Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Expression of CYP2C is inducible by light and targeted knockdown of CYP2C55 rescues light-induced cell death. Deduced amino acids of encoded human CYP2C9 and mouse CYP2C55 and CYP2C29 enzymes were aligned using Clustal Omega. Rectangular boxes represent key conserved residues for catalytic activity (gray), ligand binding (black), and heme stabilization (dashed) (see Ref. 90) (A). 661W cells on 6-well plates with/without sulfaphenazole pretreatment for 2 h at 37 °C were exposed to light (L) or remained in the dark (D). Expression of CYP2C proteins was detected from prepared cell lysates by Western blotting using anti-human CYP2C9 polyclonal antibody that cross-reacts with mouse CYP2C isozymes. The housekeeping gene β-actin was used as a loading control. Relative expression is presented by normalization with the densitometric ratio of CYP2C/β-actin in dark control cells (B). 661W cells at 50% confluence were transfected with Silencer Select predesigned siRNAs targeted to mouse CYP2C55 or CYP2C29 or mock-transfected with the scrambled control (Con.) oligos (n = 4/sample group). Cell lysates were prepared at 48 h after transfection to assess silencing efficiency by Western blotting. The densitometric ratio of CYP2C55 or CYP2C29/β-actin is presented (C). Cellular ATP indicative of cell viability was measured after light exposure (n = 4/sample group) (D). **, p < 0.01; *, p < 0.05; n.s., not significant. RLU, relative light units. In B, bars represent the relative ratio of densitometry (CYP2C/β-actin) from one representative experiment. In C, bars represent the absolute ratio of densitometry (siCYP2C55 or siCYP2C29/β-actin).

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Activity Assay, Ligand Binding Assay, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Stable expression of functional human CYP2C9-GFP fusion protein enhances cellular sensitivity to light. pCMV6-CYP2C9-GFP stably transfected 661W cells were examined by confocal microscopy showing the bright field and its switch to FITC mode to assess homogeneity of GFP-positive cells. The arrow indicates expression of CYP2C9-GFP on the surface of the cell body (A). The enzymatic activity of CYP2C9-GFP fusion protein with/without sulfaphenazole (10 μm) pretreatment was examined by a cell-based luminogenic assay that generates luminogenic signal from CYP2C9-catalyzed oxidation of the selective substrate luciferin-H. Wild-type (WT) 661W cells were controls. n = 3/luciferin-H concentration. ***, p < 0.001; *, p < 0.05 versus CYP2C9-GFP + SFZ (B). pCMV6-CYP2C9-GFP stably transfected 661W cells with/without pretreatment with sulfaphenazole (10 μm) (n = 12/treatment or non-treatment group) and wild-type cells (n = 24) were exposed to light for 4 h. Cellular ATP indicative of cell viability was measured after light (L) exposure (C). n.s., not significant. Error bars represent means ± S.D. RLU, relative light units; D, dark.

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Functional Assay, Stable Transfection, Transfection, Confocal Microscopy, Activity Assay, Concentration Assay

Journal: Nature Communications

Article Title: Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer

doi: 10.1038/s41467-023-41618-7

Figure Lengend Snippet: a Kaplan–Meier curves indicating the association between ATG16L1 transcript levels and overall survival in atezolizumab and regorafenib treated patients (IMblaze370). Median cutoff was used to determine high and low levels separately within KRAS mutant and KRAS wildtype tumors. P -values were obtained from log-rank tests. Log-rank hazard ratios (HR) are provided with 95% confidence intervals in parentheses. b Scatter plots for KRAS mutant tumors showing the correlation between ATG16L1 transcript levels and tumor microenvironment signatures for immune, stroma and epithelial cells. Pearson correlation coefficients and two-sided t-test P -values are shown ( n = 181 samples). c Immunohistochemical (IHC) staining for ATG16L1 protein in tumor biopsies obtained from IMblaze370. Dotted lines indicate tumor epithelial margins, asterisk depicts stromal component. 22 tumor biopsies were analyzed; representative micrographs are shown. Scale bar = 100μm. d , Expression of depicted lineage markers ( x -axis) in major cellular compartments (y-axis) of CRC tumor tissue, analyzed by single-cell RNA sequencing (GSE146771). e Comparison of ATG16L1 transcript levels in each cellular compartment analyzed in ( d ). f Immune cell subsets enriched in ATG16L1-low tumors within IMblaze370, as determined by CIBERSORT gene signatures. Arrows indicate T and NK cell subsets. Unadjusted P -values from two-sided t-tests are shown (KRAS mutant n = 181, KRAS wildtype n = 113 samples). Dashed lines denote significance threshold at P < 0.05. All analysis restricted to non-MSI-high tumors. * P < 0.05, ** P < 0.01, and *** P < 0.001. IMblaze370 RNAseq data have been deposited to the EGA under accession number EGAS00001005952, and GSE146771 is publicly available from GEO (see Data Availability section of Methods). Source data (including exact P -values) for panel ( f ) are provided as a Source Data file.

Article Snippet: Atg16l1 re-expression plasmid (pb-EF1_Atg16l1OE-IRES_tagBFP2) was generated with mouse Atg16l1 sequence (isoform 1, https://www.uniprot.org/uniprot/Q8C0J2 ) into pb1-EF1-IRES-tagBFP2 piggybac vector. pb-EF1_Atg16l1OE-IRES_tagBFP2 (2 μg) or its control vector pb1-EF1_tagBFP2 (2 μg) together with transposase plasmid (1 μg) was electroporated into AKPS-GFP-Luc-ATG16.ko organoids using the P1 buffer and CM137 program (Lonza).

Techniques: Mutagenesis, Immunohistochemistry, Expressing, RNA Sequencing Assay, Comparison

Journal: Nature Communications

Article Title: Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer

doi: 10.1038/s41467-023-41618-7

Figure Lengend Snippet: a Schematic of CRISPR engineering and generation of CRC organoids. b Immunoblot analysis of the indicated proteins in WT or Atg16l1 KO CRC organoids Three independent clones of each genotype are shown. c Hydrodynamic tail vein (HTV) injection of CRC organoids for liver growth model. d – h Bioluminescence imaging (BLI) signal quantification from CRC organoids implanted in the livers of immunodeficient hosts. d C57BL/6 J (BL6) immunocompetent hosts. *** P = 0.0001, **** P < 0.0001. e NOD-SCID/Gamma (NSG) immunodeficient hosts; WT control BL6 hosts shown in panel ( d ). ** P = 0.0027, *** P = 0.0001, **** P < 0.0001. f CD8 + T cell or NK cell depletion in BL6 immunocompetent hosts. Week 5 BLI data are shown. Within tumor genotypes, treatment groups are compared using Kruskal-Wallis test with Dunn’s multiple comparisons tests. Atg16l1 KO groups are also compared to their corresponding WT groups using two-sided Mann-Whitney tests; ** P = 0.0021 and **** P < 0.0001. For all Atg16l1 KO groups, n = 10 per condition. For WT groups, n = 9 for isotype control-treated mice, and n = 10 each for anti-CD8 and anti-NK1.1 treated mice. g IFN-γ KO hosts; WT control BL6 hosts shown in panel ( d ). ns, not significant. h Direct comparison across studies of BLI signal (from panels d – g ) re-plotted as values normalized to the medians of WT organoid groups (after 4 weeks of growth). For WT/untreated hosts, n = 14 (WT organoids) and n = 11 (KO organoids), **** P < 0.0001; for CD8 T cell depleted hosts, n = 10 per group, **** P < 0.0001; for NK-depleted hosts, n = 10 per group, ** P = 0.0015; for IFN-γ KO hosts, n = 11 per group, * P = 0.0233; for NSG hosts, n = 14 (WT organoids) and n = 13 (KO organoids), ** P = 0.0027. WT and Atg16l1 KO organoids were compared in each condition using two-sided Mann–Whitney tests. Atg16l1 KO groups from each immunodeficient condition were also compared to Atg16l1 KO tumor growth in WT/untreated hosts using Mann-Whitney tests; † P = 0.0048, †† P = 0.0032, ††† P < 0.0001. i , j Representative BLI images of mice (top) and tumor burden in livers (bottom) from ( i ) BL6 ( n = 14 WT; n = 11 Atg16l1 KO) hosts or ( j ) NSG ( n = 14 WT; n = 13 Atg16l1 KO) hosts. In panels ( d – h ), lower and upper hinges in box plots correspond to first and third quartiles, while whiskers extend to minima and maxima. Individual data points indicate separate mice (biological replicates). In panels ( d , e , and g ), groups were compared using two-sided Mann–Whitney tests, with P -values adjusted for multiple comparisons using the Holm-Sidak method. All data are representative of 2-3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Atg16l1 re-expression plasmid (pb-EF1_Atg16l1OE-IRES_tagBFP2) was generated with mouse Atg16l1 sequence (isoform 1, https://www.uniprot.org/uniprot/Q8C0J2 ) into pb1-EF1-IRES-tagBFP2 piggybac vector. pb-EF1_Atg16l1OE-IRES_tagBFP2 (2 μg) or its control vector pb1-EF1_tagBFP2 (2 μg) together with transposase plasmid (1 μg) was electroporated into AKPS-GFP-Luc-ATG16.ko organoids using the P1 buffer and CM137 program (Lonza).

Techniques: CRISPR, Western Blot, Clone Assay, Injection, Imaging, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer

doi: 10.1038/s41467-023-41618-7

Figure Lengend Snippet: a Visualization of eight scRNA-seq clusters in UMAP dimensions: Prolif ( n = 5154), Sensory/secretory (SS) ( n = 1956), Mature enterocyte (ME) ( n = 2378), Neuroendocrine (NE) ( n = 1046), Enterocyte progenitor (EP) ( n = 2467), IFN resp ( n = 369), Stem ( n = 1320), and Goblet/Paneth (GP) ( n = 573) clusters. b , Heatmap showing the average expression of top 10 markers for each cluster in panel ( a ). c Density plot for the WT (left) and Atg16l1 KO (right) conditions. Cells were harvested from n = 2 mice for each condition. Low and high density are denoted by blue and red, respectively. d Proportion of cells in each cluster among all cells in the respective sample (Atg16l1 KO or WT). e Barplot indicating the proportion change of each cluster in the Atg16l KO sample with respect to the WT condition. For each cluster, the y -axis shows the log-transformed value for (proportion in KO)/(proportion in WT) ratio. Adjusted P -values are derived from two-sided Pearson’s chi-squared test for two proportions as implemented in the prop.test R function, adjusted after false discovery rate correction. f GSEA for Atg16l1 KO vs WT contrasts in each cluster using MSigDB Hallmark gene sets. Only gene sets with significant results in at least two contrasts are shown (clusterProfiler, FDR-adjusted P -values for enrichment scores derived via permutation test). g GSEA of Atg16l1 KO vs WT organoids in vitro (bulk RNA-seq) for the top 100 genes marking each scRNA-seq cluster. The x -axis denotes clusters while the y-axis shows normalized enrichment scores (normalized to mean enrichment of random samples of the same size). h GSEA enrichment plot for GP cluster from panel ( g ). For panels ( g and h ), normalized enrichment scores were computed with fgsea using voom+limma derived fold changes; P -values for enrichment scores were derived via permutation test; unadjusted P -values shown. * P < 0.05, ** P < 0.01, and *** P < 0.001. (IFN interferon). Source data and exact P -values for panels ( e and g ) are provided as a Source Data file.

Article Snippet: Atg16l1 re-expression plasmid (pb-EF1_Atg16l1OE-IRES_tagBFP2) was generated with mouse Atg16l1 sequence (isoform 1, https://www.uniprot.org/uniprot/Q8C0J2 ) into pb1-EF1-IRES-tagBFP2 piggybac vector. pb-EF1_Atg16l1OE-IRES_tagBFP2 (2 μg) or its control vector pb1-EF1_tagBFP2 (2 μg) together with transposase plasmid (1 μg) was electroporated into AKPS-GFP-Luc-ATG16.ko organoids using the P1 buffer and CM137 program (Lonza).

Techniques: Expressing, Transformation Assay, Derivative Assay, In Vitro, RNA Sequencing Assay

Journal: Nature Communications

Article Title: Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer

doi: 10.1038/s41467-023-41618-7

Figure Lengend Snippet: a Visualization of ten scRNA-seq clusters in UMAP dimensions: TREM2 MF ( n = 7116), VSIG4 MF ( n = 890), MARCO MF ( n = 434), IFN responsive MONO/MF ( n = 560), MONO ( n = 2767), cDC1 ( n = 969), cDC2 ( n = 2297), CCR7 + DC ( n = 331), pDC ( n = 1115), NEUT ( n = 4770) clusters. b Heatmap showing the average expression of top 10 markers for each cluster in panel ( a ). c Density plot for the WT (left) and Atg16l1 KO (right) groups. Cells were harvested from n = 2 mice for each condition. Low and high density are denoted by blue and red, respectively. d Proportion of cells in each cluster among all cells in the respective sample (Atg16l1 KO or WT). e Barplot indicating the proportion change of each cluster in the Atg16l1 KO sample compared to the WT group. For each cluster, the y -axis shows the log-transformed value for (proportion in KO)/ (proportion in WT) ratio. P -values were calculated from two-sided Pearson’s chi-squared test for two proportions as implemented in the prop.test R function, and adjusted by false discovery rate correction. f GSEA for Atg16l1 KO vs WT contrasts in each cluster using MSigDB Hallmark gene sets. Only gene sets with significant results in at least four contrasts are shown (clusterProfiler, P -values for enrichment scores derived via permutation test, FDR-adjusted P -values < 0.05). * P < 0.05, ** P < 0.01, and *** P < 0.001. (MF macrophages, MONO monocytes, DC dendritic cells, pDC plasmacytoid dendritic cells, NEUT neutrophils). Source data and exact P -values for panel e are provided as a Source Data file.

Article Snippet: Atg16l1 re-expression plasmid (pb-EF1_Atg16l1OE-IRES_tagBFP2) was generated with mouse Atg16l1 sequence (isoform 1, https://www.uniprot.org/uniprot/Q8C0J2 ) into pb1-EF1-IRES-tagBFP2 piggybac vector. pb-EF1_Atg16l1OE-IRES_tagBFP2 (2 μg) or its control vector pb1-EF1_tagBFP2 (2 μg) together with transposase plasmid (1 μg) was electroporated into AKPS-GFP-Luc-ATG16.ko organoids using the P1 buffer and CM137 program (Lonza).

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Nature Communications

Article Title: Tumor-intrinsic expression of the autophagy gene Atg16l1 suppresses anti-tumor immunity in colorectal cancer

doi: 10.1038/s41467-023-41618-7

Figure Lengend Snippet: a GSEA using MSigDB IFNγ response hallmark gene set for differential expression between Atg16l1 KO ( n = 2) and WT ( n = 2) CRC organoids treated (in vitro bulk RNA-seq data) with p -value = 4.78 × 10 −15 (normalized enrichment score was computed with fgsea using voom+limma derived fold changes; P -value for enrichment score was derived via permutation test). b Differential expression of representative IFNγ-induced genes between Atg16l1 KO ( n = 2) and WT ( n = 2) CRC organoids in either untreated or IFNγ-treated conditions. c WT or Atg16l1 KO CRC organoids treated as indicated and stained with propidium iodide (PI). Scale bar = 1000 μm. Images are representative of 4–6 technical replicates per condition (see Source Data for Fig. 5d). d , f Cell death assayed by live imaging of WT versus Atg16l1 KO organoids ( d ), and Atg16l1 KO versus Atg16l1 + RIPK3 double KO organoids ( f ) treated with combinations of TNF + IFNγ + zVAD for 48 h. PI staining is measured by fluorescence intensity/μm 2 . Groups compared using two-way ANOVA. e Immunoblot analysis of the indicated phosphorylated and total proteins in WT or Atg16l1 KO CRC organoids stimulated with combinations of TNF + IFNγ for 4 or 18 h. * indicates non-specific bands. Data for panels c – f are representative of three independent experiments. Summary data are shown as mean ± s.e.m. Source data for panels ( d – f ) are provided as a Source Data file.

Article Snippet: Atg16l1 re-expression plasmid (pb-EF1_Atg16l1OE-IRES_tagBFP2) was generated with mouse Atg16l1 sequence (isoform 1, https://www.uniprot.org/uniprot/Q8C0J2 ) into pb1-EF1-IRES-tagBFP2 piggybac vector. pb-EF1_Atg16l1OE-IRES_tagBFP2 (2 μg) or its control vector pb1-EF1_tagBFP2 (2 μg) together with transposase plasmid (1 μg) was electroporated into AKPS-GFP-Luc-ATG16.ko organoids using the P1 buffer and CM137 program (Lonza).

Techniques: Expressing, In Vitro, RNA Sequencing Assay, Derivative Assay, Staining, Imaging, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Fishing Cat Cell Biobanking for Conservation

doi: 10.1101/2022.07.01.498384

Figure Lengend Snippet: A-B) Fishing cat fibroblasts were reprogrammed with PiggyBAC transposon carrying mouse C-myc, Klf4, Oct4 and Sox2 (MKOS) and mOrange (Or) as a reporter, together with an expression vector expressing transposase (TS). The fibroblasts expressed mOrange, indicating the successful integration of piggyBAC transposon. C-D) The fibroblasts were reprogrammed with piggyBAC transposon carrying inducible reprogramming cassette composed of human OCT4, KLF4, SOX2, C-Myc (OKSM) and human KLF2 and NANOG followed by Ires-Tdtomato. The transfection included transposase vector and piggyBAC expressing rtTA. The transfected cells were selected with puromycin and the expression of reprogramming factors were induced via Tet-on using Doxycycline. The iPSC-like colonies appeared with TdTomata expression. E-F) Reprogramming of fibroblasts using episomal vectors. The transfected cells were tested in various media including commercial HSC NutriStem and E8 medium, KOSR containing medium (KOSR) and media with 10% Fetal Bovine Serum (FBS) with LIF. The induced cells were also replated onto different matrix including mouse irradiated feeder, vitronectin and Geltrex. G-H) RNA-based reprogramming using self-replicating RNA (srRNA) expressing reprogramming factors. The testicular fibroblasts were transfected with srRNA and cultured in the presence of B18R protein before replating onto feeder cells. The iPSC-like colonies appeared in three weeks after transfection.

Article Snippet: We transfected pC6F alongside with transposase vector (pCy43, Sanger Institute) and PB rtTA vector (addgene 126034) using Lipofectamine 3000 (ThermoFisher) for overnight.

Techniques: Expressing, Plasmid Preparation, Transfection, Irradiation, Cell Culture

Journal: Journal of Virology

Article Title: A Screen for Epstein-Barr Virus Proteins That Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein

doi: 10.1128/JVI.00262-18

Figure Lengend Snippet: Effect of BKRF4 on RNF168 recruitment to dsDNA breaks. (A) U2OS 2-6-5 cells with inducible GFP-RNF168 were transiently transfected with plasmids expressing FLAG-tagged BKRF4 or BKRF4N or empty plasmid (EV), GFP-RNF168 expression was induced with doxycycline for 24 h, and cells were then treated with 10 μg/ml of etoposide for 2 h to induce DNA damage. Cells were processed for FLAG (red) and γ-H2AX (white) immunofluorescence, and GFP foci were also visualized by fluorescence microscopy. In all micrographs, dashed lines indicate the nucleus outline (as determined by DAPI staining; not shown), and insets represent ×10 magnifications of the indicated fields. Scale bar = 5 μm. (B) GFP-RNF168 foci were counted in at least 50 cells expressing the indicated FLAG-tagged protein (or EV control) for three independent experiments; distributions of the focus numbers are shown in the cluster diagram. Mean values ± SDs relative to control cells for three independent experiments are also shown in the bar graph. P values are indicated as in Fig. 1. (C) Western blots are shown for samples generated as for panel A using antibodies against GFP and FLAG (and tubulin as a loading control). Values under the lanes show expression levels of GFP-RNF168 (normalized to tubulin) relative to empty vector control.

Article Snippet: The PiggyBac (PB) transposon-based PB-TetO-GFP-RNF168 expression vector was generated in three steps. (i) The OSKM genes from PB-TAC-OSKM (kind gift from Knut Woltjen; Addgene plasmid 80481) was exchanged for a ccdB gene in a BP clonase reaction with pDONR-221 to generate PB-TAC-gateway plasmid. (ii) The IRES-mCherry reporter cassette was then removed from PB-TAC-gateway by Sfo I/ApaI restriction digestion, Klenow fill-in reaction, and ligation to generate PB-TetO-gateway. (iii) GFP-RNF168 was PCR amplified from pCDNA FRT-TO-GFP-RNF168 ( 54 ), gateway cloned into pDONR-221, and recombined into PB-TetO-gateway with LR clonase.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Fluorescence, Microscopy, Staining, Western Blot, Generated