Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Family pedigree showing a sibling pair with Fanconi anemia (red circles) who are compound heterozygous for BRCA2 variants c.2330dupA (maternal inheritance) and c.8524C>T (paternal inheritance). Family history of breast cancer (purple, all diagnosed in 60s and 70s), skin cancer (grey), and colon cancer (green, diagnosed at 40 years old). (B) Schematic of BRCA2 domain structure and key interacting proteins (C) Alignment of exon 20 BRCA2 DBD peptide sequence demonstrating it is evolutionary conserved across many species. In green are the aa residues modified by the patient variants, p.W2830_K2833del (c.8488-1G>A) and p.R2842C (c.8524C>T). Purple arrows indicate AA residues that contact DNA . (D) Immunoblot showing BRCA2 levels in WT (RA2985) control, FA-D1 (RA2525), and patient RA3105 and RA3106 LCLs. (E) Quantification of chromosome breaks following DEB treatment of WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (F-G) Cell survival assays of patient derived lymphoblast cell lines (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after MMC and PARP inhibitor olaparib (PARPi) treatment. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H) Quantification of chromosome breaks following MMC treatment of BJ wild type fibroblasts, FA-A patient fibroblasts, and HSC62 fibroblasts. (I) Cell survival of HSC62 (c.8488-1G>A) fibroblasts compared to BJ WT fibroblast, FA-A patient fibroblast, complemented FA-A patient cells (RA3087) expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV). Cells were treated with increasing concentrations of MMC. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (J) Cell survival of MMC treated HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clones 1-3. (K-L) Cell survival of BJ WT fibroblasts, and CRISPR/Cas9 targeted BJ fibroblasts: BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones (BRCA2 8488-1G>A ), c.8524C>T BJ clones (BRCA2 8524C>T) , and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cells were treated with increasing concentrations of MMC or PARPi. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, Modification, Western Blot, Control, Derivative Assay, Expressing, Plasmid Preparation, CRISPR, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A-B) Chromatograms of Sanger sequencing of DNA derived from a sibling pair and parents confirming the presence of BRCA2 c.2330dupA and c.8524C>T variants identified by whole exome sequencing (WES). (C) BRCA2 structure of the DBD illustrating the location of p.W2830_K2833del and p.R2842C patient variants at the base of the Tower domain and OB2. Structure adapted from Yang et al ., 2005. (D) Immunoblot analysis for FANCI ubiqutination following treatment with 1μM MMC for 24h of. WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (E) Metaphase for RA2985 and RA3105 following DEB treatment. (F-G) Cell survival assays of patient derived lymphoblast cell line (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after (DEB) and camptothecin (CPT) treatment. Survival assays were performed in triplicate. Cells were treated with increasing concentrations of genotoxic agents and counted after 7-10 days in culture. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H-N) Cell survival of HSC62 fibroblasts (c.8488-1G>A) to indicated agent compared to BJ WT fibroblast, FA-A patient fibroblast (RA3087), FA-A complemented patient cells expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV), RAD50 patient fibroblast or FA-D2 (BRCA2/FANCD1) patient fibroblast (FA-D1). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of indicated agent. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (O) Quantification of chromosome breaks following DEB treatment of BJ wild type fibroblasts, FA-A (FANCA) patient fibroblasts (FA-A mut ), and HSC62 fibroblasts. (P) Chromatograms of PCR amplified gDNA of CRISPR/Cas9 targeted HSC62 fibroblasts. Gene editing reverted the c.8488-1G>A variant either to homozygous WT (HSC62 WT ) or heterozygous WT (HSC62 mut/WT ) at the endogenous locus in HSC62 patient cells. The silent variant that was incorporated to destroy the CRISPR PAM sequence is indicated. (Q)cDNA analysis of HSC62 clones with either homozygous or heterozygous correction of the c.8488-1G>A variant demonstrating rescue of the 12bp deletion of exon 20 that results from alternate splicing. (R) Immunoblot showing BRCA2 levels in CRISPR/CAS9 corrected patient cell line HSC62 WT , uncorrected HSC62 cells (HSC62 mut ), and RA2630 FA-R (RAD51/FANCR) patient fibroblasts. (S-T) Cell survival of HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clone. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, Derivative Assay, Western Blot, Expressing, Plasmid Preparation, Amplification, CRISPR, Variant Assay, Clone Assay

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating that homozygous c.8488-1G>A variant in BJ fibroblast clones result in the usage of an alternative splice site donor and a 12bp deletion at the start of BRCA2 exon 20 (as seen in patient derived HSC62 fibroblasts). (B) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating the c.8524C>T missense variant and silent mutation introduced by CRISPR/Cas9 targeting. (C) Immunoblot showing BRCA2 levels in bulk BJ WT fibroblasts, BRCA2 WT fibroblast clone, c.8488-1G>A BJ clones 1-3, c.8524C>T BJ clones 1-2, and BRCA2 Trun. clones 1-2. (D-E) Cell survival of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones, c.8524C>T BJ clones, and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of CPT or aphidicolin. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (F-G) Representative images of RAD51 foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 8h post IR and 24h post MMC. Detected by immunofluorescence with anti-RAD51antibody. Quantification in and . (H) Quantification of RAD51 foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BRCA2 truncation mutant, c.8531dupA ( BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (I) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 μM MMC, detected by immunofluorescence with anti-RAD51antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. Quantified in .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Sequencing, CRISPR, Variant Assay, Clone Assay, Derivative Assay, Mutagenesis, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Immunofluorescence images of RAD51 foci, 8h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (B) Quantification of RAD51 foci 1h, 8h, and 24h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate s.d. of two independent experiments (≥200 cells per experiment). (C) Quantification of RAD51 foci 8h after 12 Gy IR of BJ WT fibroblast, wild type HSC62 - (HSC62 WT ) clones 1-3, and HSC62 uncorrected patient cell line (HSC62 mut ). (D) Quantification of RAD51 foci 24h following 1h treatment with 3 µM MMC. Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1h, 8h and 24h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), BRCA2 8488-1G>A BJ clones 2-3, BRCA2 8524C>T BJ clones 1-2, and a BRCA2 homozygous truncation mutant, c.8531dupA (BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment) (F) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8h post 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (G) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild type HSC62 clones (HSC62 WT ), and HSC62 uncorrected patient cell line (HSC62 mut ). (H) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared to luciferase control (Luc). Error bars indicate s.d. of four independent experiments. (I) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24h post 1h treatment with 3 μM MMC. BRCA2 WT , BRCA2 8524C>T , and BRCA2 8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. (J-K) MMC cell survival of BJ BRCA2 WT , BRCA2 8488-1G>A , and BRCA2 8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Immunofluorescence, Derivative Assay, Clone Assay, Mutagenesis, CRISPR, Luciferase, Control, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Images of RPA foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 24h post 1h treatment with 3 μM MMC. Detected by immunofluorescence with anti-RPA32 antibody. Quantification in . (B)Immunoblot analysis of MRE11 and CTIP siRNA depletion for cells utilized in . (C) qRT-PCR of DNA2, EXO1, WRN, and BLM expression levels of cells in . Error bars are s.d. (D) Quantification of RPA foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BJ BRCA2 truncation mutant (BRCA2 Trun. ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Representative images of RPA foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 uM MMC, detected by immunofluorescence with anti-RPA32 antibody. Third row images are individual cells enlarged to better demonstrate RPA foci. Quantified in . (F) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 μM MMC in cells depleted of DNA2 or WRN by siRNA. Error bars indicate s.d of two independent experiments. (G-H) qRT-PCR of DNA2 and WRN expression levels of cells utilized in and . Error bars indicate s.d. (I) Immunoblot analysis of WT RAD51 overexpression in BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells used in and . (J) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in cells expressing WT RAD51 or EV control. Representative data of 3 independent experiments. (K) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of SLX4 or MUS81 by siRNA. Error bars indicate s.d of two independent experiments. (L) Immunoblot analysis of MUS81 depletion for cells utilized in . (M) qRT-PCR of SLX4 expression levels of cells utilized in . Error bars indicate s.d. (N-O) qRT-PCR of DNA2 and WRN expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d. (P-Q) qRT-PCR of DNA2 and BLM expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: CRISPR, Clone Assay, Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing, Mutagenesis, Over Expression, Control

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Quantification of RPA foci 8h, 24h and 48h following 1h treatment with 3 μM MMC of FA patient derived fibroblasts compared to BJ wild type fibroblasts. Patient cells lines from FA complementation group FA-R (RAD51/FANCR) (FA-R mut ), FA-A (FANCA) (FA-A mut ), FA-L (FANCL) (FA-L mut ), FA-D2 (FANCD2) (FA-D2 mut ), FA-I (FANCI) (FA-I mut ), FA-J (FANCJ) (FA-J mut ), and FA-P (SLX4/FANCP) (FA-P mut ). FA-A patient complemented cell lines were generated by transducing WT FANCA cDNA or EV. Error bars indicate s.d. of two independent experiments. (B) FA-A patient cells expressing WT FANCA (FA-A+A) or empty vector (FA-A+EV) were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 and WRN. Quantification of RPA foci 24h following 1h treatment with 3 μM MMC. Error bars indicate s.d. of two independent experiments. (C) FA-A+EV were transfected with siRNA Luc or siRNAs targeting DNA2 and BLM. Quantification of RPA foci 24h following 1h treatment with 3 μM MMC. Error bars indicate s.d. of two independent experiments.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Derivative Assay, Generated, Expressing, Plasmid Preparation, Transfection, Control, Luciferase

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Chromatograms of BRCA2 CRISPR/Cas9 generated HEK293T clones aligned to WT. A frameshift Exon 20 BRCA2 mutant (BRCA2 Trun. ) was generated by homozygous single base pair insertion as a result of CRISPR/Cas9 targeting. BRCA2 exon 20 variants, c.8524C>T and c.8488-1G>A, and Exon 27 p.S3291A (c.9871T>G) clones were generated by targeting the respective BRCA2 exon with CRISPR/Cas9 and a 100bp ssDNA template donor. Where applicable, silent mutations are indicated. Cell lines utilized in . (B) Immunoblot showing BRCA2 levels in WT HEK293T cells and BRCA2 mutant HEK293T clones: c.8531dupA ( BRCA2 Trun ), c.8524C>T (clones 1-2), c.8488-1G>A (clones 1-3), and p.S3291A (clones 1-3). Cells utilized in . (C) qRT-PCR of BRCA2 expression levels of cells utilized in . Error bars are s.d. (D) Immunoblot of RAD51 knockdown for HEK293T cells used in . (E) SCE assay in BJ WT fibroblast and HSC62 fibroblast following depletion of BLM. (F) Representative images of SCEs in BJ WT fibroblast and HSC62 fibroblast metaphases. (G) qRT-PCR of BLM expression levels in cells described in E. Error bars indicate s.d. (H) Sister chromatid exchange (SCE) assay in BJ BRCA2 WT and BRCA2 Trun fibroblasts following treatment with MMC (0.05 μg/ml or 0.1 μg/ml). (I) Immunoblot analysis of MRE11 depletion for cells utilized in ,F. (J) qRT-PCR of DNA2 expression levels of cells utilized in ,F. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: CRISPR, Generated, Clone Assay, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing, Knockdown

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Levels of mClover positive cells were normalized to WT HEK293T (siLuc). Error bars indicate s.d. of three independent experiments performed in triplicate. (B) Sister chromatid exchange (SCE) assay in BJ WT fibroblast and HSC62 patient derived fibroblast following treatment with MMC (0.1 μg/ml or 0.2 μg/ml). (C) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun. , BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection. Cells were labeled with DNA analogs, IdU for 20 minutes and then CldU for 20 minutes. Cells were then incubated in 6 mM HU with and without MRE11 inhibitor mirin (50 uM) for 4h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate s.d. (D) Isogenic BJ fibroblast BRCA2 mutants, BRCA2 Trun. , BRCA2 8524C>T , BRCA2 8488-1G>A , and BRCA2 S3291A , were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or MRE11. Cells were treated and labeled with DNA analogs as above. Error bars indicate s.d. (E) BJ fibroblast with BRCA2 variants, BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection when depleted of RADX by shRNA or transduced with shRNA control (shCONT.). Cells were treated and labeled with DNA analogs as above. Data of two replicated plotted. Error bars indicate s.d. (F) Quantification of chromosome breaks in isogenic BJ fibroblast BRCA2 mutants following 5h of 6 mM HU and released into colcemid. Breakage was not significantly increased in BRCA2 8524C>T and BRCA2 8488-1G>A compared to BRCA2 WT .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Derivative Assay, Labeling, Incubation, Immunofluorescence, Transfection, Control, Luciferase, shRNA, Transduction

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) BJ fibroblast mutants BRCA2 8524C>T and BRCA2 8488-1G>A , were analyzed for replication fork resection when depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with control shRNA (shLuc). Cells were labeled with DNA analogs, IdU for 20 minutes and then CldU for 20 minutes. Cells were then incubated in 6 mM HU for 4h before being harvested. DNA fibers were prepared and visualized by immunofluorescence detection of IdU and CldU and measured. Error bars indicate s.d. (B) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 µM MMC in cells depleted of SMARCAL1 or ZRANB3. Error bars indicate s.d of two independent experiments. (C) Quantification of RPA foci in BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of HLTF. Error bars indicate s.d of two independent experiments. (D) MMC cell survival of isogenic BJ BRCA2 WT fibroblasts depleted of SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (E-F) MMC and CPT cell survival assay of isogenic BJ BRCA2 8488-1G>A or BRCA2 8524C>T clones depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA luciferase control (shLuc). Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: shRNA, Transduction, Control, Labeling, Incubation, Immunofluorescence, Clone Assay, Luciferase, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Immunoblot analysis of RADX depletion by siRNA for cells utilized in . (B) Immunoblot analysis of RADX depletion by two shRNAs for cells utilized in . (C-D) Comparison of levels of mClover positive cells of HEK293T BRCA2 mutants transfected with siRNAs or shRNAs targeting RADX or control (Luc or CONT). (E) Quantification of chromosome breaks in isogenic BJ BRCA2 WT and BRCA2 Trun fibroblasts following 5h of 6 mM HU and released into colcemid. (F) Quantification of chromosome breaks in isogenic BJ BRCA2 WT and BRCA2 S3291A following 5h of 6 mM HU and released into colcemid. (G) Immunoblot analysis of RPA phosphorylation 24h post 1h treatment with 3 uM MMC. BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells were depleted of either SMARCAL1 or ZRANB3 by shRNA or transduced with shRNA control (Luc). (H-I) Immunoblot analysis of shRNA depletion of SMARCAL1 and ZRANB3 for cells utilized in and .

Article Snippet: Patient-derived fibroblast cell lines ( ) and BJ foreskin normal control fibroblasts (ATCC) were transformed by expression of HPV16 E6E7 and immortalized with the catalytic subunit of human telomerase (hTERT).

Techniques: Western Blot, Comparison, Transfection, Control, Phospho-proteomics, shRNA, Transduction

Journal: Pathology, research and practice

Article Title: Spatial transcriptome analysis of lung squamous cell carcinoma arising from interstitial pneumonia provides insights into tumor heterogeneity.

doi: 10.1016/j.prp.2024.155805

Figure Lengend Snippet: Fig. 1. Spatial transcriptome analysis of lung squamous cell carcinoma (LUSC) tissue. (A) Schematic workflow of spatial transcriptome analysis. (B) Results of spatial transcriptome analysis of an LUSC tissue specimen from a patient with interstitial pneumonia. Hematoxylin and eosin staining (left), gene expression profiles of 2104 barcoded spots (middle), and Uniform Manifold Approximation and Projection (UMAP) visualization (right) are presented. Whole tissues were classified via graph-based clustering. (C) Histological image of cancerous areas, colored orange (left), and an image representing the two clusters identified as representative of LUSC (right). (D) The top seven upregulated pathways in cluster 2 compared to cluster 5 (left), and the top nine pathways upregulated in cluster 5 compared to cluster 2 (right). Only pathways where the false discovery rate (FDR) was < 1.0e-5 are presented.

Article Snippet: Among them, tissue from one patient was subjected to spatial transcriptome analysis (Visium; 10x Genomics, Pleasanton, CA, USA).

Techniques: Staining, Gene Expression

Journal: Pathology, research and practice

Article Title: Spatial transcriptome analysis of lung squamous cell carcinoma arising from interstitial pneumonia provides insights into tumor heterogeneity.

doi: 10.1016/j.prp.2024.155805

Figure Lengend Snippet: Fig. 6. Analysis of tumor-associated macrophages with spatial transcriptome data. (A) Clusters of LUSC (clusters 2, 5) and clusters histologically including inflammatory cells in the tumor microenvironment (clusters 3, 4, 7) in the transcriptome data. (B) Bubble heatmap showing the expression levels of the macrophage marker genes in clusters 3, 4, and 7. (C) Bubble heatmap showing the expression levels of the M2 macrophage marker genes in clusters 3, 4, and 7. (D) Bubble heatmap showing the expression levels of the vascular endothelial cell marker genes in clusters 3, 4, and 7.

Article Snippet: Among them, tissue from one patient was subjected to spatial transcriptome analysis (Visium; 10x Genomics, Pleasanton, CA, USA).

Techniques: Expressing, Marker

Journal: Cell death & disease

Article Title: E3 ligase TRIM8 suppresses lung cancer metastasis by targeting MYOF degradation through K48-linked polyubiquitination.

doi: 10.1038/s41419-025-07421-6

Figure Lengend Snippet: Fig. 1 TRIM8 is induced by M1 macrophages. A Venn diagram of the overlapping genes of A549 cells treated with polarized macrophage- conditioned medium (CM). A549 cells were cultured with M1 or M2 macrophage CM for 48 hours. Those genes that are upregulated by M1 macrophages and downregulated by M2 macrophages are classified as tumour suppressors (left panel). Those genes that are downregulated by M1 macrophages and upregulated by M2 macrophages are classified as oncogenes (right panel). B Heatmap of differentially expressed genes in macrophage-treated A549 cells. The red arrow indicates the top 20 genes. C Evaluation of TRIM8 mRNA expression after culture in macrophage CM by RT‒qPCR. TATA-binding protein (TBP) was used as an internal control. D The expression of TRIM8 after M1 macrophage coculture and control or TRIM8 siRNA transfection, as detected by western blotting. The TRIM8 protein levels were quantified using ImageJ, with GAPDH serving as a control. E The viability of the cells in the silencing control (siCon), siCon with M1 macrophage coculture (siCon+M1), and TRIM8-silenced (siTRIM8) groups was evaluated via PrestoBlue™reagent assays at the indicated times. F The cell migration of the siCon control, siCon with M1 macrophage coculture, and siTRIM8 cells, as determined by Transwell assay. Scale bar = 50 μm. The data are shown as the means ± SDs of at least three independent experiments, and the significance levels are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001. The data were analysed with one-way ANOVA (C, E, F).

Article Snippet: Cell culture and patient specimens The human NSCLC cell lines A549 and H358 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and CL1-0 was provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan).

Techniques: Cell Culture, Expressing, Binding Assay, Control, Transfection, Western Blot, Migration, Transwell Assay

Journal: Cell death & disease

Article Title: E3 ligase TRIM8 suppresses lung cancer metastasis by targeting MYOF degradation through K48-linked polyubiquitination.

doi: 10.1038/s41419-025-07421-6

Figure Lengend Snippet: Fig. 2 Correlation between TRIM8 expression and clinical outcome in patients with NSCLC. A TRIM8 mRNA expression levels in 601 tumour tissues and 99 normal tissues from lung cancer patients in the GEO datasets (GSE19188, GSE30219 and GSE31210). The data were quantified by the log2 method. The data were analysed with t tests. B TRIM8 mRNA expression in a Taiwanese lung cancer cohort comprising 68 lung cancer tissues and 38 adjacent normal tissues, as determined by real-time qPCR (left panel). The right panel shows TRIM8 mRNA expression in 28 pairs of tumour tissue and normal tissue samples. The data were quantified by the log2 transformation of 2−Δct values and are presented as the means ± SDs; *P < 0.05, *P < 0.01, and ***P < 0.001. The data were analysed with t tests (left panel) or paired t tests (right panel). C Kaplan‒ Meier curves for overall survival in 68 Taiwanese lung adenocarcinoma patients. Survival curves were also estimated and visualized by using published cohorts in the Kaplan‒Meier Plotter database. D Overall survival curve and univariate Cox regression analysis of 1411 NSCLC patients in the published cohort. E Overall survival curve and univariate Cox regression analysis of 528 patients with stage I NSCLC in the published cohort. F Overall survival curve and univariate Cox regression analysis of 672 lung adenocarcinoma patients in the published cohort. G Overall survival curve and univariate Cox regression analysis of 346 patients with stage I lung adenocarcinoma in the published cohort. The log-rank test was used to assess statistical significance, with a significance level set at P < 0.05.

Article Snippet: Cell culture and patient specimens The human NSCLC cell lines A549 and H358 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and CL1-0 was provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan).

Techniques: Expressing, Transformation Assay

Journal: Cell death & disease

Article Title: E3 ligase TRIM8 suppresses lung cancer metastasis by targeting MYOF degradation through K48-linked polyubiquitination.

doi: 10.1038/s41419-025-07421-6

Figure Lengend Snippet: Fig. 5 Overexpression of TRIM8 suppressed tumour metastasis in a xenograft model. A In vivo experiment in which A549, CL1-0 or H358 lung cancer cells were subcutaneously injected into a xenograft model (upper panels). The number of mice in each group was as follows: n = 3 for A549 and H358 cells and n = 4 for CL1-0 cells. Tumour weights of the mock control group and TRIM8-overexpressing group (oeTRIM8) after 49 days (A549 cells) or 56 days (CL1-0 and H358 cells) are shown in the lower panels. Scale bar = 1 cm. B Representative images of livers harvested from the mock control and oeTRIM8 animal groups (upper panels). Quantitative analysis of the total number of tumour nodules that metastasized from subcutaneous tumours to all liver regions was performed. Arrowheads indicate metastatic nodules. Scale bar = 1 cm. C Histopathological findings of the liver via subcutaneous injection of CL1-0 lung cancer cells in mice. Scale bar (upper panel) = 5 mm; scale bar (lower panel) = 50 μm. D Histopathological findings of the liver after subcutaneous injection of H358 lung cancer cells in mice. Circles show the tumour area. Scale bar (upper panel) = 5 mm; scale bar (lower panel) = 50 μm. H&E staining, ×0.4 and ×20. E Quantification of tumour volumes in solid tumour-bearing mice from the shLacZ and shTRIM8 groups over a 6-week period. The number of mice in each group was 4. F In vivo experiments were performed by subcutaneously injecting CL1-0 lung cancer cells into a xenograft model (upper panel). Quantification of tumour volumes in solid tumour-bearing mice from the shLacZ and shTRIM8 groups over a 6-week period (lower panel). Scale bars = 1 cm. G Representative images of livers harvested from animals in the shLacZ control and shTRIM8 groups (upper panel). A quantitative analysis of the total number of tumour nodules that metastasized from subcutaneous tumours to all liver regions was performed. Arrowheads indicate metastatic nodules (lower panel). Scale bars = 1 cm. The data are presented as the means ± SDs. **P < 0.01 and ***P < 0.001. The data were analysed with t tests.

Article Snippet: Cell culture and patient specimens The human NSCLC cell lines A549 and H358 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and CL1-0 was provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan).

Techniques: Over Expression, In Vivo, Injection, Control, Staining

Journal: Cell death & disease

Article Title: E3 ligase TRIM8 suppresses lung cancer metastasis by targeting MYOF degradation through K48-linked polyubiquitination.

doi: 10.1038/s41419-025-07421-6

Figure Lengend Snippet: Fig. 6 Downstream target genes regulated by TRIM8. A Volcano plot showing 2918, 92 and 353 differentially expressed genes in A549, CL1- 0 and H358 cells (mock control vs. oeTRIM8#mix transfectant), respectively. B Venn diagram showing the overlapping differentially expressed genes in A549, CL1-0 and H358 cells. C A heatmap showing differential gene expression between mock and TRIM8 transfectants in three NSCLC cell lines. D MYOF mRNA expression in different transfectants was measured via RT‒qPCR. E Silencing of MYOF expression in H358 cells with a control siRNA or MYOF-specific siRNA. The protein level was detected by western blotting with the indicated antibody. The MYOF protein levels were quantified using ImageJ, with GAPDH serving as a control. F The viability of the silencing control (siCon) and MYOF- silenced H358 cells (siMYOF) was evaluated by PrestoBlue™reagent exclusion assays at the indicated times. G Transwell migration assays were performed to evaluate the migration of siCon- and siMYOF-transfected H358 cells. Scale bar = 50 μm. The data are shown as the means ± SDs of at least three independent experiments. TBP was used as an internal control. The data were analysed with t tests; *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Cell culture and patient specimens The human NSCLC cell lines A549 and H358 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and CL1-0 was provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan).

Techniques: Control, Transfection, Gene Expression, Expressing, Western Blot, Migration

Journal: Cell death & disease

Article Title: E3 ligase TRIM8 suppresses lung cancer metastasis by targeting MYOF degradation through K48-linked polyubiquitination.

doi: 10.1038/s41419-025-07421-6

Figure Lengend Snippet: Fig. 8 TRIM8 mediated MYOF degradation through the K48-ubiquitin‒proteasome pathway. A, B C1-0 and H358 mock and oeTRIM8 transfectants were transfected with the MYOF-HA plasmid and then treated with MG132 (10 μM) for 6 h before collection. The protein expression levels were quantified using ImageJ, with GAPDH serving as a control. The relative levels of MYOF protein were normalized to GAPDH expression (lower panel). C western blot analysis of the interactions between TRIM8 and MYOF in CL1-0 cells by coimmunoprecipitation with anti-V5 antibodies. The MYOF protein levels in the input groups were quantified using ImageJ, with GAPDH serving as a control. D Western blot analysis of the expression of endogenous MYOF and TRIM8-V5 (left panel). To measure the ubiquitination of endogenous MYOF, cell lysates were analysed via immunoprecipitation with ubiquitin antibodies followed by western blotting with anti- MYOF antibodies. The relative levels of ubiquitin protein were normalized to MYOF expression (right panel). E Cells were transfected with the MYOF-HA plasmid, wild-type (WT), or K48- or K63 or K0-ubiquitin plasmid and then analysed via immunoprecipitation with anti-MYOF antibodies followed by western blotting with antibodies against the HA tag to measure the polyubiquitination of MYOF. The cells were treated with MG132 (10 μM) for 6 h before collection. The ubiquitin-HA and MYOF protein levels in input were quantified using ImageJ, with GAPDH serving as a control (left panel). The relative levels of ubiquitin-HA protein in the IP group were quantified and normalized to MYOF expression (right panel). The above data were obtained from three independent experiments. F A schematic model of TRIM8-mediated inhibition of NSCLC metastasis. The dashed line represents an unclear mechanism.

Article Snippet: Cell culture and patient specimens The human NSCLC cell lines A549 and H358 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and CL1-0 was provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Inhibition

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-primed ADSCs (vADSCs) promoted the viability, anchorage independent growth, migration, invasion, epithelial-mesenchymal transition (EMT), and stemness property of breast cancer cells. ADSCs were treated with or without visfatin, noted as vADSCs or uADSCs, respectively, at 50 and 100 ng/mL for 48 h. Then, MDA-MB-231 cells were indirectly co-cultured with uADSCs or vADSCs in a transwell system for 72 h, noted as Ctrl or V50 and V100. ( A ) After that, the MDA-MB-231 collected from the co-culture were seeded in 96-well plate for 24, 48, and 72 h for analyzing the cell viability by XTT assay. ( B ) The collected MDA-MB-231 cells were seeded in a six-well plate with Noble agar for 21 days to analyze the anchorage independent growth by soft agar colony formation assay. ( C ) The collected MDA-MB-231 cells were seeded in a 24-well transwell plate coated with or without the Matrigel for performing migration or invasion assay, respectively. ( D ) The collected MDA-MB-231 cells were seeded in a 96-well low binding plate for tumorsphere formation. ( E ) The collected MDA-MB-231 cells were analyzed for the expressions of EMT- and stemness-related proteins by western blotting. MDA-MB-231 cells only were noted as Alone. Representative images of western blot shown. The result was quantified and present as histogram. All experiments were performed in triplicate. The statistical differences were calculated by t-test from three independent experiments. p -values < 0.05 or < 0.01 were marked with “*” or “**”, respectively.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Migration, Cell Culture, Co-Culture Assay, XTT Assay, Soft Agar Assay, Invasion Assay, Binding Assay, Western Blot

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-primed ADSCs promoted the tube formation of HUVEC. ( A ) HUVEC cells were co-cultured with visfatin-treated ADSCs or untreated ADSCs, noted as V50 and V100 or Ctrl, respectively, for three days. The HUVEC cells were collected from the co-culture and seeded in a matrix gel-coated 96-well plate. The tube formation of HUVEC was observed using a microscope. The length of branches was determined by using the ImageJ software. ( B ) After the three-day co-culture in the presence or absence of GDF15 neutralizing antibody (GDF15 Nab, 5 μg/mL), the HUVEC cells were collected from the co-culture for performing the tube formation assay. The experiments were performed in triplicate.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Cell Culture, Co-Culture Assay, Microscopy, Software, Tube Formation Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-pretreated ADSCs (vADSCs) enhanced the tumor growth and metastasis in human breast cancer xenograft mouse model. ( A ) The nude mice were injected with mixture of MDA-MB-231 and untreated ADSCs (uADSCs) or vADSCs, noted as Ctrl or V50, respectively, to the mammary fat pads. The tumor volumes were measured every week after injection (Ctrl, n = 5; V50, n = 6). ( B ) After sacrificing the mice, the weight of the resected tumor was measured. ( C ) The expressions of GDF15, β-catenin, CD31, and pAKT in the tumor sections were detected by immunohistochemistry. The IHC score was calculated by multiplying the percentage of positive cells by the intensity and present as histogram. ( D ) The luciferase-expressing MDA-MB-231 were collected and injected into the tail vein of NOD/SCID mice after co-culturing with uADSCs or vADSCs, noted as Ctrl or V50, respectively (Ctrl, n = 8; V50, n = 8). The IVIS radiance signals of the mice were assessed at week 4. The representative images of high and low signal were shown. The statistical differences were calculated by t-test, *, p -value < 0.05; **, p -value < 0.01.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Injection, Immunohistochemistry, Luciferase, Expressing

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: The expressions of visfatin, GDF15, and pAKT in the specimens from breast cancer patients. ( A ) The expressions of visfatin, GDF15, and pAKT in breast cancer tissue microarray (n = 96) were detected by immunohistochemistry. The representative images of high expression levels (No. 1) and low expression levels (No. 2) were shown. The IHC score was calculated by multiplying the percentage of positive cells by the intensity, which was identified using HistoQuest Analysis Software. ( B ) The correlations between visfatin, GDF15, and pAKT according to the IHC score were calculated by using the online Pearson correlation coefficient calculator. ( C ) The correlation of serum levels of GDF15 and visfatin of breast cancer patients (n = 120) determined by ELISA was also calculated by using the online Pearson correlation coefficient calculator.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Microarray, Immunohistochemistry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin mediates its effects both directly via cAbl/STAT3 and indirectly mediated by ADSCs via GDF15/AKT on promoting malignant behavior in breast cancer. Previously, we discovered visfatin mainly produced by adipocytes promoted breast cancer cells directly through activation of c-Abl and STAT3, which was blocked by Imatinib and Stattic inhibitor, respectively (black arrow). In this study, we showed that visfatin can act via an indirect pathway by priming ADSCs, which may be recruited from the adipose tissue to tumor site or generated from autologous fat transfer, to produce GDF15 that stimulated AKT activation in breast cancer cells to promote malignant behaviors (white arrow). The effect can be blocked by the treatment of GDF15 neutralizing Ab or Wortmannin inhibitor.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Produced, Activation Assay, Generated

Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) Ewing sarcoma cell lines A673 and CHLA-10 were labeled using an APC-conjugated ICAM-1 antibody and near-IR live/dead stain. Gates were generated to analyze live cell singlets with ICAM-1 staining. % denotes the frequency of ICAM-1+ cells upon analysis of a minimum of 10,000 total events. ( B ) A673 and CHLA10 cells were treated with control or EWS-FLI1 siRNA for 72 hours. Generated cDNA was analyzed for EWS-FLI1 and RPLP0 (control) expression using RT-PCR. Relative expression normalized to control siRNA is graphed. Cells treated with control or EWS-FLI1 siRNA were also either ( C ) lysed and subjected to western blot analysis for FLI1 and a vinculin loading control or ( D ) stained and analyzed by flow cytometry for ICAM-1 surface expression ( n = 3 per cell line). Error bars reflect SD. Circles on bar graphs in B and D indicate values for individual replicates. Control versus EWS-FLI1 siRNA treated cells were compared using an unpaired t -test. * p < 0.05, *** p < 0.001.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Labeling, Staining, Generated, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) A673 ( n = 4) and CHLA-10 ( n = 4) cells were treated with control or EWS-FLI1 siRNA for 48 hours, followed by treatment with 500 U/mL IFN-γ for a subsequent 24 hours. Ewing cells were analyzed by RT-PCR analysis for ICAM-1 and RPLP0 (control) expression. Graphs represent relative increase in ICAM1 mRNA expression (values normalized to untreated control). Bars represent SD. Differences in IFN-γ treatment response between ctsi and EWFsi groups for each cell line was compared using an unpaired t -test. *** p < 0.001. ( B ) Cells were treated with IFN-γ as in (A) followed by analysis of ICAM-1 surface expression by flow cytometry. MFI (median fluorescence intensity) values for both control (ct) and EWS-FLI1 (EWF) siRNA treatment conditions in both the A673 and CHLA10 cell lines are listed. ( C ) shA673-1c cells (harboring shRNA mediated tet-repressible EWS-FLI1 vector) were treated with DMSO vehicle control or 1 microgram/mL doxycycline for 48 hours ( n = 3). RT-PCR analysis of EWS-FLI1 and RPLP0 (control) expression was performed. *** p < 0.001. ( D ) An ICAM1 LightSwitch promoter reporter assay was used to examine differences in promoter activity between shA673-1c cells + DOX (doxycycline) or DMSO control cells in the absence or presence of IFN-γ. In this system, +Dox= EWS-FLI1 ‘low’ expression. Differences in promoter activity between conditions were determined using an unpaired t -test. * p < 0.05.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Fluorescence, shRNA, Plasmid Preparation, Reporter Assay, Activity Assay

Journal: Oncotarget

Article Title: EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis

doi: 10.18632/oncotarget.26939

Figure Lengend Snippet: ( A ) A673 and CHLA-10 Ewing sarcoma cells were grown alone or in co-culture with activated T-cells for 48 hours and monitored in real time using an IncuCyte system apoptosis assay. Representative images of cells at 36 hours upon real-time monitoring for apoptosis (activated caspase 3/7 activity, red) in tumor cells in culture alone (left panels) or co-cultured with activated T-cells (right panels). The black bar denotes a length of 400 micrometers. ( B–C ) Graphs represent fold increase in apoptosis in A673 and CHLA10 upon exposure to activated T-cells ( n = 3 per cell line). Differences in apoptosis ± T-cell exposure were determined using an unpaired t -test. * p < 0.05. ( D ) A673 Ewing sarcoma cells were left untreated or treated with IgG control or ICAM-1 blocking antibody, co-cultured with activated T-cells and then monitored for caspase 3/7 activity using an IncuCyte apoptosis assay. No T-cell controls were also included . Graphs represent the max % change in tumor cell apoptosis with IgG or ICAM-1 antibody treatment compared to no antibody controls ( n = 3). * p < 0.05. ( E ) A673 ( n = 4) and ( F ) CHLA-10 ( n = 3) Ewing sarcoma cells were treated with control (ctsi) or EWS-FLI1 siRNA and placed in the in the absence (black lines) or presence (red lines) of activated T-cells at a ratio of 1:10 (T-cell : tumor cell). Caspase activity was monitored in real time using an IncuCyte apoptosis assay. Error bars represent SD.

Article Snippet: Human patient-derived Ewing sarcoma cell lines were obtained from ATCC (A673, SK-N-MC), and the Children’s Oncology Group Childhood Cancer Repository (CHLA10). shA673-1c cells (with shRNA mediated tet-repressible EWS-FLI1 expression) are a kind gift from Dr. Olivier Delattre [ , ].

Techniques: Co-Culture Assay, Apoptosis Assay, Activity Assay, Cell Culture, Control, Blocking Assay

Journal: Frontiers in oncology

Article Title: Location, location, location: mapping the lymphoma tumor microenvironment using spatial transcriptomics.

doi: 10.3389/fonc.2023.1258245

Figure Lengend Snippet: FIGURE 2 An overview of Visium (10x Genomics) and GeoMx Digital Spatial Profiler (DSP) (NanoString) technologies for FFPE tissue. 1. For Visium, (a) sections from an FFPE tissue block are placed on a Visium Gene Expression slide and then b imaged (either H&E or IF) to provide histological context and for downstream analysis. c The Visium slide contains capture areas where capture probes will bind to RNA and incorporate spatial barcodes, before d preparation of libraries for e next generation sequencing. 2. In GeoMx DSP, a FFPE blocks are sectioned onto slides and then DSP barcoded probes will bind RNA (and/or protein). b The tissue section can then be imaged and reviewed to select a region of interest, before e) measuring count expression levels within the region of interest using next generation sequencing. Created with BioRender.com.

Article Snippet: Heming et al. went on to perform spatial transcriptomics on four patient samples using Visium Spatial Gene Expression for FFPE (10X Genomics) and integrated their scRNAseq data.

Techniques: Blocking Assay, Gene Expression, Next-Generation Sequencing, Expressing