Journal: The Journal of Clinical Investigation

Article Title: Long noncoding RNA BCYRN1 promotes cardioprotection by enhancing human and murine regulatory T cell dynamics

doi: 10.1172/JCI179262

Figure Lengend Snippet: ( A ) Human iTregs were exposed to CDC-EVs or not treated (ctrl). The expression of autophagy markers (MAP1LC3B, ATG7, and SQSTM1) was assessed by Western blot (WB). ( B ) Human iTregs were exposed to ctrl-CDC-EVs or si-BCYRN1 CDC-EVs (EV with BCYRN1 knockdown), or were not treated (ctrl). Expression of autophagy markers (MAP1LC3B, ATG7, and SQSTM1) was assessed by WB. ( C ) Human iTregs were transfected with vector or OE-BCYRN1 lenti-vector, followed by assessment of autophagy markers by WB. ( D ) Biotin-labeled BCYRN1 probe was used to pull down BCYRN1-associated RNAs, followed by assessment of miR-138, negative control ( U6 and GAPDH ), and positive control (BCYRN1) expression by qPCR. ( E ) The top panel shows putative lncRNA BCYRN1 binding sites in miR-138. The bottom panel shows human iTregs that were cotransfected with WT or mutant luciferase reporters with mimic miR-138 into HEK-293T cells, followed by assessment of relative luciferase activity. ( F ) Cotransfection of miR-138 and BCYRN1 in Tregs followed by assessments of ATG7 by WB. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001 versus control group.

Article Snippet: Blots were blocked with 3% BSA in TBS-Tween 20, and probed with antibodies specific for ATG7 (1:1,000; Novus Biologicals, MAB6608), MAP1LC3B (1:1,000; Novus Biologicals, NB100-2220), SQSTM1 (1:1,000; MBL International, PM045), and ACTB (1:5,000; Thermo Scientific, MA5-15739-HRP).

Techniques: Expressing, Western Blot, Knockdown, Transfection, Plasmid Preparation, Labeling, Negative Control, Positive Control, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Control

Journal: Endocrine-Related Cancer

Article Title: Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk

doi: 10.1530/erc-16-0359

Figure Lengend Snippet: Figure 6 Effect of OID and social isolation on unfolded protein response (UPR), determined by immunoblotting UPR markers (A) GRP78; increased by OID: P = 0.016, but post hoc analysis indicated that only isolated mice fed OID had significantly higher expression of GRP78 than controls, (B) IRE1; increased by OID: P < 0.001 and social isolation: P = 0.012. (C) PERK; no significant changes, (D) CHOP; no significant changes. Effect of OID and social isolation on autophagy, determined by immunoblotting autophagy markers (H) Beclin1; increased by OID: P = 0.008, (G) Atg7; increased by social isolation: P < 0.0001. Although OID also increased Atg7 expression in group-housed mice, socially isolated mice fed control diet had higher expression than G-OID or I-OID: P for interaction = 0.002, (E) LC3II; increased by social isolation: P = 0.002. Although OID also increased LC3II expression in group-housed mice, socially isolated mice fed control diet had higher expression than G-OID or I-OID: P for interaction = 0.009, and (F) p62; downregulated by social isolation: P < 0.001. Although OID also reduced p62 expression in group-housed mice, socially isolated mice fed control diet had higher expression than G-OID or I-OID: P for interaction = 0.003. (I) LC3A/B puncta staining of mammary glands; increased by social isolation: P < 0.001. Bars marked with different letters are significantly different from each other. Means and standard error of means (s.e.m.) of 4–11 mice per group are shown; these mice were not exposed to DMBA.

Article Snippet: The following primary antibodies were used in a 1:1000 dilution: anti-LC3II, ATG7, GRP78, IRE1, PERK, CHOP and Beclin 1 antibodies were from Cell Signaling Technology, anti-β-actin antibody was from Santa Cruz Biotechnology and anti-p62 antibody was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Isolation, Western Blot, Expressing, Control, Staining

Journal: Endocrine-Related Cancer

Article Title: Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk

doi: 10.1530/erc-16-0359

Figure Lengend Snippet: Figure 8 Effect of OID and social isolation on body weight, autophagy and mammary tumorigenesis in Atg7+/− mice. Atg7+/− and their wild-type (WT) littermates were group-housed and fed control diet (G-C) or socially isolated and fed obesity-inducing diet (I-OID). Body weight gain from weaning until week 20 (n = 15–21 mice/group), (A) shown as a fold change from the body weight at weaning and (B) body weight in grams. Fold change in body weights was significantly higher in socially isolated, OID-fed mice than in group-housed mice fed the control diet: P = 0.023. (C) MPA + DMBA-induced mammary tumor incidence was significantly higher in WT mice kept in I-OID than in group-housed G-C mice (P = 0.037), but not in I-OID Atg7+/− mice, compared with G-C Atg7+/− mice (P = 0.267) (n = 16–20 mice/group). (D) Among socially isolated mice fed OID, mammary tumor multiplicity (number of tumors per mice) was significantly higher (P = 0.0014) in WT group than in Atg7+/− mice. Significant differences are marked with *. (E) LC3II or (F) p62 levels were determined in mammary glands at the end of mammary tumor monitoring period, and they were not different in group-housed WT and Atg7+/− mice. Social isolation increased LC3II levels in WT mice and reduced them in Atg7+/− mice, compared with control WT mice (P for interaction = 0.002). Social isolation reduced p62 levels in WT mice but increased them in Atg7+/− mice (P for interaction = 0.006). Bars marked with different letters are significantly different from each other. Means and standard error of means (s.e.m.) are shown.

Article Snippet: The following primary antibodies were used in a 1:1000 dilution: anti-LC3II, ATG7, GRP78, IRE1, PERK, CHOP and Beclin 1 antibodies were from Cell Signaling Technology, anti-β-actin antibody was from Santa Cruz Biotechnology and anti-p62 antibody was obtained from BD Biosciences (San Jose, CA, USA).

Techniques: Isolation, Control

Journal: Autophagy

Article Title: Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage.

doi: 10.4161/auto.21289

Figure Lengend Snippet: Figure 2. Involvement of autophagy in E2-mediated neuroprotection against FC-induced neurotoxicity in PC12 cells. (A) Atg7 silencing decreased the levels of both constitutive and FC-induced autophagy. The differentiated PC12 cells were pretreated with 100 nM Atg7 siRNA or nontargeted siRNA for 24 h followed by E2 treatment for another 24 h. Subsequently, cells were exposed to FC. (B) Atg7 silencing simulated the protective effect of E2 against the FC-induced cytotoxicity. (C) Preincubation with rapamycin significantly increased the levels of autophagy in each group except for FC-exposure group per se. The differentiated PC12 cells were pretreated with E2 followed by FC exposure. Five hours before FC exposure, the cells were preincu- bated with 25 nM rapamycin. (D) Preincubation with rapamycin significantly diminished the protective effect of E2 against FC-induced neurotoxicity. Data are expressed as means ± SD (n = 6), *p < 0.05, **p < 0.01.

Article Snippet: An equal amount of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane, incubated with antibody against LC3 antibody (1:1000), α-II spectrin antibody (1:1000, Santa Cruz Laboratories, sc-7465), ERα (1: 25) or ATG7 antibody (1:1000, Biosensis, R-161-100) followed by a second antibody, and then visualized by ECL chemiluminescence (PerkinElmer, NEL 105).

Techniques:

Journal: Autophagy

Article Title: Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage.

doi: 10.4161/auto.21289

Figure Lengend Snippet: Figure 6. Atg7 conditional knockout in DRD2 neurons abolished the sex difference in FC-induced CN injury. Knockout of Atg7 decreased the ATG7 immunoreactivity (A) and LC3 aggregation (B) in DRD2 neurons in both male and female mice. The brain tissues containing the CN from male or female mice with (Atg7+/– D rd2-Cre) or without (Atg7 F/F) Atg7 conditional knockout were sampled 2 d after FC infusion and sectioned at 10 μm thick- ness. ATG7 antibody or LC3 antibody followed by a secondary antibody conjugated to FITC and DRD2 antibody followed by a second antibody conju- gated to rhodamine were used to stain for ATG7 or LC3 (green) and DRD2 neurons (red), respectively. Simultaneously, nuclei were stained with DAPI. The nuclei surrounded by cytosolic ATG7 or LC3 in DRD2 neuron depict ATG7-containing or autophagic DRD2 neurons as indicated using arrows. (C) Knockout of Atg7 significantly decreased the FC-induced behavioral deficit in male mice and diminished the sex difference in FC-induced behavior- al deficit. Forelimb use asymmetry ratio depicting an index of behavioral deficit. (D) Knockout of Atg7 significantly decreased the level of FC-induced striatal injury in male mice and diminished the sex difference in FC-induced striatal lesion ratio. Data are expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01 compared with the sham control sex-matched group. ##p < 0.01 compared with the male sham.

Article Snippet: An equal amount of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane, incubated with antibody against LC3 antibody (1:1000), α-II spectrin antibody (1:1000, Santa Cruz Laboratories, sc-7465), ERα (1: 25) or ATG7 antibody (1:1000, Biosensis, R-161-100) followed by a second antibody, and then visualized by ECL chemiluminescence (PerkinElmer, NEL 105).

Techniques: Knock-Out, Staining, Control

Journal: Cell death & disease

Article Title: Autolysosomes and caspase-3 control the biogenesis and release of immunogenic apoptotic exosomes.

doi: 10.1038/s41419-022-04591-5

Figure Lengend Snippet: Fig. 5 Autophagy regulates the loading of LG3 on apoptotic exosome‐like vesicles (ApoExos). A Representative immunoblots and densitometric analysis of LC3, ATG7, phospho-AKT1, and total AKT1 from serum-starved (SS) HUVECs transfected with siCtrl or siATG7 or exposed to vehicle (V), wortmannin (100 nM; Wort), or bafilomycin A1 (20 nM, Baf). β-actin (ACTB) was used as a loading control. n = 3 for each condition. B HO/PI staining of apoptosis in SS HUVECs transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. n = 3–5 for each condition. ns (nonsignificant). C Immunoblots and densitometric analysis of cleaved caspase-3 in SS HUVECs transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. β-actin (ACTB) was used as a loading control. n = 3–5 for each condition. D Small particle flow cytometric quantifications of CMFDA+ AnnexinV+ ApoExos detected in the supernatant of HUVECs serum starved (SS) for 4 h and transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. n = 3 for each condition. E Representative immunoblots and densitometric analysis of LG3, 20S proteasome, LAMP2, and synthenin-1 in ApoExos purified from HUVECs serum starved (SS) for 4 h and transfected with siCtrl or siATG7 or exposed to V, Baf, or Wort. Ponceau red was used as a loading control. n = 3–5 for each condition. P values were obtained by the unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001). All values are expressed as the mean ± SEM.

Article Snippet: The antibodies used for blotting were antibodies against PARP (Cell Signaling Technology, 9542), LC3 (Novus, NB600-1384), p62 (Cell Signaling Technology, 8025), beta-actin (Sigma-Aldrich, a5441), GM130 (Abcam, ab52649), CD82 (Abcam, ab66400), TSG101 (Abcam, ab125011), tubulin (Calbiochem, cp06), TCTP (Santa Cruz Biotechnology, SC-30124), syntenin-1 (Santa Cruz Biotechnology, SC100336 or SC-515538), LAMP2 (Abcam, ab25631), 20S proteasome α3 (Santa Cruz Biotechnology, SC-67340), perlecan/LG3 (Santa Cruz Biotechnology, SC-25848), cleaved caspase-3 (Cell Signaling, 9661L), histone H3 (Cell Signaling, 9715S), ATG7 (R&D Systems, MAB6608), pSer473-AKT (Cell Signaling, 9271S), and total AKT (Cell Signaling, 9272L).

Techniques: Western Blot, Transfection, Control, Staining

Journal: International journal of oncology

Article Title: Emerging role of autophagy during ischemia-hypoxia and reperfusion in hepatocellular carcinoma.

doi: 10.3892/ijo.2012.1415

Figure Lengend Snippet: Figure 2. Effects of autophagy on HCC cell proliferation after I/H treatment. (A) Autophagy was specifically inhibited by Atg7 siRNA interference. The Atg7 knockdown was confirmed using western blot analysis. (B) Comparison of cell proliferation with or without autophagic inhibition. (C) Comparison of cell proliferation between HepG2 and Hep3B cells. To eliminate the difference in growth character between HepG2 and Hep3B cells, the proliferation rate was compared according to the ratio of the I/H group to the control group. The left panel shows the cell proliferation rate after I/H treatment and the right panel the cell proliferation rate with Atg7 interference and I/H exposure. (D) p62 accumulation after I/H treatment was measured by western blot analysis. Atg7Scr and Atg7Sh, cells transfected with control siRNA (scrambled) or Atg7 ShRNA; IH + Atg7Sh, Atg7 interference before I/H treatment; Atg7Sh, Atg7 interference without I/H treatment. Data represent the means ± SD of three independent experiments.

Article Snippet: Blots were probed with rabbit polyclonal anti-LC3, mouse monoclonal anti-HIF-α (Santa Cruz Biotechnology), mouse monoclonal anti-p62, mouse monoclonal anti-GAPDH antibodies (Santa Cruz Biotechnology), and rabbit monoclonal Atg7 antibody (Novus Biologicals, Littleton, CO, USA) overnight at 4 ̊C.

Techniques: Knockdown, Western Blot, Comparison, Inhibition, Control, Transfection, shRNA

Journal: International journal of oncology

Article Title: Emerging role of autophagy during ischemia-hypoxia and reperfusion in hepatocellular carcinoma.

doi: 10.3892/ijo.2012.1415

Figure Lengend Snippet: Figure 4. The specific generation of ROS during I/H and reperfusion. Intracellular ROS levels were measured to investigate whether the oxidative burst mediates the majority of the damage during I/H and reperfusion exposure. The ROS generations in HepG2 (A) and Hep3B (B) were measured by a fluorometry assay using intracellular oxidation of DCFH-DA and further confirmed by Atg7 interference and NAC inhibition. The comparison was carried out between data in different time-points and the control.

Article Snippet: Blots were probed with rabbit polyclonal anti-LC3, mouse monoclonal anti-HIF-α (Santa Cruz Biotechnology), mouse monoclonal anti-p62, mouse monoclonal anti-GAPDH antibodies (Santa Cruz Biotechnology), and rabbit monoclonal Atg7 antibody (Novus Biologicals, Littleton, CO, USA) overnight at 4 ̊C.

Techniques: Inhibition, Comparison, Control