Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 modulates pancreatic tumor growth. A) Immortalized human pancreatic duct cells (hTERT-HPNE and HPDE6c7), human and murine PDA cell lines, and human patient derived-organoids (PDOs) demonstrate nSMase activity. Values shown are technical replicates of one lysate per cell line. B) SMPD3 expression was analyzed in an existing human pancreas organoid RNA-Seq dataset and is depicted in normal (n=11), primary (n=35), and metastatic (n=9) human organoids. Log of normalized counts of SMPD3 expression are plotted. C) Immunocytochemistry demonstrates nSMase2 expression in two pancreatic metastatic PDOs. Scale bar is 100uM. D) Nanoparticle tracking analysis of CD63 + sEVs upon GW4869 and vehicle treatment is depicted. E) IVIS imaging results at Day 7, 14, and 19 are graphed. Eight animals were injected with each cell line. F) Images depict bioluminescence signal during IVIS imaging at day 19. G) Pancreas weight/body weight at day 19 is graphed. Average values for the KPC scrambled shRNA are 0.04540 ± 0.001635 (n=8) and KPC Smpd3 shRNA 1 are 0.02817 ± 0.0008858 (n=7). H) Rag 1 KO mice injected orthotopically with KPC Smpd3 shRNA1 and KPC Smpd3 shRNA2 cell lines lived significantly longer than Rag1 KO mice injected orthotopically with KPC scrambled shRNA cells ( P =0.0013 using log-rank test and P =0.0001 using log-rank test, respectively). The median survival of Rag 1 KO mice injected orthotopically with KPC scrambled shRNA cells (n=12) is 19 days, KPC Smpd3 shRNA 1 cells (n=6) is 31.50 days, and KPC Smpd3 shRNA 2 cells (n=6) is 36.50 days.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Derivative Assay, Activity Assay, Expressing, RNA Sequencing, Immunocytochemistry, Imaging, Injection, shRNA

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: S m pd3 regulates proliferation of PDA cells in vivo . A) SMPD3 is expressed in a panel of human pancreatic duct cells (n=2 cell lines), human PDA cells (n=12 cell lines), and mouse PDA cells (n=3 cell lines). B) qPCR demonstrates a reduction in Smpd3 expression in KPC Smpd3 shRNA 1 LucFlag (n=3) and KPC Smpd3 shRNA 2 LucFlag (n=3) cell lines when compared to the KPC scrambled shRNA LucFlag (n=3) control cell line. C) Mean ± standard deviation for tumor volume of cell lines injected subcutaneously are graphed. Results of an unpaired t test comparing KPC scrambled shRNA and KPC Smpd3 shRNA 2 cell lines at day 17 are depicted on the graph. For all cell lines, N=6 animals. D) Mean ± standard error of the mean for tumor weight at Day 17 of cell lines injected subcutaneously are graphed. For all cell lines, N=6 animals. E) Immunofluorescence images and quantification of epithelial (CK19 + , Vimentin - , Dapi + ), proliferative (Ki67 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag line (n=4) are 39.33% ± 2.046%, KPC Smpd3 shRNA 1 LucFlag line (n=5) are 28.88% ± 4.096%, and KPC Smpd3 shRNA 2 LucFlag line (n=5) are 15.83% ± 1.960. Scale bar is 50uM. F) Immunofluorescence images and quantification of epithelial (E-cadherin + , Vimentin - , Dapi + ), apoptotic (Cleaved caspase 3 + ) pancreatic cancer cells are depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.299% ± 0.2075%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.082% ± 0.2126%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=5) are 0.9832% ± 0.1307. Scale bar is 50uM. G) CTG assay or colony formation assay results for the indicated cell lines are depicted. H) Reduction of Smpd3 in epithelial pancreatic cancer cells modestly reduces fibrosis. IHC demonstrates loss of nSMase2 in epithelial pancreatic cancer cells in tumors generated using the KPC Smpd3 shRNA 1 LucFlag and KPC Smpd3 shRNA 2 LucFlag cell lines. One scale bar (1000uM) on the picrosirius red image is representative for all picrosirius red and corresponding brightfield images. One scale bar (200uM) on the nSMase2 IHC image is representative for all nSMase2 IHC images. I) Quantification of polarized light using the picrosirius red stained images is depicted. Average values for tumors generated using the KPC scrambled shRNA LucFlag cell line (n=4) are 1.734% ± 0.3760%, KPC Smpd3 shRNA 1 LucFlag cell line (n=5) are 1.319% ± 0.1328%, and KPC Smpd3 shRNA 2 LucFlag cell line (n=4) are 0.9188% ± 0.1124.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: In Vivo, Expressing, shRNA, Control, Standard Deviation, Injection, Immunofluorescence, Generated, CTG Assay, Colony Assay, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 expression is upregulated in PanIN. A) Schematic details the construct design for the Smpd3 floxed mouse. B-C) For examination of nSMase2 expression in the indicated normal murine pancreatic cell types, nSMase2 IHC sections from 3 C57BL/6J mice were scored. Preneoplasia and neoplasia lesions were scored from KPC mice (n=10 animals scored for acinar-to-ductal metaplasia (ADM), n=12 animals scored for PanIN, n=10 animals scored for primary tumor, and n=4 animals scored for metastasis). Scale bars for IHC images are 40 uM. nSMase2 expression was scored on a scale of 0-3 and is depicted in (C). The scoring system used is 0=absent, 1=low, 2=medium, 3=high. Values depicted are representative for individual animals. D) Subcellular localization of nSMase2 was scored using the same slides and animals used for expression scoring in . Results from Chi-squared tests using a 2 x 3 table are shown. E-F) H&E and IHC for nSMase2 images are displayed for the indicated genotypes.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Smpd3 ablation reduces formation of neoplasia and prolongs survival of KPC mice. A) KPC; Smpd3 f/f mice lived significantly longer than KPC; Smpd3 wt/wt mice ( P =0.0039 using log-rank (Mantel-Cox) test). The median survival of KPC; Smpd3 f/f mice (n=41) is 23.71 weeks, KPC; Smpd3 f/wt mice is 21.57 weeks (n=45), and KPC; Smpd3 wt/wt mice (n=47) is 20.00 weeks. B) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt mice (n=30), KPC; Smpd3 f/wt mice (n=27), and KPC; Smpd3 f/f mice (n=24). (C) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt (n=29), KPC; Smpd3 f/wt (n=25), and KPC; Smpd3 f/f (n=22) mice. Using Fisher’s exact tests, no significant differences were observed in the number or site of macrometastatic lesions present at end-stage in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , or KPC; Smpd3 f/f mice. If macrometastasis was present, only one site was observed per animal in these cohorts. D-E) Quantification of alcian blue staining demonstrates significantly reduced PanIN lesions in KPC; Smpd3 f/f mice (n=5) when compared to KPC; Smpd3 wt/wt mice (n=9) ( P =0.0154). H&Es used to score edema are depicted. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PanIN-bearing KPC; Smpd3 f/f mice. F) Edema scoring for KPC; Smpd3 wt/wt (n=9), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=7) mice is depicted. The scoring system used is 1=low, 2=medium, and 3=high. G-H) The percentage of pancreatic area occupied by PDA is significantly higher in KPC; Smpd3 wt/wt mice (74.16 ± 8.705) when compared to KPC; Smpd3 f/f mice (30.51 ± 17.72) ( P =0.0497) at 19-21 weeks of age. Average percent area is graphed in H (n=4 KPC; Smpd3 f/f mice, n=4 KPC; Smpd3 f/wt mice, n=5 KPC; Smpd3 wt/wt mice). Dotted lines show the PDA area in representative images. nSMase2 immunohistochemistry depicts loss of nSMase2 in pancreatic epithelium of PDA-bearing KPC; Smpd3 f/f mice at 19-21 weeks of age.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of the KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. A-F) Western blot depicts nSMase2 expression levels in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , KPC; Smpd3 f/f , KPC; Smpd3 wt/wt Mock OE, and KPC; Smpd3 wt/wt Smpd3 OE cell lines. G) Pictures of cell lines taken immediately before RNA isolation for RNA sequencing are shown. Scale bars are 100uM.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: RNA sequencing demonstrates a role for Smpd3 in modulation of cellular pathways and regulation of PDA subtype. A) Average luminescence values from 3-5 independent experiments per cell line are graphed. Average values for primary PDA cell lines generated from KPC; Smpd3 wt/wt mice (n=17 cell lines) are 98,614 ± 5,684, KPC; Smpd3 f/wt mice (n=14 cell lines) are 106,061 ± 6,699, and KPC; Smpd3 f/f mice (n=15 cell lines) are 88,460 ± 6,672. B) Average number of colonies from at least 3 independent colony formation assays per cell line are depicted. Average values from KPC; Smpd3 wt/wt mice (n=14 cell lines) are 26.02 ± 3.652, KPC; Smpd3 f/wt mice (n=14 cell lines) are 24.16 ± 2.519, and KPC; Smpd3 f/f mice (n=13 cell lines) are 21.34 ± 4.872. C) Heat maps depict expression of significantly deregulated molecules in Tumor Microenvironment Pathway and Hepatic Fibrosis Signaling Pathway in all three PPT cell lines. Molecules shown in heat maps include those molecules within the Ingenuity pathway analysis (IPA) pathway that were significantly deregulated in any comparison between PPT groups. D) IPA results show a bubble category chart of significantly deregulated pathways when comparing KPC; Smpd3 f/f PPT and KPC; Smpd3 wt/wt PPT cell lines. A positive z-score represents upregulation, and a negative z-score indicates downregulation of a pathway in KPC; Smpd3 f/f PPT when compared to KPC; Smpd3 wt/wt PPT cell lines. A gray circle depicts significant overrepresentation of a pathway, the direction of which cannot yet be determined. Select pathways are annotated. E) Picrosirius red stained images are shown for KPC; Smpd3 wt/wt (n=6), KPC; Smpd3 f/wt (n=5), and KPC; Smpd3 f/f (n=6) mice at 10-11 weeks of age. The small piece of intestine in the lower left corner of the KPC; Smpd3 f/wt image was excluded from the quantification. Scale bar is 1000uM. Inset shows brightfield image. F) Transcriptional subtyping of end-stage pancreatic tumor cell lines generated from KPC; Smpd3 wt/wt (PPT and MPT), KPC; Smpd3 f/wt (PPT), KPC; Smpd3 f/f (PPT and MPT) as well as KPC Mock OE and KPC Smpd3 OE lines is depicted. Genes used to identify subtypes according to the Moffitt classification are shown. G) Pie charts depict the number of cell lines with the classical and basal-like Moffitt subtypes. When comparing KPC; Smpd3 wt/wt PPT vs KPC; Smpd3 f/f PPT using Moffitt classification and Chi square test, p-value=0.0339.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Generated, Expressing, Comparison, Staining

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of RNA sequencing data from murine polyclonal pancreatic cancer cell lines. A) Heat map showing Integrin Signaling in all three PPT cell lines. B) Estimated counts for select genes identified from Sleuth comparison of polyclonal cell lines from end-stage PPTs of KPC; Smpd3 wt/wt (n=8), KPC; Smpd3 f/wt (n=7), and KPC; Smpd3 f/f (n=10) mice are graphed. Each box plot represents the distribution of estimated counts quantified by kallisto with 100 bootstrap samples. For each gene, the most significant protein-coding isoforms are shown. C) The top twenty pathways from IPA comparing KPC; Smpd3 f/f MPT vs KPC; Smpd3 wt/wt MPT are shown. D) A heat map including significantly differentially regulated molecules in the Pentose Phosphate Pathway are shown for KPC; Smpd3 wt/wt MPT and KPC; Smpd3 f/f MPT groups. E) The six differentially regulated pathways when comparing KPC Smpd3 OE vs KPC Mock OE are shown. F) Log of transcript per million (TPM) values for Smpd3 gene are graphed for KPC Mock OE and KPC Smpd3 OE sequenced cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: K P C ; Smpd3 f/f pancreata display significantly fewer activated stellate cells and fibroblasts when compared to both KPC; Smpd3 f/wt and KPC; Smpd3 wt/wt pancreata. A) Immunofluorescence images depict co-labeling with alpha SMA, Gfap, Desmin, and Dapi in the pancreas of 10–11-week-old mice. For KPC; Smpd3 wt/wt , n=6 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=5 animals. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel C. Scale bar is 20uM. B) Immunofluorescence images depict co-labeling with alpha SMA, Fap, Vimentin, and Dapi in the pancreas of 10–11-week-old mice. Yellow arrows in insets point to examples of quadruple positive cells that were quantified in Panel D. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, KPC; Smpd3 f/f , n=6 animals. Scale bar is 50uM. C) Quantification of alpha SMA, Gfap, Desmin, and Dapi quadruple positive cells is depicted. D) Quantification of alpha SMA, Fap, Vimentin, and Dapi quadruple positive cells is depicted. E) Pie charts depict Moffitt classification PDA subtypes for KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Analysis of lipidomics data from murine polyclonal pancreatic cancer cell lines. A) Heat map shows IPA Ceramide Signaling Pathway in all three PPT cell lines. B) Heat map shows differentially expressed lipids in comparisons amongst our murine polyclonal pancreatic cancer cell lines. Groups compared are listed as A-B where depicted upregulation or downregulation of a lipid would be in A when compared to B. C) Principal component analysis of our polyclonal murine pancreatic cancer cell lines based on lipid composition is depicted. D) Box plots showing expression of selected ceramide species in our KPC; Smpd3 wt/wt PPT, KPC; Smpd3 f/wt PPT, KPC; Smpd3 f/f PPT, KPC; Smpd3 wt/wt MPT, and KPC; Smpd3 f/f MPT cell lines. E) Box plots showing expression of selected phosphatidylcholine species in our KPC Mock OE and KPC Smpd3 OE cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Characterization of exosomes isolated from KPC; Smpd3 wt/wt PPT and KPC; Smpd3 f/f PPT cell lines. A) CD63eGFP particles per cell are graphed for KPC; Smpd3 wt/wt CD63eGFP cell lines (n=6) and KPC; Smpd3 f/f CD63eGFP cell lines (n=6). B) Fractions were subjected to WB for sEV markers to determine the fractions that exosomes are contained within using the C-DGUC exosome isolation protocol. Expected band sizes are listed for each marker. C) The percentage of animals having PDA at end-stage in addition to the differentiation status of primary pancreatic tumors at end-stage is graphed for KPC; Smpd3 wt/wt uninjected mice (n=30), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=15), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=15), KPC; Smpd3 f/f uninjected mice (n=24), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=9), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . D) The percentage of animals having macrometastasis at end-stage in addition to the site of macrometastasis at end-stage is depicted for KPC; Smpd3 wt/wt uninjected mice (n=29), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=14), KPC; Smpd3 wt/wt mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=12), KPC; Smpd3 f/f uninjected mice (n=22), KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 wt/wt PPT cell lines (n=8), and KPC; Smpd3 f/f mice injected with sEVs isolated from KPC; Smpd3 f/f PPT cell lines (n=9). Values plotted for uninjected mice are the same as those in . E) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 wt/wt are 0.04396 ± 0.006112 (n=28), KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.05477 ± 0.007678 (n=15), and KPC; Smpd3 wt/wt injected with exosomes isolated from KPC; Smpd3 f/f are 0.06809 ± 0.009409 (n=16). F) Pancreas weight/body weight is graphed for the indicated groups. Average values for uninjected KPC; Smpd3 f/f are 0.05085 ± 0.005912, KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 wt/wt are 0.06149 ± 0.01434, and KPC; Smpd3 f/f injected with exosomes isolated from KPC; Smpd3 f/f are 0.04654 ± 0.008279. G) Pancreas images at dissection for the indicated groups are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Isolation, Marker, Injection, Dissection

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: PDA cell exosomes generated through nSMase2 accelerate PDA progression. A) Schematic of exosome injection study. B) Probability of survival for the indicated groups is graphed. Log-rank test was performed between the indicated groups. C-D) Volcano plot (C) and heat map (D) depict differentially expressed exosomal miRNAs isolated from KPC; Smpd3 f/f and KPC; Smpd3 wt/wt polyclonal PDA PPT cell lines. E) Exosomal protein abundance results for KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines are depicted. The x-axis shows the log2 fold change for KPC; Smpd3 f/f over KPC; Smpd3 wt/wt and the y-axis shows −log10(q-value). Significantly upregulated proteins (q-value < 0.05) are shown in green and significantly downregulated proteins are shown in red. F) Heatmap of top 40 differentially abundant exosomal proteins comparing KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt , displaying log2 normalized expression. Red indicates lower abundance of a given protein in each sample and green indicates higher abundance. Rows and columns are arranged based on hierarchical clustering dendrograms (not displayed). The color bar at the top indicates the genotype. G) IPA results depict significantly differentially regulated pathways when comparing exosomal proteins from KPC; Smpd3 f/f versus KPC; Smpd3 wt/wt PPT cell lines.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Injection, Isolation, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: sEVs generated independently of nSMase2 promote a proinflammatory macrophage phenotype. A) Heat map depicts scaled marker expression in KPC; Smpd3 wt/wt and KPC Smpd3 f/f pancreata using our 17-marker myeloid spectral flow cytometry panel. B) t-SNE of 200,683 cells stained with our spectral flow panel depict 12 clusters identified for KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata through FlowSOM clustering. C) Heat map of scaled marker expression and number of cells for clusters shown in B is displayed. D) Bar graphs depict percent cellular subpopulations from KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreata for the indicated markers after gating in FlowJo. E) Immunofluorescence images depict co-labeling with F4/80, iNOS, and Dapi at 10-11 weeks of age. Scale bar is 20uM. F) Quantification of intralobular F4/80 + and iNOS + copositive cells in KPC; Smpd3 wt/wt (n=5 animals), KPC; Smpd3 f/wt (n=6 animals), and KPC; Smpd3 f/f (n=5 animals) pancreata at 10-11 weeks of age is depicted. G) Schematic of experiment to assess how nSMase2-mediated exosome biogenesis affects polarization of macrophages. H-J) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Generated, Marker, Expressing, Flow Cytometry, Staining, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Functional effects of PDA cell sEVs on macrophages. A) Immunofluorescence images depict co-labeling with F4/80, Ly6G, and Dapi at 10-11 weeks of age. For KPC; Smpd3 wt/wt , n=5 animals, KPC; Smpd3 f/wt , n=5 animals, and KPC; Smpd3 f/f , n=6 animals. Scale bar is 20uM. B) Quantification of interlobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. C) Quantification of intralobular F4/80 and Ly6G in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f pancreata at 10-11 weeks of age is depicted. D-F) RNA expression fold change in RAW 264.7 cells, with PBS treated normalized to 1, for each gene at the indicated time point and treatment condition is depicted. Results of unpaired t tests between groups shown at the time point written are depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Functional Assay, Immunofluorescence, Labeling, RNA Expression

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: SMPD3 is an independent prognostic factor for pancreatic cancer patient survival. A) Log2 of the normalized counts are plotted for normal human pancreatic SMPD3 expression (n=39 samples) and primary PDA SMPD3 expression (n=39 samples). B) Log2 of the normalized counts are plotted for PPT SMPD3 expression (n=231 samples) and MPT SMPD3 expression (n=158 samples) in PDA patients. C) Kaplan-Meier survival curve of the whole cohort (N = 94) with the patients dichotomized into SMPD3 high and low expression based on the median mRNA expression value. SMPD3 high group had significantly better survival (median survival of 26.5 vs 15.0 months; P = 0.0364). A (0) value indicates a censored event and a (1) value indicates a death event. D) Kaplan-Meier survival curve of the subset of the whole cohort of patients who underwent adjuvant chemotherapy (> 95% single agent gemcitabine) after surgical resection (N = 58). SMPD3 expression was significantly associated with better survival (median survival of 34.3 vs 15.9 months; P = 0.0289) indicating SMPD3 is a biomarker of adjuvant gemcitabine response. E) Kaplan-Meier survival curve of the subset of the whole cohort of patients who did not undergo adjuvant chemotherapy after surgical resection (N = 36). SMPD3 expression was not associated with survival (P = 0.8001). F-G) Kaplan-Meier curves depict patient survival based on SMPD3 expression in locally advanced primaries or metastatic PDA from the COMPASS trial for patients who received chemotherapy with modified Folfirinox or Gemcitabine plus Abraxane. SMPD3 expression was stratified into high and low with low expression defined as the maximal chi-squared statistic. H) Log2 of SMPD3 expression by hypoxia status defined by . I) Log2 of SMPD3 expression in defined Waddell structural variation subtypes in COMPASS cases is depicted. J) Kaplan-Meier curves depict patient survival based on cytoplasmic and membranous nSMase2 expression in treatment naïve primary resected PDA. Tumor, and not stromal, cell nSMase2 expression was scored in 143 patients. When comparing survival of PDA patients with low cytoplasmic nSMase2 expression (n=89, mean survival=30.1 months) and high cytoplasmic nSMase2 expression (n=54, mean survival=29.1 months), p=0.627 using the log-rank test. When comparing survival of PDA patients with low membrane nSMase2 expression (n=65, mean survival=25.6 months) and high membrane nSMase2 expression (n=78, mean survival=32.9 months), p=0.052 using the log-rank test.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Biomarker Discovery, Modification, Membrane

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: Pathways analysis of pancreatic nSMase2 expression in PDA patients based on treatment with chemotherapy in addition to the effect of nSMase2 expression on chemosensitivity and vasculature integrity. A) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs receiving adjuvant chemotherapy. The threshold line represents statistical significance. B) IPA bar chart results show the top 15 deregulated pathways when comparing PDA patients with high SMPD3 expression in treatment naïve PPTs vs low SMPD3 expression in treatment naïve PPTs not receiving adjuvant chemotherapy. C) Representative images used to score the TMA for nSMase2 low and nSMase2 high membrane and cytoplasmic labeling. D) Images depict dextran and tomato lectin, a marker of blood vessels in mice, immunofluorescence at 10-11 weeks of age in KPC; Smpd3 wt/wt and KPC; Smpd3 f/f pancreases. E) Percent pancreatic neoplastic area per field occupied by dextran is graphed for KPC; Smpd3 wt/wt (n=8) and KPC; Smpd3 f/f (n=6) mice. F) Negative log of gemcitabine IC50 for KPC; Smpd3 wt/wt , KPC; Smpd3 f/f , KPC Mock OE, and KPC Smpd3 OE cell lines is graphed. G) Quantification of total HIF1α in nSMase2 high and nSMase2 low primary pancreatic tumors of PDA patients is depicted.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Expressing, Adjuvant, Membrane, Labeling, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

doi: 10.1101/2024.09.23.614610

Figure Lengend Snippet: nSMase2 regulates PDA vasculature development. A-B) Heat maps showing VEGF Signaling and HIF1α Signaling pathways in all three PPT cell lines. C) Log of transcript per million (TPM) values for Vegfa gene are graphed for sequenced PPT cell lines. Wilcoxon test was performed. D) Pancreatic CD31 and CK19 immunolabeling at 19-21 weeks of age in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is depicted. Scale bar is 20uM. E) Quantification of pancreatic CD31 immunofluorescence in KPC; Smpd3 wt/wt , KPC; Smpd3 f/wt , and KPC; Smpd3 f/f mice is shown. F) Pancreatic CD31 and CK19 immunolabeling at 21 days post orthotopic injection of KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines into Rag1 KO mice 21 days post injection is depicted. Scale bar is 20uM. G) Quantification of pancreatic CD31 immunofluorescence in Rag1 KO mice injected with KPC LucFlag scrambled shRNA, KPC LucFlag Smpd3 shRNA 1, and KPC LucFlag Smpd3 shRNA 2 cell lines 21 days post injection is depicted. H) Immunohistochemistry of CD31 in primary tumors from PDA patients is depicted with low and high nSMase2 expression. I) Percent CD31 positive area in primary nSMase2 low (n=19) and nSMase2 high (n=34) pancreatic tumors from PDA patients is shown. J) Immunohistochemistry of HIF1α is depicted in primary tumors from PDA patients with low and high nSMase2 expression. K) Percent nuclear HIF1α epithelial tumor cells in primary nSMase2 low (n=5) and nSMase2 high (n=6) PDA patient pancreatic tumors is shown. L) Ratio of pancreas weight to body weight for gemcitabine and vehicle treated Rag1 KO mice 21 days after pancreatic orthotopic injection of the depicted cell lines is shown. For KPC scrambled shRNA vehicle treated mice, n=4, for KPC scrambled shRNA gemcitabine treated mice, n=9, for KPC Smpd3 shRNA 1 vehicle treated mice, n=10, for KPC Smpd3 shRNA 1 gemcitabine treated mice, n=10, for KPC Smpd3 shRNA 2 vehicle treated mice, n=10, and for KPC Smpd3 shRNA 2 gemcitabine treated mice, n=10. M) Kaplan-Meier survival curves show probability of survival for vehicle-injected KPC; Smpd3 wt/wt mice (n=12, median survival=20.29 weeks), gemcitabine-injected KPC; Smpd3 wt/wt mice (n=19, median survival=23.43 weeks), vehicle-injected KPC; Smpd3 f/f mice (n=15, median survival=23.29 weeks), gemcitabine-injected KPC; Smpd3 f/f mice (n=16, median survival=19.86 weeks). A black line indicates a censored animal.

Article Snippet: Removal of the Neomycin and lacZ cassettes in Smpd3 floxed mice was confirmed by genotyping with Transnetyx.

Techniques: Protein-Protein interactions, Immunolabeling, Immunofluorescence, Injection, shRNA, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: A comprehensive benchmark for multiple highly efficient base editors with broad targeting scope

doi: 10.1101/2024.12.17.628899

Figure Lengend Snippet: a , Schematic representation of base editors constructed via combination of different deaminase effectors and Cas9 variants. b , Schematic representation of the strategy for high-throughput evaluation of these ABEs and CBEs. A375 cells were firstly infected with lentiviruses at low MOI to generate expression-calibrated ABE- or CBE-expressing cell lines. The subsequent delivery of lentiviral BE evaluation library containing matched gRNA-target-barcode sequences into these cells induced base conversion at the genomically integrated target sequences. The whole fragment was PCR amplified for PE250 high-throughput sequencing to measure the editing outcomes. c , Heatmap showing the base editing window for indicated BEs. A>G/C>T indicates the spacer (gRNA) and target aligning at the same strand, while T>C/G>A means the spacer and target aligning at different strands. Values in the heatmap indicate the normalized editing efficiency (percentage to the maximal editing). d , The target position and editing efficiency relationship for default A>G/C>T conversion on the same strand or complementary target nucleotide T>C/G>A conversion on the different strands.

Article Snippet: HEK293FT, A375 (human melanoma cell line) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Construct, High Throughput Screening Assay, Infection, Expressing, Amplification, Next-Generation Sequencing

Journal: bioRxiv

Article Title: A comprehensive benchmark for multiple highly efficient base editors with broad targeting scope

doi: 10.1101/2024.12.17.628899

Figure Lengend Snippet: a , Percentage of favored, disfavored, and neutral codons across different BE sequences after human codon optimization using GeneScript’s algorithm. b , Western blot analysis for A375 cells lentivirally transduced with indicated FLAG-tagged ABEs or CBEs at a low MOI (∼0.1). ‘Control’ sample represents normal un-transduced A375 cells. GAPDH serves as a loading control. c , The editing (A>G or C>T) frequency (%) in indicated ABE- or CBE-expressing A375 cells lentivirally transduced with indivudial gRNAs targeting endogenous FANCF, ATP7B and MSTN genomic sites. Mean ± SD with n = 3 biological replicates. d , Schematic representation of the two-step strategy for constructing the BE evaluation library. e , Correlation of base editing activities on targets with default PAM patterns for indicated BEs between the two biological replicates. R means Spearman correlation coefficient and r is the Pearson correlation coefficient.

Article Snippet: HEK293FT, A375 (human melanoma cell line) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Transduction, Control, Expressing

Journal: bioRxiv

Article Title: A comprehensive benchmark for multiple highly efficient base editors with broad targeting scope

doi: 10.1101/2024.12.17.628899

Figure Lengend Snippet: a , Workflow of BE screening to identify driver mutations responsible for BRAF inhibitor (PLX-4720 or Vemurafenib) resistance in melanoma. b , The ranked list of interrogated gene mutations according to β-score in screens with indicated BEs and drug treatment. Representative hits are highlighted. c , Percentage of reads containing the intended edit ( NRAS c.182A>G) for SpRY-ABE8e-expressing A375 cells lentivirally transduced with two independent gRNAs (gRNA-1 or -2) for base editing in the presence or absence of indicated drug treatment for 14 days. Mean ± SD with n = 3. d , e , Cell number quantification ( d ) or Crystal violet staining ( e ) of SpRY-ABE8e-expressing A375 cells lentivirally transduced with two independent gRNAs (gRNA-1 or -2) targeting NRAS c.182A>G after 14 days of DMSO or indicated drug treatment. Empty vector without specific gRNA and gRNA targeting AAVS1 serves as control. Mean ± SD with n = 3 biological replicates. Unpaired two-sided t test, *** p < 0.001. f , Replenishment of PAM-mutated NRAS-Q61R but not NRAS-WT into NRAS knockout single clonal A375 cells showed significant drug resistance to PLX-4720 or Vemurafenib treatment. Cell number was counted by a hemocytometer. Mean ± SD with n = 3 biological replicates. Unpaired two-sided t test, *** p < 0.001. g , Workflow of druggable gene CRISPR knockout screens in NRAS-WT- and NRAS-Q61R-replenished NRAS knockout cells. h , The ranked list of druggable gene hits according to a differential β-score between NRAS-Q61R- and NRAS-WT-expressing cells under indicated conditions (DMSO, PLX-4720 or Vemurafenib treatment). Representative gene hits showing a preferential synthetic lethal effect with BRAF targeted drugs in resistant cells are highlighted. i , MTT assay showing the cell viability for NRAS-WT- or NRAS-Q61R-replenished NRAS knockout cells treated with PLX-4720 or JQ1 along or combination. Mean ± SD with n = 4 biological replicates. Unpaired two-sided t test, ** p < 0.01. ns means not significant.

Article Snippet: HEK293FT, A375 (human melanoma cell line) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Transduction, Staining, Plasmid Preparation, Control, Knock-Out, CRISPR, MTT Assay

Journal: bioRxiv

Article Title: A comprehensive benchmark for multiple highly efficient base editors with broad targeting scope

doi: 10.1101/2024.12.17.628899

Figure Lengend Snippet: a , Pearson’s correlation for indicated samples between the two biological replicates of ABE drug resistance screens. b , Density plot of β-score for gRNAs targeting splice sites of top essential genes in A375 cells or other gRNAs in the library of ABE screening samples (Day 21 vs Day 0) with Vehicle DMSO treatment. c , Venn diagram showing the overlap of drug-resistant variant hits across four drug resistance BE screens with two ABEs (SpCas9-ABE8, SpRY-ABE8e) and two drugs (PLX-4720, Vemurafenib). The significance of overlap between indicated groups was shown on the right with Fisher’s exact test p values. d , Western blot analysis of NRAS expression in indicated single clonal cells to confirm the knockout effect of NRAS . Three different gRNAs (A, B and C) targeting NRAS under pLenti-CRISPRv2-Puro vector were used to knockout endogenous NRAS in A375 cells and two independent single clones (#1 and #2) were expanded for each gRNA. GAPDH serves as a loading control. The red-colored sample was employed for further use. e , Western blot analysis of NRAS expression in indicated samples. Vector control, PAM-mutated NRAS-Q61R or NRAS-WT was re-introducted into NRAS knockout single clonal cells ( NRAS -KO-A #1) and two independent single clones (#1 and #2) with replenished NRAS were analyzed. The red colored clones were used in . f , Pearson’s correlation for indicated samples between the two biological replicates in druggable gene knockout screens.

Article Snippet: HEK293FT, A375 (human melanoma cell line) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Variant Assay, Western Blot, Expressing, Knock-Out, Plasmid Preparation, Clone Assay, Control, Gene Knockout

Journal: bioRxiv

Article Title: ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

doi: 10.1101/2021.07.17.452804

Figure Lengend Snippet: a , Maximum likelihood phylogeny of sarbecovirus RBDs, constructed from RBD nucleotide sequences. Node labels indicate bootstrap support values. b , Binding avidities of sarbecovirus RBDs for eight ACE2 orthologs, determined using high-throughput yeast-displayed RBD titration assays . Scale bar, bottom right. c , Alignment of tested ACE2 orthologs within RBD-contact positions (4Å cutoff in PDB 6M0J or 2AJF). d , Representative binding curves from high-throughput titrations. Underlying titration curves for individual replicate-barcoded representatives of a genotype are shown in faint gray, and the average binding across all barcodes is indicated in black. e , Biolayer interferometry binding analysis of R. affinis ACE2-Fc and biotinylated BtKY72 RBD immobilized at the surface of streptavidin biosensors. Data representative of three assays using independent preparations of RBD (biological triplicate) f , Entry of BtKY72 spike-pseudotyped VSV particles into 293T cells transiently expressing R. affinis ACE2 alleles. Each point represents the mean of technical triplicates for assays performed with independent preparation of pseudoviral particles (biological replicate). Geometric mean is indicated by horizontal line. Normalized pseudovirus western blot, and mock (no S) pseudovirus entry in R. affinis ACE2 293T cells in .

Article Snippet: HEK293T (293T) cells (ATCC CRL-11268) and 293T cells with stable transfection of human ACE2 were cultured in 10% FBS, 1% PenStrep DMEM at 37°C in a humidified 8% CO2 incubator.

Techniques: Construct, Binding Assay, High Throughput Screening Assay, Titration, Expressing, Western Blot

Journal: bioRxiv

Article Title: ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

doi: 10.1101/2021.07.17.452804

Figure Lengend Snippet: a , Representative Western blots for quantification of spike incorporation into pseudoviral particles. Anti-FLAG identifies incorporation of 3XFLAG-tagged spike, and anti-VSV-M identifies level of VSV backbone. Viral inputs into cell entry assays were normalized across pseudoviral particles by S incorporation as determined in the anti-FLAG Western blot. b , Entry into R. affinis ACE2-expressing 293T cells by mock VSV particles produced in cells in which no spike gene was transfected. c , Entry of pseudoviral particles into 293T cells not transduced to express ACE2.

Article Snippet: HEK293T (293T) cells (ATCC CRL-11268) and 293T cells with stable transfection of human ACE2 were cultured in 10% FBS, 1% PenStrep DMEM at 37°C in a humidified 8% CO2 incubator.

Techniques: Western Blot, Expressing, Produced, Transfection

Journal: bioRxiv

Article Title: ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

doi: 10.1101/2021.07.17.452804

Figure Lengend Snippet: a , Structural context of positions targeted for mutagenesis. RBD as green cartoon, with blue spheres indicating the SARS-CoV-2 residues targeted via mutagenesis. The RBD-interacting region of ACE2 is shown in gray. b , Mutational scanning measurements. For each facet, red bars mark the binding affinity of the parental RBD for indicated ACE2. Each point is the affinity of one of the mutations at the six targeted positions. See for mutation-level measurements. c , Pie charts indicating the fraction of the 14 RBD backgrounds for which the parental RBD binds the indicated ACE2 ortholog with -log 10 ( K D,app ) > 7, a single mutant binds but the wildtype does not, or no tested mutants bind. d , Binding of 1 µM human ACE2-Fc to the biotinylated parental SARS-CoV-2 RBD, BtKY72 RBD or mutant BtKY72 RBDs immobilized at the surface of streptavidin biosensors. Data representative of three assays using independent preparations of RBD (biological triplicate). e , Entry of BtKY72 spike-pseudotyped VSV (parental or mutant) in 293T cells stably expressing human ACE2. Each point represents the mean of technical triplicates in assays performed with independent preparation of pseudoviral particles (biological replicate). Geometric mean is indicated by horizontal line. Mock, VSV particles produced in cells in which no spike gene was transfected. Western blot of pseudotyped particles in , and entry into 293T cells lacking ACE2 in , Titration curves showing how mutating site 501 (SARS-CoV2-2 numbering) to tyrosine increases the affinity of the SARS-CoV-2 RBD for human ACE2, but decreases affinity in the SARS-CoV-1 Urbani RBD . g , Epistatic turnover in mutation effects. Each point represents, for a pair of RBDs, the mean absolute error (residual) in their correlated mutant affinities for human ACE2 binding versus their pairwise amino acid sequence identity. Correlations computed only for RBD pairs where the parental RBDs bind with -log 10 ( K D,app ) > 7. Blue line and shaded gray indicates LOESS mean and 95% CI trendline. Plots incorporating affinity measurements across all ACE2 ligands shown in .

Article Snippet: HEK293T (293T) cells (ATCC CRL-11268) and 293T cells with stable transfection of human ACE2 were cultured in 10% FBS, 1% PenStrep DMEM at 37°C in a humidified 8% CO2 incubator.

Techniques: Mutagenesis, Binding Assay, Stable Transfection, Expressing, Produced, Transfection, Western Blot, Titration, Sequencing

Journal: Cell systems

Article Title: Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells

doi: 10.1016/j.cels.2020.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: SKMEL28, Melanoma Cell Line , MGH Cancer Center, primary source ATCC , CLS Cat# 300337/p495_SK-MEL-28, RRID:CVCL_0526.

Techniques: Formalin-fixed Paraffin-Embedded, Recombinant, Gene Expression, RNA Sequencing, Quantitative Proteomics, Phospho-proteomics, Software, Mass Spectrometry, Targeted Proteomics, Over Expression, Knockdown, Stable Transfection, Expressing, CRISPR, High Throughput Screening Assay, Microscopy, Live Cell Imaging, Cytometry, Staining

Journal: Cell systems

Article Title: Joint analysis of chromatin accessibility and gene expression in the same single cells reveals cancer-specific regulatory programs.

doi: 10.1016/j.cels.2025.101266

Figure Lengend Snippet: Figure 1. Parallel-seq enables ultra-high throughput simultaneous profiling of accessible chromatin and gene expression in single cells (A) Parallel-seq combines combinatorial indexing and droplet overloading to measure chromatin accessibility and gene expression in the same single cells. (B) The number of UMI counts of scRNA (left) and unique fragment counts of scATAC (right) data, mapped to the human and mouse genomes. This experiment examined a mixture of K562 (human), HEK293T (human), GM12878 (human), and NIH/3T3 (murine) cells. (C) The aggregated single-cell chromatin accessibility and gene expression profiles from a Parallel-seq analysis accurately reproduced the bulk ATAC-seq (ENCFF048XLS) and nucleus RNA-seq (ENCFF631TDY) profiles in K562 cells from ENCODE, respectively. (D) Scatterplot showing the number of unique ATAC reads in peaks and RNA UMIs per cell for Parallel-seq, 103 Multiome, ISSAAC-seq, and SNARE-seq in K562. Every dot represents a single cell, with aggregated distribution shown along the x and y axes. We only compared the droplet version of ISSAAC-seq in our study. Owing to the inability to match the barcodes between scATAC and scRNA in the SHARE-seq dataset, we were unable to include the SHARE-seq dataset in the figure.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 7-AAD Viability Staining Solution BioLegend 420404 35 mm BD cell strainers BD Falcon 352235 10 mM dNTP NEB N0447L Maxima H Minus Reverse Transcriptase ThermoFisher EP0753 NEBuffer 3.1 NEB B7203S T4 DNA ligase Robustnique M0082 RNase Cocktail Enzyme Mix ThermoFisher AM2286 RNAse H Enzymatics Y9220L Chromium Next GEM Chip H 10x Genomics PN-1000162 Chromium Single Cell ATAC Reagent Kits 10x Genomics PN-1000176 SPRI beads Beckman Coulter B23318 Elution Buffer QIAGEN 19086 Qubit dsDNA HS Assay Kit ThermoFisher Q32851 Agilent High Sensitivity D1000 ScreenTape Assay Agilent 5067-5584 and 5067-5585 Deposited data Raw and analyzed data This paper GSA: HRA006440 and HRA001829; OMIX database: OMIX009208 Bulk RNA-seq and ATAC-seq data of K562 cell line Consortium et al.23 ENCODE: ENCFF631TDY and ENCFF048XLS scRNA-seq data of K562 cell line Zhao et al.24 GSE188639 scATAC-seq data of K562 cell line Granja et al.25 GSE162690 Joint scATAC-and-scRNA data of cell lines generated by 10x Multiome, and ISSAAC-seq Xu et al.15 E-MTAB-11264 Joint scATAC-and-scRNA data of cell lines generated by SNARE-seq Chen et al.12 GSE126074 Joint scATAC-and-scRNA data of cell lines generated by SHARE-seq Ma et al.14 GSE140203 Joint scATAC-and-scRNA data of cell lines generated by Paired-seq Zhu et al.13 GSE130399 Joint scATAC-and-scRNA data of cell lines generated by sci-CAR Cao et al.11 GSE117089 scRNA-seq data of T cells generated by 10x Genomics RNA kit Zheng et al.41 GSE156728 scATAC-seq data of T cells generated by 10x Genomics RNA kit Satpathy et al.3 GSE129785 WGS dataset of K562 cell line Consortium et al.23 GSE176762 H3K27ac HiChIP data of K562 cell line Mumbach et al.63 GSE101498 H3K27ac HiChIP data of lung cancer cells Liu et al.67 GSE166234 ChIP-seq data of UBP1 Partridge et al.75 GSE104247 Experimental models: Cell lines Human: HEK293T ATCC CRL-3216 Human: HeLa-S3 ATCC CCL-2.2 Human: K562 ATCC CCL-243 Mouse: NIH/3T3 ATCC CRL-1658 Oligonucleotides See Table S9 for a full list This paper N/A Software and algorithms Cutadapt v3.4 Martin91 https://cutadapt.readthedocs.io/en/stable/ STAR v.2.7.0 Dobin et al.92 https://github.com/alexdobin/STAR/releases samtools Li et al.93 http://samtools.sourceforge.net/ (Continued on next page) e2 Cell Systems 16, 101266, May 21, 2025

Techniques: High Throughput Screening Assay, Gene Expression, RNA Sequencing

Journal: Cell systems

Article Title: Joint analysis of chromatin accessibility and gene expression in the same single cells reveals cancer-specific regulatory programs.

doi: 10.1016/j.cels.2025.101266

Figure Lengend Snippet: Figure 3. Parallel-seq improves CNV and eccDNA studies by joint analysis of ATAC-seq and RNA-seq data in the same single cell (A) Comparison of CNVs detected with the examined Parallel-seq derived scRNA, scATAC, and joint scATAC-and-scRNA data (using the CNVs detected from a WGS dataset in the same K562 cell line as a reference). Color bars indicate CNV score ranges. Pearson correlation coefficients are indicated on the right.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 7-AAD Viability Staining Solution BioLegend 420404 35 mm BD cell strainers BD Falcon 352235 10 mM dNTP NEB N0447L Maxima H Minus Reverse Transcriptase ThermoFisher EP0753 NEBuffer 3.1 NEB B7203S T4 DNA ligase Robustnique M0082 RNase Cocktail Enzyme Mix ThermoFisher AM2286 RNAse H Enzymatics Y9220L Chromium Next GEM Chip H 10x Genomics PN-1000162 Chromium Single Cell ATAC Reagent Kits 10x Genomics PN-1000176 SPRI beads Beckman Coulter B23318 Elution Buffer QIAGEN 19086 Qubit dsDNA HS Assay Kit ThermoFisher Q32851 Agilent High Sensitivity D1000 ScreenTape Assay Agilent 5067-5584 and 5067-5585 Deposited data Raw and analyzed data This paper GSA: HRA006440 and HRA001829; OMIX database: OMIX009208 Bulk RNA-seq and ATAC-seq data of K562 cell line Consortium et al.23 ENCODE: ENCFF631TDY and ENCFF048XLS scRNA-seq data of K562 cell line Zhao et al.24 GSE188639 scATAC-seq data of K562 cell line Granja et al.25 GSE162690 Joint scATAC-and-scRNA data of cell lines generated by 10x Multiome, and ISSAAC-seq Xu et al.15 E-MTAB-11264 Joint scATAC-and-scRNA data of cell lines generated by SNARE-seq Chen et al.12 GSE126074 Joint scATAC-and-scRNA data of cell lines generated by SHARE-seq Ma et al.14 GSE140203 Joint scATAC-and-scRNA data of cell lines generated by Paired-seq Zhu et al.13 GSE130399 Joint scATAC-and-scRNA data of cell lines generated by sci-CAR Cao et al.11 GSE117089 scRNA-seq data of T cells generated by 10x Genomics RNA kit Zheng et al.41 GSE156728 scATAC-seq data of T cells generated by 10x Genomics RNA kit Satpathy et al.3 GSE129785 WGS dataset of K562 cell line Consortium et al.23 GSE176762 H3K27ac HiChIP data of K562 cell line Mumbach et al.63 GSE101498 H3K27ac HiChIP data of lung cancer cells Liu et al.67 GSE166234 ChIP-seq data of UBP1 Partridge et al.75 GSE104247 Experimental models: Cell lines Human: HEK293T ATCC CRL-3216 Human: HeLa-S3 ATCC CCL-2.2 Human: K562 ATCC CCL-243 Mouse: NIH/3T3 ATCC CRL-1658 Oligonucleotides See Table S9 for a full list This paper N/A Software and algorithms Cutadapt v3.4 Martin91 https://cutadapt.readthedocs.io/en/stable/ STAR v.2.7.0 Dobin et al.92 https://github.com/alexdobin/STAR/releases samtools Li et al.93 http://samtools.sourceforge.net/ (Continued on next page) e2 Cell Systems 16, 101266, May 21, 2025

Techniques: RNA Sequencing, Comparison, Derivative Assay

Journal: Cell systems

Article Title: Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells

doi: 10.1016/j.cels.2020.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: SKMEL28, Melanoma Cell Line , MGH Cancer Center, primary source ATCC , CLS Cat# 300337/p495_SK-MEL-28, RRID:CVCL_0526.

Techniques: Formalin-fixed Paraffin-Embedded, Recombinant, Gene Expression, RNA Sequencing, Quantitative Proteomics, Phospho-proteomics, Software, Mass Spectrometry, Targeted Proteomics, Over Expression, Knockdown, Stable Transfection, Expressing, CRISPR, High Throughput Screening Assay, Microscopy, Live Cell Imaging, Cytometry, Staining

Journal: Non-coding RNA Research

Article Title: Targeting NUCKS1 with a fragment of tRNAAsn(GUU) of Chinese yew for the treatment of colorectal cancer

doi: 10.1016/j.ncrna.2024.11.002

Figure Lengend Snippet: Fig. 2. tRF-T36 targets NUCKS1 and inhibits its expressions on both mRNA and protein levels through RNAi pathway. (A) Putative binding sites of tRF-T36 on the 3′ UTR region of NUCKS1 mRNA. (B) The relative luciferase activities were detected by transfecting pmiR-RB-Report h-NUCKS1-WT or pmiR-RB-Report h- NUCKS1-MUT and tRF-T36 mimic or its scramble mimic into HEK-293T cells. (C) Quantitative real-time PCR analysis of caspase-family genes associated with apoptosis in HCT-8 and LoVo cells treated with tRF-T36 mimic. (D) tRF-T36 mimic suppressed the mRNA levels of NUCKS1 in a dose-dependent manner; (E) Western blotting analysis of protein levels of NUCKS1 in HCT-8 cells treated by tRF-T36 mimic at 25 nM. Data are shown as the means ± SDs of three independent ex periments. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: HEK293T human embryonic kidney cell line, HCoEpiC human colon epithelial cell line, human CRC cell lines LoVo, HCT-8, and its taxolresistant strain HCT-8/T were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Journal: Non-coding RNA Research

Article Title: Targeting NUCKS1 with a fragment of tRNAAsn(GUU) of Chinese yew for the treatment of colorectal cancer

doi: 10.1016/j.ncrna.2024.11.002

Figure Lengend Snippet: Fig. 3. tRF-T36 mimic inhibits human CRC cell proliferation, colony formation and migration. (A) Comparison of the dose-dependent effects of tRF-T36 mimic and taxol against HCT-8, LoVo, and HCoEpiC cells. (B) Clonogenic assay of tRF-T11 mimic on HCT-8 and LoVo cells. (C) Wound healing assay of tRF-T36 mimic on HCT-8 and LoVo cells. (D) Determination of caspase-3 activity in HCT-8 and LoVo cells treated with tRF-T36 mimic. Data are presented as the means ± SDs of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: HEK293T human embryonic kidney cell line, HCoEpiC human colon epithelial cell line, human CRC cell lines LoVo, HCT-8, and its taxolresistant strain HCT-8/T were obtained from American Type Culture Collection (ATCC).

Techniques: Migration, Comparison, Clonogenic Assay, Wound Healing Assay, Activity Assay

Journal: Non-coding RNA Research

Article Title: Targeting NUCKS1 with a fragment of tRNAAsn(GUU) of Chinese yew for the treatment of colorectal cancer

doi: 10.1016/j.ncrna.2024.11.002

Figure Lengend Snippet: Fig. 4. High-throughput RNA sequencing. (A) Volcano plot of differentially expressed genes in HCT-8 and LoVo cells treated with tRF-T36 mimic or empty li posomes. (B) Heatmap of NUCKS1 gene expressions in CRC cells treated with tRF-T36 mimic or empty liposomes, n = 6 for each group. (C) Dot plot of the KEGG pathway enrichment analysis of HCT-8 and LoVo cells treated with tRF-T36 mimic. The horizontal axis represents the gene ratio, while the vertical axis represents the enriched pathway name. The color scale indicates different thresholds of the p-value, and the size of the dot indicates the number of genes corresponding to each pathway. (D) GSEA analysis showed that the enriched PI3K-Akt was suppressed in tRF-T36 mimic-treated CRC cells. (E) Heatmap of key genes expressions in PI3K- Akt pathway between CRC cells treated with tRF-T36 mimic (right) or empty liposomes (left).

Article Snippet: HEK293T human embryonic kidney cell line, HCoEpiC human colon epithelial cell line, human CRC cell lines LoVo, HCT-8, and its taxolresistant strain HCT-8/T were obtained from American Type Culture Collection (ATCC).

Techniques: High Throughput Screening Assay, RNA Sequencing, Liposomes

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: Knockdown of GGCT inhibited the proliferation and caused significant changes in gene expression profiles detected by transcriptomic highthroughput sequencing in MCF-7 cells. (A) mRNA expression of GGCT was analyzed by qRT-PCR 3 days after transfection. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. (B) Western blotting analysis of GGCT and α-tubulin 4 days after transfection of MCF-7 cells with GGCT-siRNA or non-target control siRNA. (C) The relative survival number of trypan blue-negative viable MCF-7 cells at 1, 4, 7 days post-transfection. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using one-way ANOVA followed by Tukey’s post hoc test. (D) Representative images at 4 days post-transfection. (E) Hierarchical clustering analysis of differentially expressed genes (DEGs) detected by RNA-seq analysis. The color scale indicates log10(FPKM) and intensity increases from green to red, indicating down-regulation and up-regulation, respectively. (F) Numbers of significantly up-regulated (red) and down-regulated (green) DEGs in siGGCT-transfected MCF-7 cells at days 1, 2, and 3 post-transfection, identified by RNA-seq analysis using the filtering criteria of |log₂ fold change| >1 and q-value <0.01. Scale bar: 50 μm. ANOVA, Analysis of variance; FC, fold change; FPKM, fragments per kilobase of exon model per million mapped reads; GGCT, γ-glutamylcyclotransferase; qRT-PCR, quantitative reversetranscription- polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Knockdown, Gene Expression, High Throughput Screening Assay, Sequencing, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Control, RNA Sequencing, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of DEGs in MCF-7 cells after GGCT knockdown. (A) GO enrichment analysis for biological processes (up-regulated and down-regulated genes), and KEGG pathway analysis of DEGs, using samples collected 3 days after knockdown. The top 10 biological process terms ranked according to p-Values are shown. (B) Hypothetical schema of cell cycle regulation following GGCT knockdown in MCF-7 cells, constructed based on DEGs enriched in the KEGG pathway ‘hsa04110: Cell cycle’ (http://www.kegg.jp). (C) Relative mRNA expression patterns of GGCT, TGF-β2, CDKN1A (p21 Cip1 ), and CDKN2B (p15 INK4b ) were measured with qRT-PCR. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. CDKN1A, Cyclin dependent kinase inhibitor 1A; CDKN2B, cyclin dependent kinase inhibitor 2B; DEGs, differentially expressed genes; GGCT, γ-glutamylcyclotransferase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; TGF-β2, transforming growth factor-β2.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Knockdown, Construct, Expressing, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: Up-regulation of p15 INK4b and p21 Cip1 contributes to G0/G1 cell cycle arrest and subsequent cell growth inhibition in GGCT-depleted MCF-7 cells. (A) The levels of p15I NK4b and p21 Cip1 proteins were normalized to levels of α-tubulin and the fold change relative to the control was calculated at 4 days after the indicated siRNA transfection. (B) The cell cycle distribution and (C) representative histograms in FACS analysis in MCF-7 cells 4 days after transfection with the indicated siRNAs. n=3 per group; *p<0.05; **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. (D) The number of viable cells in the trypan blue dye exclusion test and (E) representative images of MCF-7 cells at 7 days after transfection with the indicated siRNAs. Scale bar: 50 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; FACS, fluorescence-activated cell sorting; GGCT, γ-glutamylcyclotransferase; PCR, polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Inhibition, Control, Transfection, Fluorescence, FACS, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: TGF-β2/SMAD signaling axis regulates p15 INK4b and p21 Cip1 expression in GGCT-depleted MCF-7 cells. (A) mRNA expression levels of GGCT and TGF-β2 were analyzed by qRT-PCR 3 days after transfection with GGCT- and/or TGF-β2-siRNA. (B) Western blot analysis of GGCT and TGF-β2 expression 4 days after transfection with GGCT- and/or TGF-β2-siRNA. (C) Western blotting analysis of p15 INK4b , p21 Cip1 , phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3, GGCT, and α-tubulin in MCF-7 at 4 days after transfection with GGCT-siRNA and/or TGF-β2-siRNA, or non-target control siRNA. (D) Western blotting analysis of p15 INK4b , p21 Cip1 , pSMAD3, SMAD3, GGCT, and α-tubulin in MCF-7 cells at 4 days after transfection with GGCT-siRNA and/ or SMAD3-siRNA, or non-target control siRNA. (E) The number of viable cells in the trypan blue dye exclusion test and (F) representative images of MCF- 7 cells at 4 days after transfection with the indicated siRNAs. Scale bar: 200 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; pSMAD, phosphorylated SMAD; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: p15 INK4b , p21 Cip1 , and their upstream regulator TGF-β2 are involved in the induction of cellular senescence following GGCT-KD in MCF-7 cells. (A) Representative images of SA-β-Gal staining 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT, p15, and p21. Scale bar: 50 μm. (B) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. (C) Representative images of SA-β-Gal staining at 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT and TGF-β2. Scale bar: 50 μm. (D) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. n=3 per group; *p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; KD, knockdown; SA-β-Gal, senescence-associated β-galactosidase; siRNA, smallinterfering RNA; TGF-β2, transforming growth factor-β2.

Article Snippet: The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin.

Techniques: Staining, Transfection, Knockdown

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Identification and characterization of eccDNA in rat skin induced by ionizing radiation. A) Flowchart for eccDNA purification and sequencing ( n = 4 for each group). B) Quantification of unique eccDNA. C) Distribution of unique eccDNA lengths. D) Overlap of eccDNA across sample groups. E) Genomic origins of eccDNA. F) Identification of three eccDNAs using PCR and Sanger sequencing, with circle 17:44148731‐48208624 (4059.8 Kb) present in all samples (CE: crude eccDNA, EE: exonuclease‐treated eccDNA). G) Flowchart of semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. H) PCR detected five genes on circle 17:44148731‐48208624 . I) Flowchart for eccDNA pre‐treatment of rats with radiation‐induced skin injury ( n = 3 for each group). J) Vps41 protein expression in rat skin 3 days post eccDNA transfection. K) Skin damage photos at 8, 40, and 65 days post‐irradiation in eccDNA‐pre‐treated rats (4 µg injection; scale bar: 1 cm). L) Radiation damage scores and affected areas in eccDNA pre‐treated rats. M,N) Immunofluorescence and analysis of inflammatory factors (IL‐6, IL‐10, TNF‐α) in irradiated skin of eccDNA‐pre‐treated rats (scale bar: 100 µm). O) Inflammatory cytokine array detection in eccDNA‐treated WS1 cells ( n = 5 per group). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons, and limma's empirical Bayes moderated t‐statistics for high‐throughput expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Purification, Sequencing, Nucleic Acid Electrophoresis, Expressing, Transfection, Irradiation, Injection, Immunofluorescence, MANN-WHITNEY, High Throughput Screening Assay

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: eccDNA drives increased VPS41 expression. A) Diagram of Circle 17:44148731‐48208624 in rat skin. B) Validation of the PS enzyme for linear DNA removal. C) Standard curve for semiquantitative analysis of PCR by gel electrophoresis on the eccDNA gene. D) PCR analysis of RALA and VPS41 amplification in HaCaT cells post‐irradiation. E) Gel electrophoresis shows VPS41 copy number on gDNA remains unchanged after irradiation. F) Elevated mRNA and protein expression of VPS41 in HaCaT cells after irradiation. G) Vps41 protein expression in various tissues after 4 Gy (X‐ray) total body irradiation in rats. H) Vps41 expression (Log 2 Fold Change) from scRNA‐Seq data of irradiated rat skin ( n = 4 per group). I) Relative expression levels of Vps41 (Log 2 Fold Change) in different cell types in scRNA‐Seq data of irradiated rat skin. J) Immunohistochemical analysis of VPS41 expression in a patient with clinical radiation‐induced skin injury (scale bar: 100 µm). K) Significant upregulation of VPS41 mRNA levels after transfection of HaCaT cells‐derived eccDNA into WS1 cells. L) Increased VPS41 expression after HaCaT cells‐derived eccDNA transfection into HEK‐293T cells. M) PCR analysis suggests intact gene expression elements for VPS41 on eccDNA. N) Nuclear‐cytoplasmic separation and semiquantitative analysis of PCR by gel electrophoresis reveal VPS41 gene localization on eccDNA. O) Apoptosis rate of WS1 cells transfected with total eccDNA after irradiation. P) Apoptosis rate of WS1 cells transfected with purified eccDNA after irradiation. Q) UVB and paclitaxel treatment effects on VPS41 expression in skin cells (HaCaT and WS1). R) Semiquantitative analysis of PCR by gel electrophoresis of DNA damage inducers and inhibitors on eccDNA VPS41 amplification in HaCaT cells. Treatments include IR (6 Gy), UVB (20 mJ/cm 2 ), etoposide (2 µ m ), paclitaxel (20 n m ), and cisplatin (2 µ m ). P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Biomarker Discovery, Nucleic Acid Electrophoresis, Amplification, Irradiation, Immunohistochemical staining, Transfection, Derivative Assay, Gene Expression, Purification, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 upregulation confers radioprotective effects at the cellular level. A) VPS41 protein localization after EGFP‐VPS41 plasmid transfection in HaCaT and WS1 cells (Hoechst: blue, EGFP: green, LysoTracker: red; scale bar: 20 µm). B) Western blot showing VPS41 plasmid overexpression efficiency in HaCaT and WS1 cells. C) ROS levels in HaCaT cells post‐irradiation after VPS41 plasmid transfection. D,E) Effect of VPS41 plasmid transfection on γH2AX levels in HaCaT cells after irradiation (scale bar: 20 µm). F) Cell viability in HaCaT and WS1 cells post‐irradiation with VPS41 plasmid transfection. G) LDH release in HaCaT and WS1 cells following irradiation and VPS41 plasmid transfection. H) Colony formation rate in HaCaT cells post‐irradiation with VPS41 plasmid. I) Reduced apoptosis in HaCaT and WS1 cells post‐irradiation after VPS41 plasmid transfection. J) Western blot showing shVPS41 knockdown efficiency in HaCaT and WS1 cells. K) Decreased irradiated cell viability after shVPS41 infection in HaCaT and WS1 cells. L) Increased LDH release in irradiated HaCaT and WS1 cells post‐shVPS41 infection. M) Elevated apoptosis rate in irradiated HaCaT and WS1 cells after shVPS41 infection. N) Reduced colony formation in irradiated HaCaT cells after shVPS41 infection. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Plasmid Preparation, Transfection, Western Blot, Over Expression, Irradiation, Knockdown, Infection, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: Therapeutic effects of AAV‐Vps41 on radiation‐induced skin injuries in rats. A) Flowchart for skin radiation injury in rats pre‐treated with AAV‐Vps41 for 16 days ( n = 4 per group). B) Increased Vps41 protein expression in rat skin one month post AAV‐Vps41 infection. C) Photographs of skin radiation injury in AAV‐Vps41 pre‐treated rats on days 12, 44, and 72 post‐irradiation (scale bar: 1 cm). D) Radiation injury score statistics in AAV‐Vps41 pre‐treated rats. E) Analysis of the area of skin radiation injury in AAV‐Vps41‐treated rats. F) HE staining showing tissue resistance to ionizing radiation in AAV‐Vps41 pre‐treated rats (45 Gy for 72 days), scale bar 250 µm. G,H) Immunofluorescence detection and analysis of IL‐6, IL‐10, TNF‐α in irradiated skin of AAV‐Vps41 pre‐treated rats (scale bar: 100 µm). I) Schematic diagram of inflammatory factor chip detection in AAV‐Vps41 pre‐treated WS1 cells. J) GO classification of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. K) Heat Map of inflammatory factor chip results in AAV‐Vps41 pre‐treated WS1 cells. P values were calculated using different statistical methods based on data type: Mann–Whitney U test for two‐group comparisons, Fisher's exact test for GO enrichment analysis, and limma's empirical Bayes moderated t‐statistics for protein expression data. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Infection, Irradiation, Staining, Immunofluorescence, MANN-WHITNEY

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: VPS41 negatively regulates KAI1 expression through the lysosomal pathway to confer resistance to apoptosis. A) Flowchart for screening VPS41 interaction proteins via differential protein analysis and mass spectrometry after VPS41 upregulation post‐irradiation. B) Volcano plot showing proteomic analysis (VPS41 vs Vector). C) Electron microscopy analysis reveals inhibited apoptosis progression in cells with upregulated VPS41 after irradiation (scale bar: 5 µm). D) Intersection of differential proteins identified four candidates: ISG15, KAI1, IFT20, and ATPAF1. E) Co‐localization of VPS41‐EGFP and KAI1‐BFP plasmids in WS1 and HaCaT cells assessed by confocal microscopy (scale bar: 20 µm). F) Immunoprecipitation confirms VPS41 binds KAI1. G) PNGase F treatment has minimal effect on VPS41‐KAI1 interaction. H) Western Blot shows upregulation of VPS41 decreases KAI1 expression, suppressing apoptosis, while VPS41 downregulation increases KAI1 expression and enhances apoptosis. I) VPS41 and KAI1 expression changes in HaCaT cells treated with CQ (20µ m ) or MG‐132 (20 µ m ) combined with X‐ray (10 Gy). J) Analysis of KAI1 decay rate after CHX (300 µ m ) treatment and X‐ray (10 Gy) in HaCaT cells. K) Effect of VPS41 knockdown and eccDNA transfection on apoptosis rates in irradiated cells with or without KAI1 overexpression. L) Apoptosis testing shows KAI1 reverses VPS41‐mediated radiation resistance. M) LDH measurement assesses the role of KAI1 in reversing VPS41‐mediated radiation resistance. P values were calculated using different statistical methods based on data type: unpaired two‐tailed t test for differential protein analysis and one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified. [Correction added on 28 April 2025, after first online publication: figure 5 is updated in this version].

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Expressing, Mass Spectrometry, Irradiation, Plasmid Preparation, Electron Microscopy, Confocal Microscopy, Immunoprecipitation, Western Blot, Knockdown, Transfection, Over Expression, Two Tailed Test

Journal: Advanced Science

Article Title: EccDNA‐Driven VPS41 Amplification Alleviates Genotoxic Stress via Lysosomal KAI1 Degradation

doi: 10.1002/advs.202501934

Figure Lengend Snippet: The interaction between VPS41 and KAI1 is critical for the radioprotection of VPS41. A) AlphaFold 3 prediction of structural domains for VPS41 and KAI1. B) IP experiments validate interaction domains between truncated VPS41 and KAI1 after transfection of various VPS41 truncation plasmids into HEK‐293T cells. C) IP experiments verify interaction domains between truncated KAI1 and VPS41 after transfection of KAI1 truncation plasmids into HEK‐293T cells. D) Co‐transfection of VPS41_WT‐EGFP and truncated variants with KAI1‐BFP in WS1 cells, followed by confocal microscopy to assess co‐localization (scale bar: 20 µm). E) The interaction between VPS41 and KAI1 remains unaffected by CQ treatment, which inhibits endosome and lysosome fusion. F) Apoptosis assays investigate the effects of truncated VPS41 on radiation‐induced apoptosis in HEK‐293T cells. G) Apoptosis assays assess the impact of different KAI1 truncation variants on radiation‐induced apoptosis in HEK‐293T cells. H) AlphaFold 3.0 predicts interaction sites of VPS41‐1‐286 and KAI1‐Δ111‐228. I) Schematic diagram of peptide array experiment. J) ECL imaging results of KAI‐Δ111‐228 peptide array. K) Peptide array and AlphaFold 3.0 analyze protein binding sites. L) Conservation of KAI1 binding peptide containing K263 among species. P values were calculated using one‐way ANOVA followed by Bonferroni's post hoc test for multi‐group comparisons. Statistically significant differences are denoted as follows: * p < 0.05, ** p < 0.01. Data are presented as mean ± SD ( n = 3) unless otherwise specified.

Article Snippet: The HaCaT (human keratinocyte) cell line was obtained from the German Cancer Research Center (Heidelberg, Germany) as previously reported ,[ ] and the WS1 (human skin fibroblast) cell line was purchased from ATCC.

Techniques: Transfection, Cotransfection, Confocal Microscopy, Peptide Microarray, Imaging, Protein Binding, Binding Assay

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 reduces the surface levels of peptide-HLA complexes in lung cancer cells. A Outline of the experimental strategy used to measure changes in the levels of selected HLA-peptide complexes in lung cancer cells with or without TAP1 and/or TAP2 downregulation using flow cytometry. B-G A549 lung cancer cells were transfected with scrambled/control siRNA or with TAP1 and/or TAP2 targeting siRNAs and left untreated (black histograms) or stimulated with IFNγ (blue histograms) or IFNγ + TNFα (red histograms). Panels B-D show the surface levels of HLA-A2-HER2 369-377 , and panels E–G show the levels of HLA-A2-MAGE3 271-279 . H–K A549 TAP2 knockout (KO) cells were transfected with an empty vector (KO + EV) or with a vector containing FLAG-TAP2 (KO + TAP2) and left untreated (black) or stimulated with cytokines IFNγ (blue) or IFNγ + TNFα (red). I, Graphs showing the levels of TAP2 protein analyzed by flow cytometry with or without TAP2 gene elimination, J-K, Surface levels of HLA-A2-HER2 369-377 or HLA-A2-MAGE3 271-279 in A549 cells with or without TAP2 gene elimination. For panels B-G and I-K, an isotype control antibody (IgG) was used as a background signal reference. Data presented as the mean ± s.d.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels B-G, Scr transfected cells were used as a control for statistical comparison, and for I-K parental wild type (WT) cells were compared with TAP2 deleted cells (KO) and TAP2 deleted plus EV (KO + EV) or with TAP2 deleted with posterior TAP2 transfection (KO + TAP2). MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; Tx, treatment; ns, not significant

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Transfection, Control, Knock-Out, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: TAP2 downregulation protects cancer cells from tumor antigen-specific CD8 T-cell killing. A , B Schema and outline of the experimental strategy used to measure tumor antigen-specific killing of lung cancer cells by cognate CD8 + T-cells using flow cytometry, LDH release and MTT assay. C-L A549 lung cancer cells were transfected with scrambled siRNA or with TAP1 and/or TAP2 targeting siRNAs; and left untreated (black) or stimulated with IFNγ (blue) or IFNγ + TNFα (red). After treatment, target lung tumor cells were co-cultured with effector (CD8 + T-cells) cells in the ratios of 1:0, 1:2 and 1:5, respectively. Panel C shows the flow cytometry gating strategy to assess cell apoptosis in cancer cell/T-cell co-cultures using the markers CD3 (for CD8 + T-cells), EpCAM (for tumor cells) and Annexin V. Panels D-F show the percentage of EpCAM + and Annexin V + apoptotic cancer cells. Panels G-I show the percent of LDH release, and panels J-L represent the cellular viability using MTT assay. M-P A549 TAP2 knockout (KO) cells transfected with empty vector (KO + EV) or FLAG-TAP2 (KO + TAP2) and left untreated or stimulated with IFNγ or IFNγ + TNFα were co-cultured with tumor antigen specific CD8 + T-cells at different target cell (tumor): effector (CD8 + T-cell) cell ratios. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; * , p < 0.05; ** , p < 0.01; *** , p < 0.001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. For panels D-L, Scr transfected cells were used as a control for statistical comparison, and for N-P wild type (WT) compared with KO and KO + EV or with KO + TAP2 cells. si, siRNA; scr, scrambled; E, effector CD8 T cells; T, target tumor cells; Tx, treatment; ns, not significant; WT, wild type. See also supplementary Fig. S5-S6

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, MTT Assay, Transfection, Cell Culture, Knock-Out, Plasmid Preparation, Control, Two Tailed Test, Comparison

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Downregulation of TAP2 alters intracellular immunomodulatory pathways via SOCS1 upregulation and TAP2 downregulation in human NSCLC is due to epigenetic changes. A A549 cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs followed by targeted transcriptomic analysis. Graph shows differentially expressed genes in TAP2 silenced versus control cells ranked based on low (blue) to high (red) transcript expression. B , C SOCS1 protein expression by flow cytometry in A549 cells. B, Cells were transfected with scrambled siRNA or with TAP2 targeting siRNAs C, Cells were transfected with empty vector or with the full-length TAP2 including a FLAG octapeptide. D-G, A549 cells were transfected with TAP1/2 siRNAs as indicated in 3A and stimulated with IFNγ ( D , E ) or with IFNγ plus TNFα ( F , G ). Volcano plots representing differential expression of IFNγ pathway signature genes. H A549 cells were transfected with TAP1/2 siRNAs followed by stimulation with IFNγ plus TNFα. Phosphoprotein levels were measured and ranked based on low (blue) to high (red) expression in TAP2 silenced vs control cells. I-M Normal adjacent to tumor (NAT) and lung tumor tissues surgically resected from NSCLC patients with low TAP2 protein levels. I Schematic of the strategy for obtaining and analyzing single cell preparations. J Volcano plot showing the differential gene expression of paired lung cancer relative to NAT using RNA sequencing. K Fold change of the mRNA expression of TAP2 and SOCS1 in tumor samples relative to NAT, L Heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region. M Predicted transcription factor (TF) binding sites with the highest affinity scores (log2 count) for the TAP2 promoter region. Data are presented as the mean ± s.d. ** , p < 0.01 determined by two-tailed unpaired Student’s t-test. FC, fold change; MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; TFs, transcription factors. See also supplementary Fig. S7

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Control, Expressing, Flow Cytometry, Plasmid Preparation, FLAG-tag, Quantitative Proteomics, Gene Expression, RNA Sequencing, Binding Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Myeloid cell-derived IL-4 reduces TAP2 expression via epigenetic remodeling in lung cancer. A TCGA NSCLC cohort analysis of TAP2 expression stratified by the median IL-4 mRNA levels. B A549 cells were treated with IL-4 for 0-24 h and TAP2 protein levels were measured using flow cytometry. C-I A549 cells were treated with IL-4, IL-4 + IFNγ or IL-4 with IFNγ + TNFα. C, TAP2 protein levels measured by flow cytometry, D, SOCS1 protein levels by flow cytometry, E, Surface levels of HLA-A2-HER2 369-377 complexes and F, HLA-A2-MAGE3 271-279 complexes measured by flow cytometry. G-I A549 cells (target) were treated with IL-4 ± IFNγ + TNFα and incubated with effector tumor antigen-specific CD8 + T-cells to measure, G, cancer cell killing by Annexin V positivity, H, LDH release and I, cellular viability using MTT assay. J-M Fluorescence images and signal measurement from multiplexed spatial analysis of protein and mRNA transcripts in NSCLCs from Cohorts #1 and #2. J-K, Representative captions of cytokeratin (CK, green), IL-4 mRNA (red), CD11b mRNA (white), TAP2 protein (yellow) and nuclei (blue). L, expression levels of IL-4 mRNA measured selectively in CK + tumor cells or in CD11b + myeloid cells. M, expression levels of TAP2 protein in CK + tumor cells stratified by the median IL-4 mRNA expressed in CD11b + myeloid cells. N-R A549 cells were treated with IL-4 ± IFNγ + TNFα and analyzed using ATAC-seq. N, ATAC-seq promoter peak enrichment values of the TAP2 gene. O, comparative analysis of the TAP2 ATAC-seq promoter peak enrichment after different cytokine treatments in A549 cells. P, heatmap of ATAC-seq analysis representing chromatin accessibility in the TAP2 promoter region after cytokine treatments. Q, promoter peak enrichment values of the SOCS1 gene. R, comparative analysis of the SOCS1 ATAC-seq promoter peak enrichment after cytokine treatments. Promoter regions were considered as the DNA sequences between the gene Transcription Start Site (TSS, + 3 kb) and Transcription End Site (TES, -3 kb). S-U Autologous single cell suspension cultures including cancer and immune cells from primary NSCLC tissues stimulated with IFNγ + TNFα followed by incubation with IL-4Rα (αIL-4R) or PD-1 blocking antibodies (αPD-1). S, levels of CD8 + /CD25 + T-cells measured by flow cytometry. T, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions. U, percentage of EpCAM + /Annexin V + apoptotic cancer cells in autologous cell suspensions with selective elimination of CD3 + T-cells (TECS). Isotype (IgG) was used as a background signal control. Data are presented as the mean ± s.d. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test with a Holm-Bonferroni correction for multiple comparisons. B-I and S-U, untreated cells were used as a control. E, effector CD8 + T-cells; FPKM, fragments per kilobase million; ns, not significant; MFI, mean fluorescent intensity; T, tumor; S, stroma. See also supplementary Figs. S8-S12

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Incubation, MTT Assay, Fluorescence, Suspension, Blocking Assay, Control, Two Tailed Test

Journal: Molecular Cancer

Article Title: IL-4 mediated TAP2 downregulation is a dominant and reversible mechanism of immune evasion and immunotherapy resistance in non-small cell lung cancer

doi: 10.1186/s12943-025-02276-z

Figure Lengend Snippet: Restoration of TAP2 protein expression induces tumor cell surface antigenicity in lung cancer cells. A Schematic showing the strategy for high throughput screening of pharmacologic agents using TAP2 and PD-L1 immunostaining in A549 cells. B-D Representative plots showing the upregulation of TAP2 protein (red) or PD-L1 protein (blue) or both (green) in A549 cells treated with individual compounds from the Pharmakon 1600 library (B), Enzo 640 FDA library (C) and Enzo epigenetic library (D). The scores for each compound were calculated as TAP2 protein level fold change and normalized to the vehicle control (DMSO) treated group. E Summary of TAP2 protein inducer compounds selected based on representation of all 3 libraries. F-G Dose–response curves to determine EC 50 values for TAP2 protein upregulation in A549 cells using selected compounds shown in E. H Experimental outline for SAHA/Vorinostat treatment and analysis of A549 cells. I Levels of TAP2 protein measured by flow cytometry. J surface HLA-A2-HER2 369-377 complexes and K surface HLA-A2-MAGE3 271-279 levels measured by flow cytometry. L Levels of apoptotic cancer cell death (Annexin V staining) and cell viability (LDH release and MTT assay) of parental A549 cells co-incubated with tumor antigen-specific effector CD8 + T-cells using different effector to target cell ratios (1:0, 1:2 and 1:5) with or without treatment with 3.5 µM SAHA. An isotype control antibody (IgG) was used as a background signal reference. Data are presented as the mean ± s.d.; *p < 0.05; **p < 0.01; ****, p < 0.0001 determined by two-tailed unpaired Student’s t-test. FC, fold change; ns, non-significant; No-Tx, no treatment; MFI, mean fluorescent intensity; TKI, tyrosine kinase inhibitor. See also supplementary Fig. S13

Article Snippet: The lung adenocarcinoma cell lines A549 (HLA-A2 + , KRAS mutant), H1975 (HLA-A2 − , EGFR mutant), and H520 (HLA-A2 − , KRAS/EGFR wild type) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, High Throughput Screening Assay, Immunostaining, Control, Flow Cytometry, Staining, MTT Assay, Incubation, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA NONHSAT143692.2 is involved in oxidative DNA damage repair in the lens by regulating the miR-4728-5p/OGG1 axis

doi: 10.3892/ijmm.2020.4707

Figure Lengend Snippet: Results of high-throughput lncRNA sequencing. (A) Scatter plot of sequencing results. (a) In the UVB group, there were 1,410 upregulated mRNAs, and 3,270 downregulated mRNAs; (b) 295 lncRNAs were upregulated, while 2,177 lncRNAs were downregulated. (c) In the H 2 O 2 group, 1,211 mRNAs were upregulated, while 3,756 mRNAs were downregulated; (d) in addition, 44 lncRNAs were upregulated, while 344 lncRNAs were downregulated. (B) Expression levels of NONHSAT143692.2 and NONHSAT173383.1, which exhibited the most significant differences in the 2 groups.

Article Snippet: High-throughput lncRNA sequencing was performed by Sinotech Genomics.

Techniques: High Throughput Screening Assay, Sequencing, Expressing