Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: MAP1LC3C shRNA sequences.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: shRNA

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: Quantitative real-time PCR primer sequences.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Real-time Polymerase Chain Reaction

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: TaqMan probes.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques:

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: A) MAP1LC3C expression is decreased in tumors (n = 501) compared to normal lung tissue (n = 49) (median in grey, ****p < 0.0001). Heat maps ranked for MAP1LC3C expression for B) myeloid and C) lymphoid immune cells’ gene signatures, D) HLA class I and E) HLA class II, HLA chaperones and CIITA , F) immune checkpoints on APCs/tumor cells and G) immune checkpoints on T-cells. FKPM, Fragments per Kilobase of transcript per million Mapped reads. Spearman (r) correlation are indicated in .

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Expressing

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: Spearman correlation (r) values between MAP1LC3C and cancer immunity genes expression are indicated in LUSC. * : 0.5 < r < 0.6; **: r > 0.6., all p-values are < 0.0001.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Expressing

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: Heat map of MAP1LC3C expression and HLA class II ( HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DRB1 and HLA-DRB5 ), HLA chaperone ( HLA-DMA ) and CIITA (left to right) expression (Z-score normalized data). B) Average mRNA expression level obtained from single-cell transcriptomics of NSCLC human tumors analysis. Data were extracted from lung squamous cell carcinoma (n = 2) and lung adenocarcinoma (n = 5) (MoMØDC, Monocytes - Macrophages - Dendritic cells; RBC, red blood cells; pDC, plasmacytoid DCs). TPM, Transcripts Per Million.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Expressing, Single-cell Transcriptomics

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: A) Quantitative PCR of MAP1LC3C in control and 2 independent SW900 MAP1LC3C knockdown cell lines. B) Confirmation of MAP1LC3C knockdown by immunoblot analysis C-G) Quantitative PCR of HLA class II (HLA-DRA, HLA-DPA1, HLA-DPB1 ), HLA chaperone ( HLA-DMA) and CIITA mRNA expression in control and 2 independent MAP1LC3C deficient SW900 cell lines (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). H) Immunofluorescent staining of pan-HLA class II (HLA-DP/DQ/DR) in SW900 control cells or MAP1LC3C deficient cells following IFNγ treatment (50 ng/ml, 24h). Scale bar: 36.8μM. I) Histograms of flow cytometry analysis and quantification of HLA-DR1 and J) pan-HLA class II protein cell surface expression following IFNγ treatment (*p < 0.05, **p < 0.01). K) Immunoblot analysis and quantification of pan-HLA class II (HLA-DP/DQ/DR) after IFNγ (50 ng/ml, 24h) and CQ (5 µ g/ml, 24h) exposure (****p < 0.0001). J-K) Data are representative of three or more independent experiments and values are expressed as mean ± SD (A-I) and as median ± SD (J-K).

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Real-time Polymerase Chain Reaction, Control, Knockdown, Western Blot, Expressing, Staining, Flow Cytometry

Journal: PLOS ONE

Article Title: MAP1LC3C repression reduces CIITA- and HLA class II expression in non-small cell lung cancer

doi: 10.1371/journal.pone.0316716

Figure Lengend Snippet: Quantitative PCR of A) MAP1LC3C mRNA in engineered cells, with and without exposure to doxycycline/CITTA-induction (doxyxycline 1µg/mL, 48h). B) CIITA and C-E) HLA class II and F) HLA chaperone ( HLA-DMA ) expression following CIITA overexpression. (ns: non-significant, * p < 0.05); **p < 0.01). G) Immunoblot analysis and quantification of pan-HLA class II protein levels following CIITA overexpression (**p < 0.01). Quantitative PCR of H) HDAC target gene (c-Myc) , I-J) HLA class II , K) HLA chaperone ( HLA-DMA ) and L) CIITA mRNA expression following HDAC inhibition (Vorinostat 1 µ M, 24h) (ns: non-significant, * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). M) Immunoblot analysis and quantification of pan-HLA class II (HLA-DP/DQ/DR) protein following HDAC inhibition (Vori).Data are representative of three or more independent experiments and values are expressed as mean ± SD.

Article Snippet: MAP1LC3C , Hs01374916_m1.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Western Blot, Inhibition

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: Figure 5. CALCOCO1 interacts with MAP1LC3C and other LC3/GABARAP family members. (A) Cartoon of domains and motifs found in human and mouse CALCOCO family members. (B) Immunoprecipitation (IP) analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, resolved by SDS-PAGE, and probed with anti-HA or anti-DDK antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (C) IP analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs expressed in HEK293 cells that were treated overnight with chloroquine (CQ) to inhibit lysosomal flux. HA-tagged protein complexes were IP’d with anti-HA antibody and probed with anti-DDK or mixed antibodies against HA, MAP1LC3A/B, and MAP1LC3C. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (D) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with wild type (WT) CALCOCO1-MYCDDK, CLIR mutants L140A and V142A, and LIR mutant W47A in HEK293 cells. CALCOCO1W47A was transfected at 2 different concentrations to match WT expression levels. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, and probed with anti-HA or anti-CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as noted. (E) IP analyses of CALCOCO1-MYCDDK with endogenous MAP1LC3B. WT CALCOCO1-MYCDDK, 2 concentrations of the CALCOCO1W47A, and GFP-DDK (control) were expressed in HEK293 cells, then treated for 18 h with 100 nM MLN0128 and 100 uM CQ. CALCOCO1-MYCDDK complexes were immunoprecipitated with anti- Flag® M2 affinity gel (MilliporeSigma, A2220) and probed with anti-DDK or MAP1LC3B antibodies. Expressions of CALCOCO1-MYCDDK, GFP-DDK, ACTB and MAP1LC3B in cell lysates are shown with antibodies as noted. (F) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with WT CALCOCO1-MYCDDK, CLIR mutants (L140A and V142A), and R12H mutant in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody and probed with anti-HA or anti- CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies, as noted.

Article Snippet: Primary antibodies used in this study include: Abcam antibodies for CALCOCO1/CoCoA (ab70564); ABD SeroTech antibodies GAPDH (HCA272); BD Biosciences antibodies for SQSTM1 (610832) and RPS6KB1 (611261); Cell Signaling Technology antibodies for ATG3 (3415), ATG5 (8540), ATG7 (8558), MAP1LC3A/B (12741), MAP1LC3C (14723), ULK1 (6439 and 8054), p-EIF4EBP1 T37/46 (2855), p-EIF4EBP1 S65 (9456), EIF4EBP1 (9644) p-AKT1 S473 (4060), AKT1 (9272) p-RPS6KB T389 (9234), p-ACACA S79 (11818), ACACA (3662) DYKDDDDK-tag (14793), MYC-tag (2276), ACTB (beta-Actin; 3700), LMNA (lamin A/C; 4777); Novus antibodies for MAP1LC3B (NB100-2220); Protein Tech antibodies for RETREG1 (21537-1-AP), GABARAPL2 (18724-1-AP), MAP1LC3C (18726-1-AP); Santa Cruz Biotechnology antibodies for CALCOCO1 (sc-515670), HAtag (sc-805), GST-tag (sc-138), TUBA (alpha-tubulin; sc5286); MilliporeSigma antibody for Flag M2 (F1804).

Techniques: Immunoprecipitation, SDS Page, Mutagenesis, Transfection, Expressing, Control