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: Oncology Reports

Article Title: Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway

doi: 10.3892/or.2018.6744

Figure Lengend Snippet: Apoptotic rates of U87 and U251 transduced with RNF138-siRNA and negative control lentivirus. (A) Transfection with RNF138-siRNA in U87 and U251 cells significantly increased cell apoptosis compared with negative control groups by flow cytometry detection. (B) Quantification of flow cytometry data. Quadrant R2 and X4 were apoptotic cells. Data are presented as the mean ± standard deviation for three independent experiments. **P<0.01. RNF138, ring finger protein 138; siRNA, small interfering RNA; 7-AAD, 7-aminoactinomycin D.

Article Snippet: Bcl2 associated X apoptosis regulator (BAX; 1:1,000; cat. no. sc-4239 WB; Santa Cruz Biotechnology, Inc.), apoptosis regulator Bcl2 (Bcl2; 1:1,000; cat. no. sc-23960; Santa Cruz Biotechnology, Inc.); vascular endothelial growth factor (VEGF; 1:1,000; cat. no. sc-365578) and hypoxia-inducible factor-1α (HIF-1α; 1:1,000; cat. no. sc-13515); Santa Cruz Biotechnology, Inc.), cleaved caspase3 (cat. no. 9664), caspase3 (cat. no. 9662), Erk1/2 (cat. no. 4695), phospho (p)-Erk1/2 (cat. no. 4370), matrix metalloproteinase 2 (MMP2; cat. no. 40994), E-cadherin (cat. no. 14472) and vimentin (cat. no. 5741) were acquired from Cell Signaling Technology, Inc. and RNF138 (cat. no. DF4025; Affinity Biosciences, Cincinnati, OH, USA) at a dilution of 1:1,500.

Techniques: Transduction, Negative Control, Transfection, Flow Cytometry, Standard Deviation, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway

doi: 10.3892/or.2018.6744

Figure Lengend Snippet: Protein levels of apoptotic-associated factors in U87 and U251 cells transduced with RNF138-siRNA and negative control lentivirus. (A) Expression of caspase3, cleaved caspase3, BAX and Bcl2 were detected by western blot analysis. β-actin served as the loading control. Densitometry analysis of (B) caspase3, (C) cleaved caspase3, (D) BAX and (E) Bcl2, relative to β-actin expression in U87 and U251 cells with ImageJ analysis. Data are presented as the mean ± standard deviation for three independent experiments. **P<0.01. RNF138, ring finger protein 138; siRNA, small interfering RNA; BAX, Bcl2 associated X apoptosis regulator; Bcl2, Bcl2 apoptosis regulator; NS, not significant.

Article Snippet: Bcl2 associated X apoptosis regulator (BAX; 1:1,000; cat. no. sc-4239 WB; Santa Cruz Biotechnology, Inc.), apoptosis regulator Bcl2 (Bcl2; 1:1,000; cat. no. sc-23960; Santa Cruz Biotechnology, Inc.); vascular endothelial growth factor (VEGF; 1:1,000; cat. no. sc-365578) and hypoxia-inducible factor-1α (HIF-1α; 1:1,000; cat. no. sc-13515); Santa Cruz Biotechnology, Inc.), cleaved caspase3 (cat. no. 9664), caspase3 (cat. no. 9662), Erk1/2 (cat. no. 4695), phospho (p)-Erk1/2 (cat. no. 4370), matrix metalloproteinase 2 (MMP2; cat. no. 40994), E-cadherin (cat. no. 14472) and vimentin (cat. no. 5741) were acquired from Cell Signaling Technology, Inc. and RNF138 (cat. no. DF4025; Affinity Biosciences, Cincinnati, OH, USA) at a dilution of 1:1,500.

Techniques: Transduction, Negative Control, Expressing, Western Blot, Control, Standard Deviation, Small Interfering RNA