Journal: bioRxiv

Article Title: Development of the ULK1-Recruiting Chimeras (ULKRECs) to enable proximity-induced and ULK1-dependent degradation of mitochondria

doi: 10.1101/2024.04.15.589474

Figure Lengend Snippet: ULKRECs rely on ULK1 for the redirection of autophagy components to mitochondria and subsequent mitophagic degradation (A) Western blot of mouse embryonic fibroblasts (MEF) WT and MEF ULK1/2 -/- treated with 1 μM ULKRECs, 5 μM CCCP or both and probed for ULK1, Mfn2 and Actin – quantification of the relative intensity of Mfn2 shown in (B), normalised to Actin. Representative immunofluorescence of MEF WT (C) and MEF ULK1/2 -/- (D) treated with 5 μM CCCP, 1 μM ULKRECs or both and immunostained for Alexa 488 ATP5a and Alexa 647 LC3B. Quantification of the colocalised LC3 to ATP5a spot area shown in WT vs ULK1/2 -/- treated MEF cells shown in (E). Data shown are mean ± SD, n = 3, *p<0.05, **p<0.01, ***p<0.001, Two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Plates were incubated overnight at 4°C with 1:400 LC3B (D11) XP Rabbit mAb Alexa Fluor 647 Conjugate (Cell Signalling Technologies, #65299) and 1:400 ATP5a (Cell Signalling Technologies, #18023) lightning-linked to Alexa Fluor 488.

Techniques: Western Blot, Immunofluorescence

Journal: eLife

Article Title: Glutamine metabolism modulates chondrocyte inflammatory response

doi: 10.7554/elife.80725

Figure Lengend Snippet: Figure 4. Glutamine deprivation promotes autophagy, and ammonia inhibits autophagy. (A) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) in the presence or absence of chloroquine (10 μM) for 24 hr. Protein expressions of p62 (62kDa) and LC3-II (17kDa) were measured by immunoblotting, with representative image displayed. Bands quantified in supplemental figure. (B) Primary murine chondrocytes were plated on coated cover slips cultured in glutamine containing or glutamine free media for 12 hr. Cells were treated with chloroquine (10 μM) for 6 hr. Cells were fixed with 4% formaldehyde in PBS, and immunofluorescence (IF) was performed for LC3B and p62. Cells were mounted on slides and imaged with representative images displayed. (C) Primary chondrocytes were cultured in media containing 4 mM or 0 mM glutamine. Cells were supplemented with ammonium chloride at the indicated concentrations. After 6 hr, cells were treated with IL-1β (10 ng/mL) for 24 hr. Immunoblotting was performed for p62 and LC3B to display autophagosome processing. Image displays representative experiment. Bands quantified in supplemental figure. (D) Primary chondrocytes were cultured in media containing 4 mM or 0 mM glutamine. Cells were supplemented with glutamate (200 μM). After 6 hr, cells were treated with IL-1β (10 ng/mL) for 24 hr. Immunoblotting was

Article Snippet: Cells were incubated with anti- LC3b (12741, CST, Danvers, MA, USA; RRID:AB_2617131) or anti- p62 (2C11, Abnova, Taiwan, RRID:AB_437085) antibodies at 1:100 concentration in antibody dilution buffer (1× PBS, 1% BSA, and 0.1% saponin) overnight at 4°.

Techniques: Cell Culture, Western Blot, Immunofluorescence

Journal: PLoS ONE

Article Title: Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

doi: 10.1371/journal.pone.0093334

Figure Lengend Snippet: (A) Representative electron microphotographs showing autophagosomes in the ischemic penumbra of cerebral cortex of sham, vehicle or TAT-14-3-3ε pre-treated animals 24 h after reperfusion. Autophagosomes are indicated by arrows. Scale bar = 1 μm. (B) Western blot analysis for the expression of LC3B, p62 and Beclin-1 in cerebral hemisphere. (C, D and E) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to β-actin. The data are presented as the mean ± SD (n = 5 per group). # p <0.05 and ## p <0.01 versus sham group. * p <0.05 versus vehicle group.

Article Snippet: Polyclonal rabbit anti-LC3B antibody (1∶1000, Santa Cruz, CA, USA), polyclonal rabbit anti-Beclin-1 antibody (1∶1000, Santa Cruz, CA, USA) and polyclonal rabbit anti-p62/SQSTSM1 antibody (1∶500, Abgent, CA, USA) were used for immunoreactivity detection.

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

doi: 10.1371/journal.pone.0093334

Figure Lengend Snippet: Rats were treated with an i.c.v of saline or 3-MA (600 nmol) at the end of 2h ischemia, or RAP (35 pmol, i.c.v.) combined with TAT-14-3-3ε (10 mg/kg, i.v.) 2 h before 2 h MCAO. Rats were then subjected to 24 h reperfusion, after which all animals were sacrificed. (A) Western blot analysis of the LC3B, p62 and Beclin-1 protein expression in the ischemic cerebral hemisphere. (B, C and D) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to the loading control (β-actin). Bar represents mean ± SD (n = 4 per group). ## p <0.01 compared with sham group. * p <0.05 compared with vehicle group. § p <0.05 compared with RAP + TAT-14-3-3ε group.

Article Snippet: Polyclonal rabbit anti-LC3B antibody (1∶1000, Santa Cruz, CA, USA), polyclonal rabbit anti-Beclin-1 antibody (1∶1000, Santa Cruz, CA, USA) and polyclonal rabbit anti-p62/SQSTSM1 antibody (1∶500, Abgent, CA, USA) were used for immunoreactivity detection.

Techniques: Saline, Western Blot, Expressing, Quantitation Assay, Control