Journal: Cell Reports

Article Title: Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation

doi: 10.1016/j.celrep.2021.109899

Figure Lengend Snippet: v-ATPase is required for M2-induced LC3 lipidation (A) Immunoprecipitation (IP) analysis of endogenous ATG16L1 in HCT116 cells infected for 16 h with PR8. Control 1, lysate from PR8-infected cells incubated with beads to control for unspecific binding; control 2, beads and ATG16L1 antibody. (B) The M2-induced interaction between ATG16L1 and the v-ATPase depends on K490 of the ATG16L1 CTD. HCT116 ATG16L1 −/− cells reconstituted with WT or K490A mutant FLAG-muATG16L1 were infected with PR8 for 16 h followed by IP with anti-FLAG antibody. (C) Induction of ATG16L1-v-ATPase interaction by M2 depends on M2 ion channel activity. HCT116 ATG16L1 −/− FLAG-muATG16L1 reconstituted cells were infected with MUd for 16 h. Amantadine was added at 3 days p.i. IP as in (B). (D) Tet-ON M2 cells stably expressing mCherry or mCherry-SopF following treatment with dox or mock treated. M2 expression was detected using the M2-specific antibody 14C2. Black arrow indicates mCherry-SopF-expressing cell; white arrow indicates mCherry-SopF-negative cell. (E) LC3B lipidation analysis in Tet-ON M2 cells stably expressing either mCherry or mCherry-SopF after treatment with either Torin 1 (250 nM for 3 h), dox, or VPS34 IN-1 pretreatment (1 μM for 30 min) followed by monensin (100 μM for 1 h). (F) HCT116 stably expressing mCherry or mCherry-SopF treated as in (E) or infected with PR8 for 16 h (MOI of 10 PFU per cell). (G) Quantification of (E) (right panel) and (F) (left panel). The graph shows fold change in the LC3II/LC3I ratio relative to mCherry DMSO. Bars show mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: VPS34 inhibitor 1 , Selleckchem , Cat#S8456.

Techniques: Immunoprecipitation, Infection, Control, Incubation, Binding Assay, Mutagenesis, Activity Assay, Stable Transfection, Expressing

Journal: Cell Reports

Article Title: Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation

doi: 10.1016/j.celrep.2021.109899

Figure Lengend Snippet:

Article Snippet: VPS34 inhibitor 1 , Selleckchem , Cat#S8456.

Techniques: Virus, Recombinant, Reverse Transcription, Random Hexamer, CRISPR, Control, Knock-Out, Software

Journal: International Journal of Nanomedicine

Article Title: Enhancing of Nanocatalyst-Driven Chemodynaminc Therapy for Endometrial Cancer Cells Through Inhibition of PINK1/Parkin-Mediated Mitophagy

doi: 10.2147/IJN.S329341

Figure Lengend Snippet: Illustration of nMIL-100 (Fe) nanocatalyst for tumor chemodynamic therapy and the underlying mechanism of mitophagy. The nMIL-100 (Fe) NPs were internalized into the cytoplasm of EC cells (Step 1) and acted as Fenton reagents catalyzed H2O2 to generate highly oxidative ·OH in slightly acid TME (Step 2). Owing to the excessive toxic ·OH around normal mitochondria, the depolarization of mitochondrial membrane potential (Δψ) was subsequently initiated, which further induced mitochondrial oxidative damages and ultimate cell apoptosis (Step 3a). During this process the loss of Δψ forced PINK1 to be stagnated on the outer membrane of mitochondria (OMM). Step 3b) PINK1 further recruited the upregulated cytosolic Parkin in response to ·OH pressure and eventually activated mitophagy for cellular rejuvenation (Step 4). As a solution, suppressing mitophagy using both Parkin siRNA transfection and Mdivi-1 addition (a mitophagy inhibitor) observably amplified ·OH therapeutic effects and exacerbated cell damage.

Article Snippet: Mitochondrial division inhibitor 1 (Mdivi-1) was from MedChemExpress.

Techniques: Transfection, Amplification

Journal: International Journal of Nanomedicine

Article Title: Enhancing of Nanocatalyst-Driven Chemodynaminc Therapy for Endometrial Cancer Cells Through Inhibition of PINK1/Parkin-Mediated Mitophagy

doi: 10.2147/IJN.S329341

Figure Lengend Snippet: PINK1/Parkin mediated-mitophagy activation by nMIL-100 (Fe) and H 2 O 2 nanosystem. ( A ) The fluorescence images of GFP-LC3B-transfected ISK cells after treatment with 100 μg/mL nMIL-100 (Fe) and 50 μM H 2 O 2 for 4 h or 6 h followed by Mito-Tracker (red) incubation (scale bar is 20 µm). The colocalization between green spots and red mitochondria tracker was elevated by Plot Profile tool. ( B ) Quantifications of the mitochondrial membrane potential (MMP) ΔΨm (JC-1 red/green fluorescence intensity ratio) after different treatment including control, 100 μg mL −1 nMIL-100 (Fe) alone, 50×10 −6 M H 2 O 2 alone, a combination of both or a pretreatment of Mdivi-1 (5×10 −6 M, 1 h) before the both additions for 24 h. ( C ) Western blot analysis of PINK1, Parkin, LC3-I and LC3-II expression in ISK and KLE cells treated with 100 μg mL −1 nMIL-100 (Fe) and 50×10 −6 M H 2 O 2 for different time intervals, using GAPDH as loading control. ( D ) Parkin expression in mitochondrial and cytosolic enriched fractions of ISK and KLE cells after100 μg mL −1 nMIL-100 (Fe) and 50×10 −6 M H 2 O 2 treatment for 6 h was determined by Western blot, using GAPDH and COX IV as cytosolic and mitochondrial loading control respectively. ( E ) The images of immunofluorescence in ISK cells undergoing the same treatments as the aforementioned in ( D ) followed by co-staining with Mito-Tracker (red) and Hoechst 3342 (blue) (scale bar is 20 µm). The MFI of Parkin and the colocalization (white straight lines) between green Parkin and red mitochondria tracker was analyzed by Plot Profile tool. Data were represented as mean ± SD (n=3). * p < 0.05, ** p < 0.01, ns, not significant.

Article Snippet: Mitochondrial division inhibitor 1 (Mdivi-1) was from MedChemExpress.

Techniques: Activation Assay, Fluorescence, Transfection, Incubation, Western Blot, Expressing, Immunofluorescence, Staining

Journal: International Journal of Nanomedicine

Article Title: Enhancing of Nanocatalyst-Driven Chemodynaminc Therapy for Endometrial Cancer Cells Through Inhibition of PINK1/Parkin-Mediated Mitophagy

doi: 10.2147/IJN.S329341

Figure Lengend Snippet: Effects of mitophagy on nMIL-100 (Fe) and H 2 O 2 nanosystem mediated oxidative damage. ( A ) Western blot analysis of PINK1, Parkin, P62, cPARP, cCaspase-3, LC3-I and LC3-II expression in ISK and KLE cells treated with control, 100 μg mL −1 nMIL-100 (Fe) alone, 50×10 −6 M H 2 O 2 alone, a combination of both or a pretreatment of 3-MA (2×10 −6 M, 1 h) accompanied by the both additions for 24 h, GAPDH protein were served as an internal control. ( B ) Relative cell growth inhibition percentage of indicated ISK and KLE cells on the 24 h and 48 h after the aforementioned combination or a 3-MA (2×10 −6 M, 1 h) addition before the combination treatments. ( C ) Western blot for ATG 7-siRNA 1–3 knockdown efficiencies and the Parkin protein expression of ISK cells at 48 h after transfection. ( D ) The corresponding expression of apoptosis-related molecules in KLE cells transfected with or without control siRNA or ATG 7-siRNA followed by the aforementioned combination. ( E ) The relative cell viability of ISK and KLE cells after the same treatments as in ( D ) for 24 h or 48 h. ( F ) The protein levels of Parkin and its downstream molecules including Miro1, Mfn1 and Mfn2 after transient knockdown or overexpression its PRKN gene in ISK and KLE cells respectively. ( G – I ) Detection of active caspase 3 and relative cell growth inhibition ratio of ISK cells by the aforementioned combination additions for 24 h after different types pretreatments with Mdivi-1 (5×10 −6 M, 1 h), Parkin-siRNA or Parkin-pcDNA transfection. Data were represented as mean ± SD (n=3). * p < 0.05, ** p < 0.01, *** P < 0.005, **** P < 0.001, ns, not significant.

Article Snippet: Mitochondrial division inhibitor 1 (Mdivi-1) was from MedChemExpress.

Techniques: Western Blot, Expressing, Inhibition, Transfection, Over Expression