS cells stably expressing GFP-MYH10 were transiently transfected with mRFP-OPTN, -CALCOCO2, or -SQSTM1. Scale bar: 20 μm. (C and F) Cells stably expressing YFP-PRKN were stained with MitoTracker (red) and antibodies against SQSTM1 (C) or OPTN (F) and treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 20 μM) and bafilomycin A 1 (BafA1, 100 nM) for 2 h. White arrowheads indicate PRKN-positive and SQSTM1- (C) or OPTN-negative (F) mitochondria. Scale bar: 20 μm. (D-H) Number and relative percentage of PRKN- and SQSTM1- (D and E) or OPTN-positive (G and H) mitophagosomes, which was calculated as the number of SQSTM1- or OPTN-positive mitophagosomes divided by the total number of PRKN-positive damaged mitochondria. Values are mean ± SEM of n >15 randomly selected cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by paired two-tailed t test.

Article Snippet: GFP-SQSTM1 , Sino Biological , HG17112-ANG.

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Stable Transfection, Staining, Two Tailed Test

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: GFP-SQSTM1 , Sino Biological , HG17112-ANG.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Representative immunohistochemistry showing the expression levels of ANXA1, SQSTM1, and p-AKT (S473) in the NPCs with different metastatic potentials. Scale bar = 50 μm. b Correlation analysis of ANXA1 and SQSTM1expressin in 127 NPCs based on immunohistochemistry scores (Spearman’s correlation test). c ANXA1 positively regulating SQSTM1 expression. Western blot analysis showing the expression levels of SQSTM1 in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. d ANXA1 regulating SQSTM1 expression through autophagy. Western blot analysis showing the expression levels of SQSTM1 in the ANXA1 knockdown 5–8F cells and scramble shRNA control cells treated with BAF or CQ (left) or transfected with BECN1 or ATG5 siRNA (right). Scr, scramble. Vector, an empty vector; KD, knockdown; OE, overexpression; BAF, bafilomycin A1; CQ, chloroquine

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Immunohistochemistry, Expressing, Western Blot, Control, Knockdown, shRNA, Transfection, Plasmid Preparation, Over Expression

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Western blot analysis showing the expression levels of BECN1, SQSTM1 and LC3-II in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. b Electron microscopic examination showing autophagic vacuoles ( red arrows ) in the cytoplasm of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 5 μm. c ( top ) Immunofluorescent staining showing the number of LC3 puncta in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Cells was stained by indirect immunefluorescence using anti-LC3 antibody and observed by confocal microscopy (bottom). The number of LC3 puncta per cell was quantified. Scale bar = 10 μm. d ANXA1 knockdown enhances autophagic flux (left). Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells treated with BAF (middle and right). The number of EGFP-LC3 puncta in the ANXA KD1 5–8F cells and scramble shRNA control cells treated with BAF. Cells were transfected with 1 μg of EGFP-LC3 plasmid for 24 h, and BAF treatment for an additional 24 h, thereafter the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. e Autophagy induction by ANXA1 knockdwon follows an autophagic pathway dependent of BECN1 and ATG5. (left) Western blot analysis showing the LC3-II levels in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. (middle and right) The number of EGFP-LC3 puncta in the ANXA KD 5–8F cells and scramble shRNA control cells transfected with BECN1 or ATG5 siRNA. Cells were cotransfected with 1 μg of EGFP-LC3 plasmid and siRNA against BECN1 or ATG5 for 24 h, and the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. Mean ± SD and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns, no significance

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Control, Staining, Confocal Microscopy, Knockdown, shRNA, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a The differential phospho-kinases in the ANXA1 KD 5–8F cells and control cells identified by phospho-kinase antibody array (left). The levels of p-AKT (S473 and T308) and p-ERK1/2 (T202/Y204) are shown (right). Mean ± SD ( n = 2 replicates) and statistical significance are denoted; ** P < 0.01; ns, no significance. b Western blot analysis showing the levels of p-AKT (T308 and S473) and p-ERK1/2 (T202/Y204) in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. c Immunofluorescent staining showing the levels and membrane translocation of p-AKT (S473) in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 50 μm. d (top) Western blot analysis showing the expression levels of BECN1, SQSTM1 and LC3-II in the ANXA1 OE 6–10B cells and vector control cells treated with LY294002 or MK2206. (middle and bottom) The number of EGFP-LC3 puncta in the ANXA OE 6–10B cells and vector control cells treated with LY294002 or MK2206. Cells were transfected with 1 μg of EGFP-LC3 plasmid for 24 h, and LY294002 or MK2206 treatment for an additional 24 h, thereafter the number of EGFP-LC3 puncta was examined and quantified by fluorescence microscopy. Scale bar = 10 μm. Mean ± SD and statistical significance are denoted; ** P < 0.01; ns, no significance. e Western blot analysis showing the expression levels of SQSTM1 and LC3-II in the ANXA1 KD 5–8F cells and scramble shRNA control cells treated with U0126

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Control, Ab Array, Western Blot, Staining, Membrane, Translocation Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy, shRNA

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a Scratch wound-healing showing the migration of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 100 μm. b Matrigel invasion assay showing the invasion of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Mean ± SD ( n = 3 replicates) and statistical significance are denoted; *** P < 0.001. Scale bar = 100 μm. c The in vivo metastasis assays of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells using the experimental lung metastasis model in nude mice ( n = 10 mice each). The representative photography of lungs (top) and H&E staining of lung sections (middle) from each group showing metastatic tumors (white arrows), and the numbers of surface lung metastases per mouse are shown (bottom). Mean ± SEM ( n = 10) and statistical significance are denoted; *** P < 0.001. Scale bar = 100 μm. d Representative immunohistochemistry showing the expression of ANXA1, SQSTM1, p-AKT (S473), E-cadherin, N-cadherin, and Vimentin in the mice lung metastases of ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. Scale bar = 100 μm. Mean ± SD and statistical significance are denoted; ** P < 0.01

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Migration, Control, Invasion Assay, In Vivo, Staining, Immunohistochemistry, Expressing

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: a QRT-PCR showing the mRNA expression levels of ANXA1, E-cadherin, N-cadherin, Vimentin, and Snail in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. b Western blot analysis showing the protein expression levels of E-cadherin, N-cadherin, Vimentin and Snail in the ANXA1 KD 5–8F cells, ANXA1 OE 6–10B cells and their control cells. c Western blot analysis showing the protein expression levels of E-cadherin, N-cadherin, Vimentin, and Snail in the EphA2 KD 5–8F cells and scramble shRNA control cells transfected with BECN1 siRNA or ATG5 siRNA (left), or treated with 3-MA (right). d Western blot analysis showing the Snail protein expression level in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with SQSTM1 expression plasmid, and ANXA1 OE 6–10B cells and vector control cells transfected with SQSTM1 siRNA. e QRT-PCR showing the Snail mRNA expression level in the ANXA1 KD 5–8F cells and scramble shRNA control cells transfected with SQSTM1 expression plasmid, and ANXA1 OE 6–10B cells and vector control cells transfected with SQSTM1 siRNA. Mean ± SD ( n = 3 replicates) and statistical significance are denoted; * P < 0.05; ** P < 0.01; *** P < 0.001; no, no significance

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, shRNA, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Annexin A1-suppressed autophagy promotes nasopharyngeal carcinoma cell invasion and metastasis by PI3K/AKT signaling activation

doi: 10.1038/s41419-018-1204-7

Figure Lengend Snippet: In this model, ANXA1 activates PI3K/AKT signaling, leading to BECN1 and ATG5-dependent autophagy inhibition; autophagy inhibition makes SQSTM1 accumulation, which inhibits the degradation of Snai1; the increased Snai1 induces EMT-like alterations, and then promotes NPC cell migration and invasion and metastasis

Article Snippet: SiRNAs against BECN1 (sc-29797), ATG5 (sc-41445) and SQSTM1 (sc-29679), purchased from Santa Cruz, were used to silence the BECN1, ATG5 and SQSTM1 genes in the indicated cells in according to the manufacturer’s protocol.

Techniques: Inhibition, Migration

Journal: The Journal of Biological Chemistry

Article Title: Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis *

doi: 10.1074/jbc.M115.675371

Figure Lengend Snippet: Nrf2/p62 plays a critical role for cell survival and cell death resistance in the AsT cells. The basal expression levels of Nrf2 and p62 were measured in the AsT cells and non-transformed BEAS-2B cells (A and F). The normal and AsT cells were exposed to various concentrations of As3+ (0–20 μm) for 24 h (B and G) or various times (0–24 h) with 20 μm As3+ (C and H), and then the levels of Nrf2, Keap1, p62, heme oxygenase-1 (HO-1), and NQO1 were detected. To diminish Nrf2 or p62 levels, cells were transfected with siRNA specific to Nrf2 or p62. After overnight transfection, cells were exposed to 20 μm As3+ for an additional 24 h (D and I), and an apoptosis assay was performed (E and J). The expression levels of Nrf2 or p62 were quantified and are represented in the lower panels. The results represent the mean ± S.E. (error bars) of three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent a significant difference between the experiments (ANOVA and Scheffé's test). S, short exposure; L, long exposure; Cont, control.

Article Snippet: Four unique human 29-mer shRNA constructs in retroviral GFP vector for Nrf2 (TG311194) and p62 (TG309121) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, Transformation Assay, Transfection, Apoptosis Assay

Journal: The Journal of Biological Chemistry

Article Title: Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis *

doi: 10.1074/jbc.M115.675371

Figure Lengend Snippet: Nrf2 and p62 have a positive feedback loop, and the binding activities of Nrf2 to the ARE regions of the p62, Bcl-2, and Bcl-xL promoters were increased in the arsenic-exposed AsT cells. Cells were transfected with siRNA specific to p62 or Nrf2. After 12 h of transfection, the cells were treated with 20 μm As3+ for an additional 24 h. The expression levels of p62, Nrf2, heme oxygenase-1 (HO-1), and Bcl-2/Bcl-xL were analyzed (A and D). The expression levels of Nrf2/p62 and Bcl-2/Bcl-xL were quantified and are represented in A (lower panel) and D (lower panel), respectively. For the ChIP assay, cells were treated with As3+ (20 μm) for 6 h, fixed with formaldehyde, and cross-linked, and then chromatin was isolated. The chromatin was immunoprecipitated (IP) with an anti-Nrf2 antibody or control mouse IgG. The Nrf2 binding to the p62, Bcl-2, and Bcl-xL promoters was analyzed by normal real time PCR (B and E) or quantitative real time PCR (C and F) with a primer specific for the ARE region of the promoter. The data represent the percent input and are normalized to each control. The results are expressed as the mean ± S.E. (error bars) relative to the control of triplicate experiments. *, p < 0.05 and ***, p < 0.001 versus the untreated transformed control cells; ###, p < 0.001 versus the As3+-treated transformed cells (ANOVA and Scheffé's test). Actin was used as a loading control. Cont, control.

Article Snippet: Four unique human 29-mer shRNA constructs in retroviral GFP vector for Nrf2 (TG311194) and p62 (TG309121) were purchased from OriGene Technologies, Inc.

Techniques: Binding Assay, Transfection, Expressing, Isolation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis *

doi: 10.1074/jbc.M115.675371

Figure Lengend Snippet: AsT cells are characterized as having autophagy deficiency. The basal expression level of autophagy-related proteins (ATG3, ATG5, and ATG7) were measured (A). Cells were exposed to various concentrations of As3+ (0–20 μm) for 24 h, and then the levels of LC3, ATG3, ATG5, and ATG7 were detected (B). The cells were transfected with the GFP-LC3 plasmid, treated with As3+, and visualized (C), and the number of GFP-LC3 punctum-positive cells was counted (D). To analyze autophagy flux, the cells were preincubated with bafilomycin A1 (Baf) (100 nm) or wortmannin (Wort) (100 nm) for 1 h before treatment with As3+. After 24-h incubation, the levels of LC3-I and LC3-II were detected (E). The expression level of LC3-II was quantified and is represented in F. The expression levels of LC3-I and LC3-II were further analyzed after exposure to rapamycin (10 nm) or in starvation condition (G) for further confirmation of autophagy deficiency of the AsT cells. The binding affinity of Bcl-2 with Beclin1 was analyzed by immunoprecipitation (K). The ubiquitination of p62 was further analyzed using anti-ubiquitin after performing immunoprecipitation (IP) (J). In addition, the cells were transfected with the mCherry-EGFP-LC3 plasmid and treated with As3+. The yellow puncta (mCherry+/GFP+) and red puncta (mCherry+/GFP−) were visualized using fluorescence microscopy (H), and quantification of puncta is represented in I. For the inhibition assay, cells were incubated with As3+ (20 μm) for 24 h in the presence and absence of rapamycin (Rapa) (100 nm) or wortmannin (100 nm). Thereafter, cell viability was determined (L). The shRNA Beclin1-transfected stable BEAS-2B cells were treated with As3+ (0–20 μm) for 24 h, and the apoptotic cells (M) and fluorescent punctum-positive cells (N) were determined. The results are shown as the mean ± S.E. (error bars) in triplicate or three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent a significant difference between the experiments (ANOVA and Scheffé's test). Scale bars, 20 (C) and 50 μm (H). Cont, control.

Article Snippet: Four unique human 29-mer shRNA constructs in retroviral GFP vector for Nrf2 (TG311194) and p62 (TG309121) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Binding Assay, Immunoprecipitation, Fluorescence, Microscopy, Inhibition, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis *

doi: 10.1074/jbc.M115.675371

Figure Lengend Snippet: Nrf2 and p62 regulate intracellular ROS levels. ROS are a primary regulator of Nrf2 and p62 expression and the main mediator of apoptotic and autophagic cell death. AsT cells were transfected with siRNA specific to Nrf2 or p62. After overnight transfection, ROS levels were measured using fluorescence microscopy (A), a fluorescence microplate reader (B), and ESR (C and D). Cells were transfected with siRNA specific to p62 or Nrf2. After 12 h of transfection, the cells were exposed to 20 μm As3+ for an additional 24 h. The expression levels of catalase and SOD2 were measured (E). In addition, the catalase-, SOD1-, and SOD2-overexpressing stable BEAS-2B cells were exposed to As3+ (20 μm). The expression levels of p62, Nrf2, cleaved (c) caspase-3, and LC3 were analyzed (F), and then cell viability was determined (G). The results are shown as the mean ± S.E. (error bars) in triplicate experiments or three separate experiments. **, p < 0.01 and ***, p < 0.001 versus the unexposed BEAS-2B cells or the vehicle control (ANOVA and Scheffé's test). Scale bars in A, 100 μm. CAT, catalase; Cont, control; DCF, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate.

Article Snippet: Four unique human 29-mer shRNA constructs in retroviral GFP vector for Nrf2 (TG311194) and p62 (TG309121) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, Transfection, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis *

doi: 10.1074/jbc.M115.675371

Figure Lengend Snippet: Antioncogenic or oncogenic role of Nrf2 and its critical role in lung adenocarcinoma development. BEASE-2B cells in which Nrf2 or p62 was knocked down with shRNA transfection were exposed to 100 nm As3+ for 6 months, and soft agar (A) and xenograft assays (B) were performed. AsT cells were transfected with shRNA specific for either Nrf2 or p62, and then a soft agar assay (E) and clonal assay (F) were performed. Human lung adenocarcinoma tissues (stage IA or IIA) were homogenized, and Western blotting analysis (D) or immunohistochemical staining (C) was performed. The results are shown as the mean ± S.E. (error bars) in three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus the As3+-exposed vehicle control or the untreated vehicle control cells (ANOVA and Scheffé's test). Actin was used as an internal control. 1550, stage IIA; 1294, stage IIA; 1048, stage IA. G, scheme showing the dual role of Nrf2 in As3+-exposed human bronchial epithelial cells. Scale bars in C and E, 50 μm. CAT, catalase; Cont, control; N, normal; T, tumor.

Article Snippet: Four unique human 29-mer shRNA constructs in retroviral GFP vector for Nrf2 (TG311194) and p62 (TG309121) were purchased from OriGene Technologies, Inc.

Techniques: shRNA, Transfection, Soft Agar Assay, Clone Assay, Western Blot, Immunohistochemical staining, Staining

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Knockdown of MYH10 suppresses neurodegeneration caused by FTD-associated mutant CHMP2B in iPSC-derived cortical neurons. (A) Schematic of the experimental timeline of the differentiation of cortical neurons from iPSCs and the lentiviral transduction strategy. (B) Viability of iPSC-derived cortical neurons transfected with CHMP2B WT , CHMP2B intron5 , and control or MYH10 shRNAs. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey post-hoc test for multiple comparisons. (C) Western blot analysis of the lysates of iPSC-derived cortical neurons with anti-MYH10, anti-SQSTM1, anti-CHMP2B, anti-ACTB/β-actin, or anti-LC3 antibodies. (D-F) Quantification of MYH10 knockdown (D) and autophagic flux based on the ratio of LC3-II (E) or SQSTM1 (F) relative to ACTB in iPSC-derived cortical neurons. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by one-way ANOVA with Tukey post-hoc test for multiple comparisons.

Article Snippet: mRFP-SQSTM1 , Sino Biological , HG17112-ANR.

Techniques: Mutagenesis, Derivative Assay, Transduction, Transfection, Western Blot

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 is essential for autophagosome formation. (A) Autophagic flux assay. Lysates of wild-type (WT) or MYH10 knockout (KO) U2OS cells were subjected to amino acid starvation in the absence or presence of bafilomycin A 1 (BafA1, 100 nM) for 2 h and analyzed by western blot with antibodies against MYH10, SQSTM1, ACTB, or LC3. (B-C) Quantification of autophagic flux based on the difference of LC3-II (B) or SQSTM1 (C) relative to ACTB in WT or MYH10 KO U2OS cells. Values are mean ± SEM of 4 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. (D) Representative immunofluorescence images of LC3- and SQSTM1-positive autophagosomes in wild-type (WT) or MYH10 knockout (KO) U2OS cells after amino acid starvation in the absence or presence of bafilomycin A 1 (BafA1, 100 nM) for 2 h. Scale bar: 20 μm. (E-F) Number of LC3- (E) or SQSTM1-positive (F) puncta per cell. Cells were counted with the ITCN plugin using FIJI. Values are mean ± SEM of n > 15 randomly selected cells. ****P < 0.0001, ns, not significant by two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.

Article Snippet: mRFP-SQSTM1 , Sino Biological , HG17112-ANR.

Techniques: Flux Assay, Knock-Out, Western Blot, Immunofluorescence

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: MYH10 recruits autophagic receptor proteins to autophagosomes. (A) Co-immunoprecipitation analysis of MYH10 and autophagic receptors. HEK293T cells were transfected with vectors expressing proteins tagged with Flag-MYH10 and green fluorescent proteins (GFP, GFP-OPTN, GFP-CALCOCO2, or GFP-SQSTM1), and were subjected to co-immunoprecipitation with anti-Flag agarose beads in the absence or presence of amino acid starvation for 2 h. Western blotting was done with anti-Flag antibody or anti-GFP antibody. (B) Cellular localization of GFP-MYH10 with monomeric red fluorescent protein (mRFP)-autophagic receptors (OPTN, CALCOCO2, and SQSTM1). U2OS cells stably expressing GFP-MYH10 were transiently transfected with mRFP-OPTN, -CALCOCO2, or -SQSTM1. Scale bar: 20 μm. (C and F) Cells stably expressing YFP-PRKN were stained with MitoTracker (red) and antibodies against SQSTM1 (C) or OPTN (F) and treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 20 μM) and bafilomycin A 1 (BafA1, 100 nM) for 2 h. White arrowheads indicate PRKN-positive and SQSTM1- (C) or OPTN-negative (F) mitochondria. Scale bar: 20 μm. (D-H) Number and relative percentage of PRKN- and SQSTM1- (D and E) or OPTN-positive (G and H) mitophagosomes, which was calculated as the number of SQSTM1- or OPTN-positive mitophagosomes divided by the total number of PRKN-positive damaged mitochondria. Values are mean ± SEM of n >15 randomly selected cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by paired two-tailed t test.

Article Snippet: mRFP-SQSTM1 , Sino Biological , HG17112-ANR.

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Stable Transfection, Staining, Two Tailed Test

Journal: Autophagy

Article Title: Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

doi: 10.1080/15548627.2023.2169309

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: mRFP-SQSTM1 , Sino Biological , HG17112-ANR.

Techniques: Protease Inhibitor, Electron Microscopy, Silver Staining, Plasmid Preparation, Negative Control, Subcloning, Recombinant, Expressing, shRNA

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a targets several autophagy related genes. ( a ) Relative mRNA levels (normalized by GAPDH) of BECN1, SQSTM1, ATG16L1 in MCF7 or ( c ) MDA-MB-231 cells overexpressing NC or miR-20a were analyzed by qPCR. ( b ) MCF7 or ( d ) MDA-MB-231 cells transfected with NC, miR-20a, LNA-NC or LNA-20a were either untreated or treated with EBSS for 4 h. The protein levels of ATG16L1, BECN1 and SQSTM1 were analyzed by immunoblotting. ( e ) Schematic illustration of the predicted miR-20a pairing sites in BECN1, SQSTM1 and ATG16L1 3′UTRs. Luciferase reporters containing wild type (WT) or mutated (MUT) BECN1, SQSTM1 or ATG16L1 3′UTR fragments were co-transfected with NC or miR-20a into MCF7 cells. Relative luciferase activity was normalized to NC (** P <0.01).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a increases intracellular ROS levels and DNA damage. ( a ) Immunofluorescence of γH2AX in MCF7 cells overexpressing NC or miR-20a, γH2AX fluorescence intensity was quantified with Image J software (*** P <0.001). ( b ) Protein expression of γH2AX and GAPDH were detected by immunoblotting in MCF7 cells overexpressing miRNAs or LNA inhibitors. ( c ) Comet assay in MCF7 cells overexpressing NC or miR-20a. Tail moment (TM) of at least 50 cells in three independent experiments were analyzed by the specific software CASP (** P <0.01). ( d ) MCF7 cells overexpressing miRNAs or siRNAs were cultured in normal medium or EBSS medium for 4 h. Intracellular ROS levels were determined by Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. ( e ) The expression levels of γH2AX, BECN1, SQSTM1, ATG16L1, LC3 and ACTB in MCF7 cells transfected with siRNAs were determined by western blotting. ( f ) Relative γH2AX protein expression (normalized by ACTB) was determined by Image J densitometric analysis. ( g ) DQ Red BSA fluorescence intensity in MCF7 cells transfected with siRNAs against ATG16L1, BECN1 or SQSTM1 (* P <0.05, ** P <0.01). ( h ) miR-20a and plasmids (BECN1-GFP, FLAG-ATG16L1, HA-SQSTM1) were co-transfected into MCF7 cells, cells were cultured in normal medium or EBSS medium for 4 h, samples were collected for immunoblotting analysis. ( i ) Relative γH2AX protein expression (normalized by ACTB) was determined by Image J densitometric analysis. ( j ) Proteolytic activity was analyzed in MCF7 cells expressing miR-20a and plasmids (* P <0.05).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Immunofluorescence, Fluorescence, Software, Expressing, Western Blot, Single Cell Gel Electrophoresis, Cell Culture, Transfection, Activity Assay

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: Expression of miR-20a and its target genes in breast cancer specimens. ( a ) Expression of miR-20a in normal breast ( n =30) and breast tumor tissues ( n =30) was determined by in situ hybridization. ( b ) miRNA in situ hybridization and HE staining in tissues from a triple-negative breast cancer patient. ( c ) Immunohistochemical analysis of γH2AX, BECN1, SQSTM1 and ATG16L1 in triple-negative breast cancer tissues and adjacent normal tissues. ( d ) Immunoblotting analysis of SQSTM1, ATG16L1, BECN1 and OPTN in normal mammary tissues and triple-negative breast cancer tissues. Densitometric ratios of BCEN1, SQSTM1, OPTN and ATG16L1 versus ACTB were quantified by Image J software.

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing, In Situ Hybridization, Staining, Immunohistochemical staining, Western Blot, Software

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: Higher expression of miR-20a is associated with downregulation of BECN1 , ATG16L1 and SQSTM1 . ( a ) Correlation between miR-20a and BECN1, SQSTM1, and ATG16L1 was determined using Spearman coefficient analysis in TCGA breast cancer samples ( n =500). ( b ) The expression of BECN1, SQSTM1 and ATG16L1 in triple-negative breast cancers ( n =82) and other subtypes ( n =391).

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing

Journal: Oncogene

Article Title: MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability

doi: 10.1038/onc.2017.193

Figure Lengend Snippet: miR-20a promotes tumorigenesis in a xenograft mouse model. ( a ) Genomic instability (fraction of copy-number altered genome and mutation counts) was detected in TCGA breast cancer patients ( P <0.001, n =347). ( b ) MDA-MB-231 cells stably expressing NC or miR-20a were injected into nude mice. miR-20a promotes tumorigenesis compared with NC at day 8 of injection. ( c ) miR-20a promotes tumor growth in vivo . Tumor growth curve was determined by measuring the width and length every day (* P <0.05, ** P <0.01, *** P <0.001). ( d ) Oncogenic miR-20a directly targets ATG16L1, BECN1 and SQSTM1, inhibits autophagic flux and lysosomal proteolytic activity, increases ROS levels and accumulates DNA damage, which likely contribute to genome instability and tumor-initiating capacity.

Article Snippet: SQSTM1 plasmid was purchased from Addgene (Cambridge, MA, USA; 28027); BECN1-GFP and FLAG-ATG16L1 plasmids were purchased from GeneChem Co., Ltd (Shanghai, China).

Techniques: Mutagenesis, Stable Transfection, Expressing, Injection, In Vivo, Activity Assay