Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 1. CCPG1 Is an LIR Motif-Containing Interactor of Human ATG8 Orthologs (A) Schematic of CCPG1 structure (NTD, N-terminal amino acids 1–230; TM, transmembrane anchor). (B) GST or GST fusions of ATG8 orthologs (LC3B, LC3C, and GABARAP) were used in affinity precipitation (AP) of transfected myc-CCPG1 from HEK293 cells. (C) GST or GST-GABARAP (mtLDS, LIR-docking site mutant) were used in AP of transfected myc-CCPG1 NTD from HEK293 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Transfection, Mutagenesis

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 2. CCPG1 Is a FIP200-Interacting Protein (A) A549 NTAP (FLAG-HA)-CCPG1 cells were immunoprecipitated for tagged CCPG1 using anti-HA antibody and immunoprecipitates subjected to LC-MS/MS and CompPASS analysis (see the STAR Methods and Table S1). Interacting proteins at a cut-off of WDN score 0.8 are shown here. (B) A549 cells stably expressing NTAP empty vector () or NTAP-CCPG1 (+) were immunoprecipitated for tagged CCPG1 with anti-FLAG beads and im- munoblotted for indicated proteins. (C) A549 cells were EBSS starved or left untreated for 1 hr, prior to lysis and endogenous immunoprecipitation of CCPG1 and subsequent immunoblotting (IgG, negative control IgG). (D) HEK293 cells were transfected with FLAG-FIP200 and indicated variants of full-length (FL) GFP-CCPG1 (DNTD, amino acids 231–757). Immunoprecipitation was performed with GFP-Trap and immunoblotting performed with indicated antibodies. (E) Recombinant FIP200 was incubated with either glutathione Sepharose beads alone, or with pre-purified GST or GST-CCPG1 NTD bound beads. Affinity precipitation (AP) followed by immunoblotting was then performed to assess direct interaction. See also Figure S1 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Negative Control, Transfection, Recombinant, Incubation

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 3. Identification of a Linear Peptide Motif in CCPG1 for Binding to FIP200 C-Terminal Region (A) A 15-mer peptide array (peptides 1–55) was probed with recombinant FIP200. Bound FIP200 was detected by indirect immunodetection. Peptide sequences corresponding to binding regions A–C are shown below the array. (B and C) HEK293 cells were transfected with FLAG-FIP200 and indicated myc-tagged deletions or truncations of CCPG1 NTD prior to anti-myc immunopre- cipitation and immunoblotting (EV, empty vector). (D) Sequence alignment of the region from amino acids 97 to 118 of human CCPG1 against vertebrate orthologs (upper) or of regions amino acids 99–113 and 17– 31 of human CCPG1 (lower). Conserved S/T and acidic residues are blue, hydrophobic residues are red. Asterisks indicate evolutionary conservation of residues. Black boxes indicate residues identical between FIR1 and FIR2. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Binding Assay, Peptide Microarray, Recombinant, Immunodetection, Transfection, Western Blot, Plasmid Preparation, Sequencing

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 4. CCPG1 Is Recruited into Autophagosomes from the ER (A) A549 cells were transfected with siCtrl or siCCPG1 and, at 24 hr post-transfection, either left untreated or starved for 1 hr in EBSS, then stained for endogenous CCPG1. Cells with CCPG1 foci were scored (n = 3, ± SEM, *p < 0.05, two-tailed paired sample t tests). Scale bar, 20 mm. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Transfection, Staining, Two Tailed Test

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 5. CCPG1 Is a UPR-Inducible Gene that Remodels the ER (A) A549 cells were treated with indicated ER stressors for 16 hr (Tun, tunicamycin, 2.5 mg/mL and Thaps, thapsigargin, 0.5 mM). qRT-PCR was performed for CCPG1 (n = 3, ± SEM, *p < 0.05, one-way ANOVA followed by Tukey’s post-hoc test). (B) HeLa cells were treated with indicated ER stressors (DTT, 0.5 or 2 mM, and Tun at 1 or 2.5 mg/mL, or Thaps at 0.5 mM) for 16 hr and then immunoblotted.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Quantitative RT-PCR

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 6. Defective Proteostasis in the Pancreas of Ccpg1 Hypomorphic Mice (A and B) Whole pancreata from littermate 6-week-old WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice were immunoblotted for CCPG1 or subjected to RNA extraction and qRT-PCR for Ccpg1 (n = 3 pairs, ± SEM, ***p < 0.001, two-tailed t test). (C and D) Fifty mg of whole pancreata from littermate pairs of 6-week-old WT and Ccpg1 hypomorphic mice were homogenized in SDS. Insoluble protein was pelleted, washed and extracted in 8 M urea +10 mM DTT. Pellet samples were normalized according to protein concentration in the soluble fraction and subjected to label-free LC-MS/MS quantification. A median absolute deviation analysis is presented as a heatmap here to show species changing significantly between pairs of mice (pairs joined by connecting brackets). Secretory enzymes are in red, ER luminal chaperones/oxidoreductases are in blue. (E and F) Detergent soluble and insoluble samples prepared as above were immunoblotted and ratios of insoluble to soluble protein species obtained via densitometry (n = 3 pairs, ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t tests). See also Figure S5 and Table S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: RNA Extraction, Quantitative RT-PCR, Two Tailed Test, Protein Concentration, Liquid Chromatography with Mass Spectroscopy

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 7. Loss of Cell Polarization and ER Homeostasis, and Consequent Tissue Injury, in Ccpg1 Hypomorphic Exocrine Pancreata (A) The acinar unit of the exocrine pancreas. Polarized acinar cells secrete condensed enzyme (zymogen) granules into ducts from their apical stores. These enzymes are initially synthesized in the expansive rough ER (rER), which occupies the basolateral regions of the cell. (B) CARS imaging or immunohistochemical staining for the ER (protein disulfide isomerase, PDI) in pancreatic tissue from 6-week-old littermate WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice. Punctate CARS signals indicate protein or lipid inclusions. Scale bars, 20 mm. (C) Transmission electron microscopy (TEM) of pancreata from 6-week-old littermate pairs. Scale bar, 5 mm. Analysis of percent cytosolic area occupied by osmophilic protein granules was performed in ImageJ (n = 4 pairs, ± SEM, *p < 0.05, two-tailed t test). (D) High magnification TEM of a Ccpg1 hypomorphic mouse reveals that the rER is distended and many supernumerary inclusions are in fact intracisternal granule-like structures (arrows in zoomed inset). Scale bar, 1 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Synthesized, Imaging, Immunohistochemical staining, Staining, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Stem Cells Translational Medicine

Article Title: Functional Human and Murine Tissue‐Engineered Liver Is Generated from Adult Stem/Progenitor Cells

doi: 10.5966/sctm.2016-0205

Figure Lengend Snippet: Human LOU generate human TELi. (A): Human LOU prepared from fresh liver biopsy samples. (B–C): hTELi in murine host at 4 weeks. (D): H&E of cellular hTELi at 4 weeks. (E): Immunofluorescence costaining for ALB and α‐SMA. (F): Costaining for ALB and CK19. (G): Costaining for ALB and PCNA. (H): Human antigen β2M. (I): IgG staining control. Nuclei stained with DAPI (blue). Scale bar = 25 μm. Images represent three to four independent experiments. (J): Quantification of human ALB in murine plasma by enzyme‐linked immunosorbent assay. Number in parentheses is number of implanted TELi in each mouse. Sham, n = 4; TELi(2), n = 4, TELi(6), n = 13. ∗, p < .05 compared with TELi(2). (K): Liquid chromatography‐mass spectrometric quantification of human specific metabolite, 4‐OH‐debrisoquine derived from debrisoquine in vivo in mice with hTELi. One hour and 4 hours on the x‐axis indicate time after debrisoquine administration in mice. Sham, n = 4‐6; TELi, n = 7–11. ∗, p < .05 compared with 4‐hour sham. One‐hour TELi to 1‐hour sham was not significant ( p = .08). Abbreviations: ALB, albumin; α‐SMA, α‐smooth muscle actin; β2M, β2‐microglobulin; DAPI, 4′,6‐diamidino‐2‐phenylindole; H&E; hematoxylin and eosin; hTELi, human tissue‐engineered liver; LOU, liver organoid units; PCNA, proliferating cell nuclear antigen; TELi, tissue‐engineered liver.

Article Snippet: Human albumin in the plasma was quantified by human albumin enzyme‐linked immunosorbent assay kit (Bethyl Laboratories, Montgomery, Texas, https://www.bethyl.com ) according to the manufacturer's instructions.

Techniques: Immunofluorescence, Staining, Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Liquid Chromatography, Derivative Assay, In Vivo

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A) Flow cytometry analysis of tetrameric FGL-1 FD::streptavidin Phycoerythrin (SA-PE) binding to LAG-3+ Jurkat cells across a concentration range (1 – 16 µM). (B) Comparison of FGL-1 FD tetramer binding to LAG-3+ Jurkat cells versus wild-type (WT) Jurkat cells (LAG-3 low) (n = 3). Data are presented as median fluorescent intensity (MFI), normalized to LAG-3+ Jurkat cells MFI at each concentration. Error bars represent standard deviation (SD). (C) NF-κB::eGFP induction and (D) IL-2 production in LAG-3+ Jurkat T cells stimulated with anti-CD3/CD28-coated beads +/− FGL-1 FD or mouse IgG1 isotype control (mIgG1) for 24 hours (n = 4, n = 3, respectively). NF-κB::eGFP induction data is shown as percent MFI normalized to anti-CD3/CD28 stimulation (set to 100%). Error bars represent standard deviation (SD). (E) Expression of T cell activation markers: PD-1, CD69, CD25, ICOS, 4-1BB and OX40 in LAG-3+ Jurkat T cells, either unstimulated or stimulated with anti-CD3/CD28 +/− FGL-1 FD (n = 4). Data are shown as MFI ± SEM, normalized to the anti-CD3/CD28 condition. (F) Control expression of activation markers in WT (LAG-3 low) Jurkat T cells under identical conditions (n = 4). Data represent MFI ± SEM from triplicate wells across two independent experiments. Statistical significance was performed using one or two-way ANOVA, followed by Tukey or Šídák’s post hoc tests, respectively. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant p > 0.05.

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Flow Cytometry, Binding Assay, Concentration Assay, Comparison, Standard Deviation, Control, Expressing, Activation Assay

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: (A, B) Label-free LC–MS/MS analysis of phosphotyrosine (pY) peptides in LAG-3+ Jurkat cells, either unstimulated or stimulated for 5 minutes with anti-CD3/CD28 +/− FGL-1 FD (n = 4), (A) number of unique pY peptides identified per condition (B) Log 2 intensity distribution of detected pY peptides. Statistical analysis performed using two-way ANOVA with Tukey’s post hoc tests. Solid black line = median; dashed lines = interquartile range. **** p < 0.0001, ns = not significant (p > 0.05) (C) Venn diagram showing overlap of pY peptides detected in cells stimulated with anti-CD3/anti-CD28 versus anti-CD3/anti-CD28 + FGL-1 FD from one representative experiment. (D – F) Volcano plots illustrating differential log 2 fold changes (log 2 FC) in pY intensity differences, across the three conditions: (D) anti-CD3/CD28 vs. unstimulated; (E) anti-CD3/CD28 + FGL-1 FD vs. unstimulated; (F) anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. Red = significantly upregulated phosphosites (log 2 FC ≥ 1.5); blue = significantly downregulated phosphosites, (log 2 FC ≤ 1.5); grey = unchanged phosphosites (log 2 FC) < 1.5).

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Functional enrichment analysis of proteins with upregulated pY sites (log 2 FC ≥ 1.5, red), and downregulated pY (log 2 FC ≤ 1.5; blue) in response to anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation, relative to unstimulated cells and stimulation without FGL-1 FD. Color intensity indicates –Log 10 (p-value) of pathway enrichment; X denotes non-enriched pathways. ( B ) Log 2 fold change (log 2 FC) analysis of TCR signaling-associated pY peptides following anti-CD3/CD28 and anti-CD3/CD28 + FGL-1 FD stimulation compared to unstimulated cells. ( C ) Heatmap of log 2 FC phosphorylation differences in TCR signaling-related pY peptides across three conditions: anti-CD3/CD28 vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. unstimulated; anti-CD3/CD28 + FGL-1 FD vs. anti-CD3/CD28. ( D ) Bar graphs showing log 2 FC values of TCR signaling pY sites in CD3 ITAMS, ZAP-70, LAT, ERK1/2, TEC and distal molecules upon FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( E ) Time-course western blot analysis of phosphorylation at CD3ζ Y142, ZAP70 Y319, and ERK 1/2 Y204/Y187 in LAG-3+ Jurkat cells unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Quantification represents phospho/total protein signal intensity, normalized to unstimulated (US) values (n = 3). ( F ) Bar graphs showing log 2 FC values of CD28 signaling pY sites: CD28 pY191, pY209, and PI3K P85A pY467, in response to FGL-1 FD stimulation relative to stimulation without FGL-1 FD. ( G ) Western blot analysis of CD28 Y191 phosphorylation in LAG-3+ cell lysates: unstimulated, stimulated with anti-CD3/CD28 +/− FGL-1 FD. Band intensities were quantified and normalized to total CD28 and unstimulated control (n = 5). Statistical significance was determined using a two-way ANOVA with Šídák’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Functional Assay, Phospho-proteomics, Western Blot, Control

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD3 (green) and LAG-3 (red) in unstimulated and anti-CD3/CD28-stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Representative confocal images depicting colocalization of CD28 (green) and LAG-3 (red) under the same conditions: unstimulated and anti-CD3/CD28. Scale bar: 5 µm. (C) Violin plots of Manders’ Overlap Coefficient (MOC) quantifying colocalization of CD3 and CD28 with LAG-3 in unstimulated and stimulated cells (n = 66). Median values are indicated by solid black lines; interquartile ranges by dashed black lines. Statistical significance was assessed using Kruskal-Wallis ANOVA, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative fixed-cell confocal microscopy images showing colocalization of CD28 (red) and Lck (pY394, cyan) in unstimulated, anti-CD3/CD28-stimulated, and anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (B) Violin plots quantifying CD28 and Lck pY394 colocalization using Manders’ overlap coefficient (MOC) and Pearson’s correlation coefficient (PCC) (n ≥ 50). (C) Representative confocal images showing colocalization of CD28 (green) and total Lck (red) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated LAG-3+ Jurkat cells. Scale bar: 5 µm. (D) Colocalization analysis of CD28 and total Lck, measured by MOC and PCC (n ≥ 60). (E) Representative confocal images of CD28 (red) and Lck pY394 (cyan) in unstimulated, anti-CD3/CD28 +/− FGL-FD stimulated Jurkat cells. Scale bar: 5 µm. (F) Colocalization analysis of CD28 and Lck pY394, determined by MOC and PCC (n ≥ 75). (G) Comparison of MOC and PCC values for CD28 and Lck pY394 colocalization between unstimulated and stimulated LAG-3+ Jurkat cells compared to WT (LAG-3 low) Jurkat cells (n ≥ 75). Median values are shown in solid black lines; interquartile ranges as dashed black lines. Statistical analysis was performed using Kruskal-Wallis ANOVA test, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant (p > 0.05).

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: FGL-1 binding to LAG-3 inhibits T cell activation via disruption of CD28 and TCR signaling

doi: 10.1101/2025.08.05.668721

Figure Lengend Snippet: ( A ) Representative confocal images showing colocalization of CD28 and active Lck (pY394) in primary T cells from three healthy donors, pre-activated to induce LAG-3 expression. Images display LAG-3+ CD3+ cells from three healthy donors under unstimulated, stimulated with anti-CD3/CD28 +/− FGL-FD. Scale bar: 5 µm. (B) Quantitative colocalization analysis of CD28 and Lck pY394 expressed as Manders’ Overlap Coefficient (MOC) values and log (MOC), in CD3+/LAG-3+ cells across three donors (3 donors x n ≥ 50 per condition). Median values are shown as solid black lines, and interquartile ranges as dashed black lines. Statistical significance was assessed using Kruskal-Wallis ANOVA, followed by Dunnett’s post hoc tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = non-significant (p > 0.05).

Article Snippet: Jurkat T cells or primary T cells were added to poly-L-lysine-coated chambered coverslips (Lab-Tek, Thermo Fisher) pre-coated with 5 μg/mL human anti-CD3 antibody (clone SK7, Miltenyi Biotec), 5 μg/mL anti-CD28 antibody (clone L293, Miltenyi Biotec), and 20 μg/mL FGL-1 FD or human IgG isotype control antibody in 1× PBS.

Techniques: Expressing

Journal: Nature communications

Article Title: GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

doi: 10.1038/s41467-021-27056-3

Figure Lengend Snippet: Fig. 2 GAPDH binds to EV surface via G58 domain. a Western blot showing binding of G58 peptide to HEK293T (designated as 293T) and MSC EVs. The second domain of TARBP protein was attached to G58 peptide for detection by anti-TARBP2 antibody. The experiment was independently repeated two times. b NTA profile showing the size distribution of HEK293T EVs after binding to the G58T protein. Inset is the scatter plot representing size (mean) of EVs. Each dot is a mean of three reading frames taken at different timepoints. Data are shown as mean ± s.d (n = 9 independent biological experiment) Statistical differences were determined by unpaired two-sided Student’s t-test, (ns = non-significant). c Agarose-gel-shift assay of EVs after incubation with either G58T (G58 + dsRBD) protein or dsRBD of TARBP2 protein. siRNA alone was used as negative control to determine interaction of EVs with siRNA. A gradual decrease in the intensity of siRNA reflects entrapment of siRNA near the wells due to interaction with G58T EVs. Lack of siRNA binding to dsRBD treated EVs confirms G58 peptide mediated binding of protein to EV surface. The experiment was independently repeated three times. d–f High- resolution single EV analysis by Imaging Flow Cytometry (IFC) to determine localization of GAPDH and G58 peptide on EVs. d Represents method validation by using either non-labelled HEK293F derived EVs or neon GFP labelled HEK293:CD63-neon GFP derived EVs as biological reference material. e Detection of GAPDH on HEK293F, HEK293F/CD63-GFP and MSCs EVs, using alexa fluor 647 labelled anti-GAPDH antibody. f G58 peptide binding on EVs expressing GAPDH on their surface. EVs were incubated with alexa fluor 488 (af488) labelled G58 peptide and af647 anti-GAPDH antibody. Experiment (d–f) were independently repeated three times. FACS sequential gating/sorting strategies is provided as Supplementary Fig. 9. g Distribution of secreted GAPDH-GFP protein in the cell-culture media. Media from HEK293T cells expressing GAPDH-GFP protein were processed to isolate EVs from proteins by gel-filtration chromatography. Both EVs and protein fractions contained GAPDH-GFP protein, indicating vesicular and non-vesicular modes of GAPDH secretion. Data are shown as mean ± s.d (n = 3 independent biological experiments). Source data are provided as a Source Data file.

Article Snippet: In brief, 25 μl of EVs at a concentration of 1 × 1012 particles/ml were incubated overnight at 4 °C with 400 pmol AlexaFluor488 labelled G58 peptide and/or with AlexaFluor647-labelled rabbit anti human GAPDH antibodies (abcam, ab204480, clone EPR16884) or APC-labelled mouse anti human CD63 antibodies (Miltenyi Biotec, 130–100–182; clone H5C6) at a final concentration of 8 nM in v-bottom 96 wells (ThermoFisher Scientific).

Techniques: Western Blot, Binding Assay, Agarose Gel Electrophoresis, Shift Assay, Incubation, Negative Control, Imaging, Flow Cytometry, Biomarker Discovery, Derivative Assay, Expressing, Cell Culture, Chromatography

Journal: Cell Reports Methods

Article Title: MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

doi: 10.1016/j.crmeth.2025.101120

Figure Lengend Snippet: Development of an enrichment strategy for MR1-dependent antigen discovery by protein-metabolite cross-linking and de novo MR1 antigen discovery (A) Schematic of a recombinant platform to express fully functional, C-terminally-tagged single-chain MR1/β2M (scMR1) molecules with either lysine or alanine at position 43, developed for high-specificity MR1 enrichment. The alpha 1, 2, 3, and transmembrane (TM) domains of MR1 are depicted. (B) MR1 staining of A549 (left) and MM909.24 (right) cell lines, either wild type (WT), MR1 knockout (MR1 KO), and MR1 KO cells transduced with scMR1 (MR1 KO + scMR1-WT) and mutant scMR1 (MR1 KO + scMR1-K43A). Numbers in the left-hand corner are the MR1-specific Allophycocyanin (APC) mean fluorescence intensities (staining with anti-MR1 26.5 antibody clone). Gates are set for viable, single cells. (C) Overnight activation assay with MAIT cell TCR-T (primary CD8 + T cells transduced with A-F7 MAIT TCR) versus M. smegmatis -infected and uninfected A549 cells followed by a tumor necrosis factor (TNF) ELISA confirming MAIT cell recognition of scMR1 in the presence of endogenous antigen. Error bars depict the standard deviation of duplicate conditions. (D) Enrichment efficiency obtained from MM909.24 cells stably transduced with scMR1-K43 molecules based on protein abundances obtained by LC-MS/MS (MR1 and β2M are highlighted separately as red dots that overlap). (E) Proof of concept for the detection of MR1/Ac-6-FP cross-link in a cell-based system. scMR1-transfected MM909.24 melanoma cells pulsed with 50 μM Ac-6-FP for 16 h were subjected to the cross-linking workflow. The graph shows extracted ion chromatograms for DSVTRQ K EPRAPW and DSVTRQ K EPRAPW bound to Ac-6-FP, respectively, including the three most abundant isotopes (M, M+1, and M+2) for each of the two peptide variants. (F) Schematic of the data analysis workflow employed to detect DSVTRQ K EPRAPW cross-linked to unknown ligands. Peptide sequence ladder ions unaffected by the ligand (y1–y6 and b1–b6) were used as reporter ions. MS/MS spectra containing these ions were subsequently shortlisted. Subtraction of the theoretical DSVTRQ K EPRAPW peptide mass generates Δ-mass values for cross-linked ligands that can be corrected for the mass of the free ligand, which can be queried for candidate compounds using tools such as CEU mass mediator. (G and H) Applied to scMR1-transfected MM909.24 pulsed with 50 μM Ac-6-FP for 16 h, the data analysis pipeline successfully detected spectra indicating the presence of other ligands bound to MR1, such as 6-formylpterin (XIC, G) and methylglyoxal (XIC, H). See also and and .

Article Snippet: Anti CD8 REA734, APC conjugated , Miltenyi Biotec , RRID: AB_2659237.

Techniques: Recombinant, Functional Assay, Staining, Knock-Out, Transduction, Mutagenesis, Fluorescence, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Transfection, Sequencing, Tandem Mass Spectroscopy

Journal: Cell Reports Methods

Article Title: MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

doi: 10.1016/j.crmeth.2025.101120

Figure Lengend Snippet: The B6 vitamers pyridoxal and PLP activate Jurkat cells expressing the A-F7 MAIT TCR and the MC.7.G5 TCR, as well as primary CD8 + T cells expressing the A-F7 MAIT TCR (A) Jurkat cells with no TCR or transduced with A-F7 MAIT TCR were co-incubated overnight with A549 WT and A549 MR1 KO cell lines treated with pyridoxal at 100, 10, and 1 μg/mL or loaded with M. smegmatis (MOI: 1:300). Cells were stained for CD69 expression with mean fluorescence intensity (MFI) displayed. Background MFI of Jurkat cells with A549 WT or MR1 KO alone with no pyridoxal or M. smegmatis was subtracted. Jurkat cells with A-F7 were gated on co-marker rCD2 + . Data display duplicate conditions ( E). (B) Jurkat cells with no TCR or transduced with A-F7 MAIT TCR were co-incubated overnight with A549 WT and the following compounds: 5-A-RU (converts to MAIT ligand 5-OP-RU in cells and was added in the absence of exogenously applied methylglyoxal, which increases potency), pyridoxal and PLP at 100, 10, 1, 0.1, 1 × 10 −2 , 1 × 10 −3 , and 1 × 10 −4 μg/mL. Cells were stained for CD69 expression with MFI displayed. Background MFI of Jurkat cells with A549 cells alone with no pyridoxal was subtracted. Jurkat cells expressing the A-F7 TCR were gated on the rCD2 co-marker. Assay was performed in triplicate ( F), and curves were fitted using a four-parameter logistic model. Points indicate mean values, with error bars depicting standard deviation. EC 50 values with a 95% confidence interval (CI) and R 2 are indicated, with the results reproducible over two assays ( G). (C) Primary CD8 + T cells from three healthy donors with no TCR transduction or expression of the A-F7 TCR to generate TCR-T cells, were co-incubated for 4 h with A549 WT cells ± pre-treatment with 100 μg/mL of pyridoxal, and then reactivity measured via T107 assay. T cells were also incubated alone or with CD3/CD28 Dynabeads, with the latter acting as a positive control. Cells were gated on lymphocytes, viable CD3 + , single cells, rCD2 + /CD8 + (or CD8 + for the untransduced), and then TNF + versus CD107a + for reactivity. For the pyridoxal conditions, background reactivity toward A549 cell lines with no pyridoxal has been subtracted. For reactivity toward CD3/CD28 Dynabeads, the reactivity for the T cell-alone condition has been subtracted ( H). (D) Jurkat cells with no TCR or transduced with MC.7.G5 TCR were co-incubated overnight with C1R cells ± pyridoxal at 100, 10, 1, 0.1, and 1 × 10 −2 μg/mL. Cells were stained for CD69 expression with MFI displayed. Background MFI of Jurkat cells alone with no pyridoxal was subtracted. Jurkat cells with MC.7.G5 TCR were gated on co-marker rCD2 + . Assay was performed in triplicate ( I), and curves were fitted using a four-parameter logistic model. Points indicate mean values, with error bars depicting standard deviation. EC 50 values with a 95% CI and R 2 are indicated ( J). See also and .

Article Snippet: Anti CD8 REA734, APC conjugated , Miltenyi Biotec , RRID: AB_2659237.

Techniques: Expressing, Transduction, Incubation, Staining, Fluorescence, Marker, Standard Deviation, Positive Control