Journal: Autophagy

Article Title: The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore

doi: 10.1080/15548627.2021.1896157

Figure Lengend Snippet: MOAP1 is dispensable during the early steps of autophagy signaling involving initiation and elongation of phagophore. (A and B) Formation of ATG5 or p-ATG16L1 puncta is unaffected in the absence of MOAP1 in cells treated with EBSS. The MOAP1 +/+ and MOAP1 -/- cells were subjected to EBSS treatment for the indicated time periods. The cells were then harvested for immunofluorescence staining by detecting the presence of GFP-ATG5 (A) or p-ATG16L1 puncta (labeled with Cy3) (B) to monitor the formation process of autophagosome during autophagy signaling. Scale bar: 10μm. ns, not significant (Student’s t test). (C) Loss of MOAP1 does not affect conjugation of ATG12–ATG5-ATG16L1 complex. The MOAP1 +/+ and MOAP1 -/- HeLa cells were harvested under basal condition or after 2 h of EBSS treatment before being subjected to immunofluorescence staining. GFP-ATG5 or Cy3 labeled-p-ATG16L1 puncta were used as markers for detecting the formation of ATG5-12-16 L1 complex. Scale bar: 5μm. (D) Loss of MOAP1 does not affect the interaction between ATG5-12 and ATG16L1 complex. Plasmid encoding MYC-ATG5 or ATG12 was transfected into the MOAP1 +/+ or MOAP1 -/- HeLa cells. After 24 h, the transfected cells were subjected to immunoprecipitation to evaluate the interaction between ATG16L1 and ATG12–ATG5. Arrows: indicated protein signal; Asterisk: IgG signal. (E) LC3 conjugation process induced by EBSS treatment is unaffected by the absence of MOAP1 . Total cell lysates from the MOAP1 +/+ and MOAP1 -/- HeLa cells were harvested after 6 h of EBSS treatment. BECN1, ATG7, ATG5-12 and LC3 are the markers for the upstream signaling events during autophagic induction whereas SQSTM1 and NBR1 are the autophagy substrates. (F) Low number of LC3 puncta in the MOAP1-deficient EBSS-treated cells. The MOAP1 +/+ and MOAP1 -/- HeLa cells were subjected to EBSS treatment for 30 or 60 min as indicated. At the indicated time points, the treated and untreated MOAP1 +/+ or MOAP1 -/- cells were subjected to immunofluorescence staining. The number of LC3 puncta was then quantified by ImageJ analysis. Scale bar: 10μm. ***p < 0.001, ns, not significant (Student’s t test). (G) Low level of autophagy flux in the absence of MOAP1 during starvation. The MOAP1 +/+ and MOAP1 -/- HeLa cells were transfected with plasmid encoding the GFP-LC3-RFP-LC3G reporter prior to EBSS treatment for 2 h. The level of fluorescence signals was then measured and the ratio of GFP:RFP was then calculated by FACS analysis. Scale bar: 5μm. **p < 0.01, ns, not significant (Student’s t test)

Article Snippet: The primary antibodies used were as follows: ACTB (Sigma-Aldrich, SAB5500001), ATG16L1 (Abcam, ab187671), ATG16L1 (phospho-S278; Abcam, ab192542), ATG5 (nanoTools, 0262–100), ATG7 (Abcam, ab133528), BECN1 (Cell Signaling Technology, 3495), cl-CASP3 (Cleaved-Asp175; Cell Signaling Technology, 9661), FLAG (Sigma-Aldrich, F3165), GST (Santa Cruz Biotechnology, sc-138), HA (Abcam, ab9110), His (Santa Cruz Biotechnology, clone H-3), HSPD1/HSP60 (Santa Cruz Biotechnology, sc-13115), LMNA/lamin A/C (Santa Cruz Biotechnology, sc-376248), LAMP1 (Cell Signaling Technology, 9091), LC3B (Cell Signaling Technology, 2775), MOAP1 (Sigma-Aldrich, HPA000939), MYC (Santa Cruz Biotechnology, sc-40), NBR1 (Novus Biologicals, H00004077-M01), SQSTM1/p62 (Abcam, ab56416), ABCD3/PMP70 (Abcam, ab109448), SEC22B (Abcam, 181076), STX17 (MBL International, PM076), TFR2 (Abcam, 185550), TOMM20 (Santa Cruz Biotechnology, sc-11415), TUBA/tubulin (Santa Cruz Biotechnology, sc-8035), ULK1 (Cell Signaling Technology, 8054), ULK1 (phospho-S317; Cell Signaling Technology, 12753), WIPI2 (Abcam, ab105459).

Techniques: Immunofluorescence, Staining, Labeling, Conjugation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Fluorescence

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: a Phase 1 Clinical Trial

doi: 10.1158/1078-0432.CCR-16-0600

Figure Lengend Snippet: (A) Simplified iNKT preparation protocol overview. (B) Clinical Trial Protocol Flow Chart. (C) Analysis of patient leukapheresis product PBMC iNKT by 3 methods, Vα24-Vβ11 antibodies, Vα24-6B11 antibodies, and αGalcer-analogue loaded-CD1d tetramer, shown with Vβ11 antibody. Representative patient #1. Last panel on the right shows negative control unloaded-CD1d tetramer for comparison.

Article Snippet: Disclosure of Potential Conflicts of Interest : MAE and SPB have licensed the 6B11 antibody to NKT Therapeutics and are shareholders.

Techniques: Negative Control, Comparison

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: a Phase 1 Clinical Trial

doi: 10.1158/1078-0432.CCR-16-0600

Figure Lengend Snippet: Immune cells were monitored before, during and after iNKT infusions, as shown using gating in figure S2. (A) iNKT levels (Vα24+6B11+). (B) Dendritic cells (lineage-negative CD45+ DR+). (C) Activated CD4 T cells (CD69+). (D) Treg (CD3+CD4+CD25+ FoxP3+). (E) NK cell levels (CD56+CD3-negative). (F) Proportion of activated (CD69+) NK cells. (G) CD14+ monocyte levels. (H) Activated (CD69+) monocytes.

Article Snippet: Disclosure of Potential Conflicts of Interest : MAE and SPB have licensed the 6B11 antibody to NKT Therapeutics and are shareholders.

Techniques: