Journal: Nature Communications

Article Title: MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

doi: 10.1038/ncomms14448

Figure Lengend Snippet: ( a ) Schematic diagram for isolation of extracellular vesicles (EVs) from colon cancer CT26 cell line with multimodal imaging report. CT26 cells stably transduced with a lentiviral vector expressing membrane-bound Gaussia luciferase (GlucB) and biotin ligase (BirA). ( b ) Venn diagram summarizing unique and shared exosomal miRNAs detected in the tissues of naïve colon, primary colon cancer and metastatic mouse colon cancer in the liver using miRNA microarray data ( n =5 mice per group). ( c ) Microarray data visualization by scatter plot comparing exosomal miRNAs detected in primary colon cancer ( x axis) and metastatic colon cancer in the liver at day 3 ( y axis) after a CT26 cell intrasplenic injection. ( d ) Heat map depicting changes in miRNAs with a statistically significant ( P <0.05) change in the exosomal miRNAs from normal mouse colon, primary colon cancer tissue and metastatic colon cancer in the liver at days 3, 7 and 14 after injection of CT26 cells ( n =3 mice per group). All tumour-derived exosomes were isolated with streptavidin magnetic beads. Microarray analysis results ( e ) and qPCR verification ( f ) of selected exosomal miRNAs from the source as described in d . ( g ) qPCR analysis of the plasma- (left panel) or faeces- (right panel) derived exosomes from the source are depicted in d . * P <0.05 versus naïve colon; # P <0.05 versus primary colon cancer (two-tailed t -test). Data are representative of three independent experiments (error bars, s.e.m.).

Article Snippet: The BALB/c syngeneic colon carcinoma CT26 cell line, human colonic epithelial SW620 cell line and human embryonic kidney 293 cells (American Type Culture Collection, Rockville, MD, USA) were grown at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM, Life technology) supplemented with 10% heat-inactivated EV-depleted fetal bovine serum (FBS), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin.

Techniques: Isolation, Imaging, Stable Transfection, Transduction, Plasmid Preparation, Expressing, Membrane, Luciferase, Microarray, Injection, Derivative Assay, Magnetic Beads, Clinical Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

doi: 10.1038/ncomms14448

Figure Lengend Snippet: ( a ) Schematic diagram of the putative binding sites of miR-193a in the wild-type (WT) Caprin1 3′ untranslated regions (UTR). The miR-193a seed matches in the Caprin1 3′UTR are mutated at the positions as indicated. CDS, coding sequence. ( b ) Potential miR-193a binding sites on Caprin1 (in grey) are broadly conserved among vertebrates. ( c ) Expression of miR-193a and candidate target gene Caprin1 as well as downstream genes (Ccnd2, c-myc) in CT26 cells assessed by qPCR following transfection of miR-193a mimic and control scramble miRNA. ( d ) Expression of candidate miR-193a and candidate target genes Caprin1 as well as downstream genes (Ccnd2, c-Myc, G3bp1) in CT26 cells assessed by western blot, following transfection of miR-193a mimic and control miRNA for 72 h. ( e ) Proliferation of CT26 cells with miR-193a and potential target Caprin1 knock down. Cell viability was detected from day 0 to 5 after transfection. ( f ) Luciferase activity assays of wild-type (WT) and mutated Caprin1 3′UTR luciferase reporters after co-transfection with miR-193a mimic, miRNA mimic control (scramble), anti-sense miR-193a or anti-sense negative control RNA in CT26 cells. The luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of transfected control mimic miRNA was set as a relative luciferase activity of 1. ( g ) Survival of BALB/c mice after intrasplenic injection of CT26 cells with miR-193a overexpression ( n =6 mice per group). ( h ) The cell cycle phase analysis of CT26 cells transfected with miR-193a mimic for 72 h using PI. The percentage of cells in the G1, S, and G2 phases are shown in the bar graph. ( i ) BrdU incorporation assay for cell cycle analysis of CT26 transfected with miR-193a mimic and control miRNA. Error bars represent s.e.m. * P <0.05 and ** P <0.01 (two-tailed t -test). Each data point was measured in triplicate (error bars, s.e.m.).

Article Snippet: The BALB/c syngeneic colon carcinoma CT26 cell line, human colonic epithelial SW620 cell line and human embryonic kidney 293 cells (American Type Culture Collection, Rockville, MD, USA) were grown at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM, Life technology) supplemented with 10% heat-inactivated EV-depleted fetal bovine serum (FBS), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin.

Techniques: Binding Assay, Sequencing, Expressing, Transfection, Control, Western Blot, Knockdown, Luciferase, Activity Assay, Cotransfection, Negative Control, Injection, Over Expression, BrdU Incorporation Assay, Cell Cycle Assay, Two Tailed Test

Journal: Nature Communications

Article Title: MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

doi: 10.1038/ncomms14448

Figure Lengend Snippet: ( a ) Biotin-miR-193a complex was pulled down from whole cell extracts using streptavidin beads and then analysed by electrophoresis followed by Coomassie blue staining (left panel). MALDI-TOF analysis of tryptic peptides (right panel) from the band indicated (left panel). ( b ) Western blot analysis expression of MVP proteins from before (top panel) and after streptavidin pulldown (bottom panel) of lysates of CT26 cells transfected with Bio-miR-193a or control miRNA. ( c ) MVP knockout (KO) CT26 cells were generated using the CRISPR/Cas9 system. qPCR-quantification of mature miR-193a, MVP, Caprin1, CyclinD and c-MYC expressed in CT26 cells (left panel) and CT26 exosomes (right panel) after the cells were treated as indicated. ( d ) Western blot analysis showing the level of MVP, Caprin1, CCND2 and c-MYC in cell lysates treated as indicated. ( e ) Proliferation of MVP KO CT26 cells treated as indicated. Cell viability was detected from day 0 to 5 after transfection. ( f ) Schematic representation (left panel) of treatment schedule as indicated. Representative livers (middle top panel) (metastatic nodules shown by arrows) and H&E-stained sections of livers (middle bottom panel, × 400 magnification, scale bar 200 μm) from tumour-bearing BALB/c mice ( n =5 per group). Liver weight (right, top panel) and number of metastatic foci in liver (right bottom panel) were quantitatively analysed. ( g ) Mature miR-193a in tumour tissue (left panel) and tumour exosomes (middle panel) was quantified by qPCR. Survival analysis of BALB/c mice after intrasplenic injection of CT26 cells treated as indicated (right panel) ( n =9 per group). ( h ) Representative images of xenografts in SW620 tumour-bearing nude mice (left panel) ( n =5 mice per group). Changes of tumour volumes in an SW620 xenograft model (right panel). Liver tumour volume was used to evaluate tumour size using the following formula: nodule volume=(width) 2 × length/2. ( i ) qPCR-quantification of mature miR-193a in exosomes and tissues of tumour in SW620 xenograft mice. * P <0.05 (two-tailed t -test); NS represents non-significance. Each data point was measured in triplicate (error bars, s.e.m.).

Article Snippet: The BALB/c syngeneic colon carcinoma CT26 cell line, human colonic epithelial SW620 cell line and human embryonic kidney 293 cells (American Type Culture Collection, Rockville, MD, USA) were grown at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM, Life technology) supplemented with 10% heat-inactivated EV-depleted fetal bovine serum (FBS), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin.

Techniques: Electrophoresis, Staining, Western Blot, Expressing, Transfection, Control, Knock-Out, Generated, CRISPR, Injection, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/elife.67750

Figure Lengend Snippet: Figure 1. Oxaliplatin-resistant (OxR) colorectal cancer (CRC) cell lines exhibit enhanced sensitization to TRAIL-mediated apoptosis via the intrinsic pathway and mitochondrial permeabilization. (A) Oxaliplatin-resistant SW620, SW480, HCT116, and HT29 colon cancer cell lines demonstrate similar or enhanced sensitivity to TRAIL compared to their parental counterparts after 24 hr of treatment. N = 3 (biological replicates); n = 9 (technical replicates). (B) IC50 values were calculated using a variable slope four-parameter nonlinear regression. (C) Representative Annexin-V/PI flow plots comparing SW620 parental and OxR cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. The four quadrants represent viable cells (bottom left), early apoptosis (bottom right), necrosis (top left), and late apoptosis (top right). (D) Representative flow plots of JC-1 assay after treatment with 1000 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. (E) Mitochondrial depolarization Figure 1 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) SW620 adenocarcinoma, colorectal, Dukes’ type C ATCC #CCL-227 RRID:CVCL_0547 L15 Media Cell line (Homo sapiens) SW480 adenocarcinoma, colorectal, Dukes’ type B ATCC #CCL-228 RRID:CVCL_0546 L15 Media Cell line (Homo sapiens) HT29 adenocarcinoma, colorectal ATCC #HTB-38 RRID:CVCL_0320 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 carcinoma, colorectal ATCC #CCL-247 RRID:CVCL_0291 McCoy’s 5A Media Cell line (Homo sapiens) SW620 OxR adenocarcinoma, colorectal, Dukes’ type C Kobe Pharmaceutical University #CCL-227 RRID:CVCL_4V77 L15 Media Cell line (Homo sapiens) SW480 OxR adenocarcinoma, colorectal, Dukes’ type B MD Anderson Cancer Center Characterized Cell Line Core #CCL-228 RRID:CVCL_AU18 L15 Media Cell line (Homo sapiens) HT29 OxR adenocarcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #HTB-38 RRID:CVCL_ 5949 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 OxR carcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #CCL-247 RRID:CVCL_4V73 McCoy’s 5A Media Chemical compound, drug Oxaliplatin MedChemExpress Cat# HY-17371 CAS No: 61825-94-3 Commercial assay or kit MTT Assay Kit Abcam Cat# ab211091 Peptide, recombinant protein Recombinant Human s TRAIL/Apo2L PeproTech Cat# 310-04 Antibody Mouse AntiTNFRSF10A Recombinant Antibody (clone mAY4) Creative Biolabs Cat# HPAB-1616-FY Mapatumumab (0.01–10 mg/ml) Commercial assay or kit FITC Annexin-V Apoptosis Detection Kit I BD Pharmingen Cat# 556547 Includes propidium iodide Software, algorithm FlowJo v10.7.1 FlowJo RRID:SCR_008520 Commercial assay or kit JC1 – Mitochondrial Membrane Potential Assay Kit Abcam Cat# ab113850 Commercial assay or kit RNeasy Plus Mini Kit Qiagen Cat# 74134 Commercial assay or kit RT2 First Strand Kit Qiagen Cat# 330404 Commercial assay or kit RT2 Profiler PCR Human Apoptosis Array Qiagen Cat# PAHS-012Z Continued on next page Greenlee et al. eLife 2021;10:e67750.

Techniques: Fluorescence

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/elife.67750

Figure Lengend Snippet: Figure 2. Microarray profiles show that parental and oxaliplatin-resistant (OxR) colorectal cancer (CRC) cell lines have similar expression of apoptotic transcripts while OxR derivatives have significantly downregulated CASP10. (A, B) Volcano plots of RT-PCR Apoptosis Profiler arrays demonstrate downregulation of CASP10 in OxR phenotypes. N = 3. (C) CRISPR/Cas9 knockout of caspase-10 in SW620 parental cells was confirmed via western blot. sgRNA/Cas9 ribonucleoprotein complexes reduced caspase-10 expression by 93% compared to cells treated with Cas9 alone. (D) CASP10 knock- out (KO) cells demonstrate slight decreases in viability when treated with TRAIL compared to Cas9 control. Data are presented as mean ± SD. N = 3 (n = 9). (E) Representative Annexin-V/PI flow plots comparing SW620 parental (Cas9 only) and CASP10 KO cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. (F) Depletion of caspase-10 did not have a significant effect on TRAIL sensitization (unpaired two-tailed t-test). Data are presented as mean + SEM. N = 3 (n = 9). The online version of this article includes the following source data and figure supplement(s) for figure 2:

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) SW620 adenocarcinoma, colorectal, Dukes’ type C ATCC #CCL-227 RRID:CVCL_0547 L15 Media Cell line (Homo sapiens) SW480 adenocarcinoma, colorectal, Dukes’ type B ATCC #CCL-228 RRID:CVCL_0546 L15 Media Cell line (Homo sapiens) HT29 adenocarcinoma, colorectal ATCC #HTB-38 RRID:CVCL_0320 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 carcinoma, colorectal ATCC #CCL-247 RRID:CVCL_0291 McCoy’s 5A Media Cell line (Homo sapiens) SW620 OxR adenocarcinoma, colorectal, Dukes’ type C Kobe Pharmaceutical University #CCL-227 RRID:CVCL_4V77 L15 Media Cell line (Homo sapiens) SW480 OxR adenocarcinoma, colorectal, Dukes’ type B MD Anderson Cancer Center Characterized Cell Line Core #CCL-228 RRID:CVCL_AU18 L15 Media Cell line (Homo sapiens) HT29 OxR adenocarcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #HTB-38 RRID:CVCL_ 5949 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 OxR carcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #CCL-247 RRID:CVCL_4V73 McCoy’s 5A Media Chemical compound, drug Oxaliplatin MedChemExpress Cat# HY-17371 CAS No: 61825-94-3 Commercial assay or kit MTT Assay Kit Abcam Cat# ab211091 Peptide, recombinant protein Recombinant Human s TRAIL/Apo2L PeproTech Cat# 310-04 Antibody Mouse AntiTNFRSF10A Recombinant Antibody (clone mAY4) Creative Biolabs Cat# HPAB-1616-FY Mapatumumab (0.01–10 mg/ml) Commercial assay or kit FITC Annexin-V Apoptosis Detection Kit I BD Pharmingen Cat# 556547 Includes propidium iodide Software, algorithm FlowJo v10.7.1 FlowJo RRID:SCR_008520 Commercial assay or kit JC1 – Mitochondrial Membrane Potential Assay Kit Abcam Cat# ab113850 Commercial assay or kit RNeasy Plus Mini Kit Qiagen Cat# 74134 Commercial assay or kit RT2 First Strand Kit Qiagen Cat# 330404 Commercial assay or kit RT2 Profiler PCR Human Apoptosis Array Qiagen Cat# PAHS-012Z Continued on next page Greenlee et al. eLife 2021;10:e67750.

Techniques: Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction, CRISPR, Knock-Out, Western Blot, Control, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/elife.67750

Figure Lengend Snippet: Figure 7. TRAIL-conjugated liposomes neutralize circulating tumor cells (CTCs) from the blood of patients with metastatic, oxaliplatin-resistant colorectal cancer. (A) Liposomes were synthesized using a thin-film hydration method, followed by extrusion and his-tag conjugation of TRAIL and E-selectin protein. Patient blood samples were treated in a cone-and-plate viscometer under circulatory shear conditions with either control liposomes, TRAIL liposomes, soluble TRAIL, or oxaliplatin. (B) Effects of TRAIL liposomes and control treatments on the number of viable CTCs, normalized to Figure 7 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Homo sapiens) SW620 adenocarcinoma, colorectal, Dukes’ type C ATCC #CCL-227 RRID:CVCL_0547 L15 Media Cell line (Homo sapiens) SW480 adenocarcinoma, colorectal, Dukes’ type B ATCC #CCL-228 RRID:CVCL_0546 L15 Media Cell line (Homo sapiens) HT29 adenocarcinoma, colorectal ATCC #HTB-38 RRID:CVCL_0320 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 carcinoma, colorectal ATCC #CCL-247 RRID:CVCL_0291 McCoy’s 5A Media Cell line (Homo sapiens) SW620 OxR adenocarcinoma, colorectal, Dukes’ type C Kobe Pharmaceutical University #CCL-227 RRID:CVCL_4V77 L15 Media Cell line (Homo sapiens) SW480 OxR adenocarcinoma, colorectal, Dukes’ type B MD Anderson Cancer Center Characterized Cell Line Core #CCL-228 RRID:CVCL_AU18 L15 Media Cell line (Homo sapiens) HT29 OxR adenocarcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #HTB-38 RRID:CVCL_ 5949 McCoy’s 5A Media Cell line (Homo sapiens) HCT116 OxR carcinoma, colorectal MD Anderson Cancer Center Characterized Cell Line Core #CCL-247 RRID:CVCL_4V73 McCoy’s 5A Media Chemical compound, drug Oxaliplatin MedChemExpress Cat# HY-17371 CAS No: 61825-94-3 Commercial assay or kit MTT Assay Kit Abcam Cat# ab211091 Peptide, recombinant protein Recombinant Human s TRAIL/Apo2L PeproTech Cat# 310-04 Antibody Mouse AntiTNFRSF10A Recombinant Antibody (clone mAY4) Creative Biolabs Cat# HPAB-1616-FY Mapatumumab (0.01–10 mg/ml) Commercial assay or kit FITC Annexin-V Apoptosis Detection Kit I BD Pharmingen Cat# 556547 Includes propidium iodide Software, algorithm FlowJo v10.7.1 FlowJo RRID:SCR_008520 Commercial assay or kit JC1 – Mitochondrial Membrane Potential Assay Kit Abcam Cat# ab113850 Commercial assay or kit RNeasy Plus Mini Kit Qiagen Cat# 74134 Commercial assay or kit RT2 First Strand Kit Qiagen Cat# 330404 Commercial assay or kit RT2 Profiler PCR Human Apoptosis Array Qiagen Cat# PAHS-012Z Continued on next page Greenlee et al. eLife 2021;10:e67750.

Techniques: Liposomes, Synthesized, Conjugation Assay, Shear, Control

Journal: The Journal of Cell Biology

Article Title: Mesenchymal–epithelial interactions in the skin

doi: 10.1083/jcb.200311122

Figure Lengend Snippet: Differential expression of leupaxin, DKK1, and DKK3 by palmoplantar (PP) and by nonpalmoplantar (NP) fibroblasts. Representative differences in gene expression patterns of leupaxin, DKK1, and DKK3 between palmoplantar fibroblasts and nonpalmoplantar fibroblasts as measured by microarray (top; quantitative results are summarized in and ). (middle) RT-PCR confirms the expression patterns of leupaxin, DKK1, and DKK3 in palmoplantar and in nonpalmoplantar fibroblasts. These data are representative of five independent experiments. (bottom) Real-time PCR to quantitate the expression of leupaxin, DKK1, and DKK3 after normalization of the target gene to GAPDH. Data are reported as means ± SD.

Article Snippet: Scanning of the two fluorescent intensities of the cDNA chip was performed by a standard two-color microarray scanner (model GenePix 4000A DNA; Axon Instruments, Inc.).

Techniques: Quantitative Proteomics, Gene Expression, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Analysis of The Cancer Genomic Atlas (TCGA) Colon Adenocarcinoma (COAD) database. Comparative analysis of TXNIP transcript expression between adjacent normal tissue and cancer tissues. (B-C) Kaplan-Meier analysis of overall survival (B) and distant metastasis-free survival (C) in CRC patients with different TXNIP mRNA expression levels. Wilcoxon rank-sum test p value indicated.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Expressing

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) DLD1 cells (A) or HCT15 cells (B) were treated with 10 µM oxaliplatin for 48h and surviving cells were analysed by RNA sequencing. A volcano plot (log2 FC versus negative log of P value) was used to visualize statistically significant gene expression changes (fold ≥1.5 and adjusted P value <0.05). TXNIP is labelled. The number of DE genes is indicated in the upper left. 3 biological replicates per group. (C-D) Western blotting analysis of TXNIP expression in DLD1 cells (C) or HCT15 cells (D) treated with oxaliplatin at different time points. β-ACTIN was used as an internal reference. (E-F) Western blotting analysis of TXNIP expression in DLD1 cells (E) or HCT15 cells (F) treated with oxaliplatin at different doses for 48h. (G-H) Immunoblot analysis of TXNIP in DLD1 cells (G) or HCT15 cells (H) treated with N-acetyl-L-cysteine (1.25mM) or oxaliplatin (10µm) or the combinational treatment for 48h. (I-J) Quantification of MLXIP (MondoA), TXNIP and ARRDC4 mRNA in DLD1 cells (I) or HCT15 cells (J) upon knockdown of MLXIP by siRNA after treatment with 10µm oxaliplatin treatment for 48h. (K-L) Immunoblot analysis of TXNIP expression in MondoA-knockout DLD1 cells (K) or HCT15 cells (L) after 10µm oxaliplatin treatment for 48h. (M) MondoA occupancy on the promoters of TXNIP in DLD1 cells treated with 10µm oxaliplatin or the combinational treatment with NAC (1.25mM) for 48h. Results shown, excluding A and B, are representative of three independent experiments. All values were expressed as mean ± SEM. Two-tailed Student’s t test; **p<0.01, ***p < 0.001, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: RNA Sequencing, Gene Expression, Western Blot, Expressing, Knockdown, Knock-Out, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) Assessment of TXNIP mRNA expression in DLD1 cells (A) or HCT15 cells (B) treated with oxaliplatin by q-RT-PCR analysis. Cells were treated with 10µM oxaliplatin and harvested at indicated time points. (C-D) q-RT-PCR analysis of TXNIP mRNA in DLD1 cells (C) or HCT15 cells (D) treated with oxaliplatin for 48h at indicated concentrations. (E-F) q-RT-PCR analysis of TXNIP mRNA in two different PDTOs treated with 10µm oxaliplatin for indicated time periods. (G-H) q-RT-PCR analysis of TXNIP mRNA in DLD1 (G) or HCT15 (H) spheroids treated with 10µm oxaliplatin for indicated time periods. (I-J) Western blotting analyses of TXNIP post oxaliplatin treatment (10µm) in two different PDTOs for 48h. (K-L) Western blotting of TXNIP in DLD1 (K) or HCT15 (L) spheroids treated with 10µm oxaliplatin for 48h. Results shown are representative of three independent experiments. All values were expressed as mean ± SEM. *p<0.1, **p<0.01, ***p < 0.001, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) DLD1 cells (A) and HCT15 cells (B) were treated with 10µm oxaliplatin with ROS measured at 48h. (C) qRT-PCR analysis of TXNIP mRNA in DLD1 cells (left panel) or HCT15 cells (right panel) treated with N-acetyl-L-cysteine (NAC) (1.25mM) or oxaliplatin (10µm), or combinational treatment, for 48h. (D) Overlapping DEGs (>4-fold change; Padj<0.05) from live DLD1 and HCT15 cells, after 48h of 10µm oxaliplatin treatment, as determined by RNA sequencing. (E) Heatmap showing 23 overlapping transcripts from D, in DLD1 cells (left panel) and HCT15 cells (right panel). (F-G) qRT-PCR analysis of ARRDC4 mRNA in DLD1 cells (F) and HCT15 cells (G) treated with with NAC (1.25mM) or oxaliplatin (10µm), or combinational treatment, for 48h. (H) Immunoblot analysis of MondoA expression in DLD1 cells after 10µm oxaliplatin treatment for 48h. (I) Effects of oxaliplatin treatment (10µm for 48h) on subcellular localization of MondoA assessed by cell fractionation and immunoblotting, in DLD1 cells. LAMIN A - a nuclear marker, GAPDH - a cytoplasmic marker. Results shown are representative of three independent experiments. All values were expressed as mean ± SEM. **p<0.01, ***p < 0.001, ****p < 0.0001, vs. Control

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Quantitative RT-PCR, RNA Sequencing, Western Blot, Expressing, Cell Fractionation, Marker, Control

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Pearson correlation coefficient scores and p values showing the relationship between TXNIP transcript expression and different immune marker transcript expression; including T cell markers ( CD247, CD4, CD8A ), antigen presentation markers ( B2M, CD80, CD86, BATF3 ) and cytokines ( IFNG, TNF, CXCL9, CXCL10, IL2, IL15, CCL2, CCL3, CCL4, CCL5, IL18, IL1B ) from the TCGA COAD dataset. (B-C) Effects of oxaliplatin (10µm for 48h) on subcellular localization of TXNIP assessed by cell fractionation and immunoblotting in DLD1 cells (B) and HCT15 cells (C). (D-E) Immunoblot analysis of cleaved caspase 1(p20) (D) and IL-1β (E) in control (NTC) and TXNIP-KO (TKO) DLD1 cells with/ without 10µm oxaliplatin treatment for 48h. Results shown are representative of three independent experiments.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Expressing, Marker, Immunopeptidomics, Cell Fractionation, Western Blot, Control

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Proteomic analysis of the conditional media from TXNIP-KO (TKO) and control (NTC) DLD1 cells as assessed by mass spectrometry. Heatmap illustrating differentially expressed proteins (left panel) and table showing the top six upregulated proteins in conditional media from TKO cells (right table). (B) 105 plex cytokine arrays incubated with conditional media from TKO and NTC cells with or without 10µM oxaliplatin treatment for 48h. The respective GDF15 spot is highlighted (red box). (C-D) Immunoblotting of TXNIP and GDF15 in NTC and TKO DLD1 cells (C) and NTC and TKO HCT15 cells (D) with or without drug treatment (10µm oxaliplatin for 48h). (E-F) Pooled densiometry data from 3 repeats of C and D. Standard error bars shown. (G) GDF15 concentration in conditional media for E were determined by ELISA. Standard error bars are shown. (H) Immunoblot of TXNIP and GDF15 in NTC (TKO-) and TKO (TKO+) PDTOs: CRC001 (left panel), CRC002 (right panel). (I) Immunofluorescent detection of GDF15 in NTC and TKO DLD1 cells with or without 10µm oxaliplatin treatment for 48h as assessed by confocal microscopy. DAPI (blue), Epcam (green), GDF15 (red). (J) Immunoblotting of MondoA, TXNIP and GDF15 in MondoA-knockdown (siMondaA) and control (NTC) DLD1 cells. Results shown are representative of three independent experiments. All values were expressed as mean ± SEM. Two-tailed Student’s t test; *p<0.05, **p<0.01, ***p < 0.001, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Control, Mass Spectrometry, Incubation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) Immunoblotting of GDF15 in DLD1 cells after treatment with 10µm oxaliplatin at indicated time points. (A); after treatment of different dosages of oxaliplatin for 48 hours (B). (C-D) Immunoblotting of TXNIP and GDF15 in control (NTC) and TXNIP-overexpressing (TXNIPa) DLD1 cells with or without 10µm oxaliplatin treatment for 48h (C); pooled densiometric data from C (D). Standard error bars are shown n=3. (E) Quantitation of immunofluorescence from (GDF15 levels relative to cell area) from 3 independent experiments. (F) Immunoblotting of TXNIP and GDF15 in TXNIPa or NTC cells treated with oxaliplatin (10µm) or combined treatment with oxaliplatin and NAC (1.25mM) for 48h. Results shown are representative of three independent experiments. All values were expressed as mean ± SEM. *p<0.1, **p<0.01, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Western Blot, Control, Quantitation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Analysis of The Cancer Genomic Atlas (TCGA) Colon Adenocarcinoma (COAD) database. Comparative analysis of expression of GDF15 between adjacent normal tissue and cancer tissues. Wilcoxon rank-sum test p value indicated. (B) GDF15 transcript expression in single epithelial cells derived from matched primary CRC tumors and adjacent normal colon (n=10 pairs). (C-D) Kaplan-Meier analysis of overall survival (C) and distant metastasis-free survival (D) in CRC patients with different GDF15 mRNA expression levels.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Detection of GDF15 in both tumor and adjacent normal tissue (ANT) samples from patients with primary colorectal cancer. Magnification ×200. (B) Statistical analysis of GDF15 IHC score between ANT and tumor tissue (n=42). (C-D) Correlations of TXNIP and GDF15 protein(cohort of 42 CRC patients) (C) and TXNIP and GDF15 transcripts (TCGA COAD) (D). Pearson correlation coefficients (R ) are indicated. (E) Sequential sections from colorectal tumor samples collected pre- and post-neo-adjuvant chemotherapy. Detection of TXNIP and GDF15 by IHC. (F) GDF15 expression in 11 paired treatment-naïve (Pre-T) tumor samples and oxaliplatin-based neo-adjuvant chemotherapy treated tumor samples (Post-T). (G) GDF15 mRNA levels in samples from F (aggressive cases highlighted in red). (H-I) Kaplan–Meier analysis of overall survival in CRC patients with different GDF15 staining scores from a cohort of 42 CRC patients (H) and CRC tumor tissue microarray (n=94) (I). Results shown are representative of three independent experiments. All values were expressed as mean ± SEM. *p<0.05, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Adjuvant, Expressing, Staining, Microarray, Control

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) Immunoblot of TXNIP and GDF15 expression in NTC, GDF15 knockout (GKO), TKO, GDF15 and TXNIP knockout (GTKO) DLD1 cell lines after 48h of oxaliplatin treatment (10µm). (B) Immunoblot of GDF15 expression in GDF15-CRISPRa (GDF15a) DLD1 cell line in the presence of 10µm oxaliplatin for 48h. Results shown are representative of three independent experiments.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Western Blot, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) PBMCs were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of fresh supernatant from indicated cell lines (NTC,GKO,TKO,GTKO; GDF15a). Live cells were counted using trypan blue and a haemocytometer. n=10 (A) and n=5 (B). (C-F) Labelled PBMCs were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of fresh supernatant from indicated cell lines, before being stained with anti-CD3 and anti-CD8 (C-D) or anti-CD4 (E-F) antibodies and measured by flow cytometry. Normalised proliferation on gated CD3 + CD8 + or CD3 + CD4 + cells is shown. n=6. (G-H) Normalised IFNγ concentrations in the supernatant of cells from C-F. (I-J) PBMCs were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of fresh supernatant from NTC or GDF15a cell lines (I) or media alone or 5ng/ml recombinant human TGFβ1 (J). Cells were stained with anti-CD3, anti-CD4 antibodies extracellularly before intranuclear staining of Foxp3 was performed. % of CD4 + Foxp3 + cells are shown. n=10 (I) and n=5 (J). (K) Immunohistochemistry using anti-GDF15 and anti-Foxp3 antibodies on serial sections from colorectal cancer cases. (L) Correlations of indicated immune transcripts (normalised for PTPRC[CD45] expression) and GDF15 transcripts from TCGA COAD dataset. Thick line indicates R value >0.1 and dashed line indicates transcription factor. (M) Pooled data from K showing Foxp3 + cell counts in GDF15 low and GDF15 high populations; median split. n=32. (N-O) Isolated naïve CD4 cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of indicated cell line supernatant and either isotype control (10µg/ml) or anti-CD48 (10µg/ml) as indicated. These cells were then co-cultured with anti-CD3 stimulated proliferation dye labelled responder PBMCs for 4 days, before cells were stained for CD3, CD8 and CD4. Normalised proliferation dye (MFI) of the indicated responder population is shown. n=9. All values were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. Control.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Staining, Flow Cytometry, Recombinant, Immunohistochemistry, Expressing, Isolation, Control, Cell Culture

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A-B) Immunoblot analysis of TXNIP and GDF15 expression after 48h of 10µm oxaliplatin treatment in colorectal cancer cell lines, including DLD1, HCT15, HT29, SW48 (A, derived from primary site), and DiFi, LIM1215 (B, derived from secondary site). (C) Ratio of GDF15/TXNIP for cell lines in A-B treated as indicated and measured using densiometry. (D-E) Microarray data showing the correlation between GDF15 and TXNIP mRNA expression in primary (D) or metastatic (E) CRC tumors. R and p values shown (Pearson’s). (F-G) Immunoblot analysis of TXNIP and GDF15 expression after 48h of 10µm oxaliplatin treatment in oxaliplatin-resistant (OXAR) cells: DLD1-OXAR (F) and HCT15-OXAR (G). (H) Ratio of GDF15/TXNIP for cell lines in F-G treated as indicated and measured using densiometry. (I-J) Microarray data showing the correlation between GDF15 and TXNIP mRNA expression in primary tumors that respond (responder; I) or do not respond (non-responder; J) to FOLFOX chemotherapy. R and p values shown (Pearson’s). (K) Ratio of GDF15/TXNIP for primary tumours in and treated as indicated as measured using densiometry. (L) Receiver operating characteristic (ROC) curve showing area under the curve and p values for the use of pre-treatment GDF15/TXNIP ratio in predicting responsiveness to oxaliplatin (O; responder [n=23] and non-responder [n=14]) using publicly available data. (M) Pooled pre-treatment data (ratio of GDF15/TXNIP) from C, H, K with ‘aggressive’ classed as secondary site, resistant to oxaliplatin and aggressive and ‘non-aggressive’ primary site, sensitive to oxaliplatin and non-aggressive (N) Post-treatment GDF15/TXNIP ratio divided by pre-treatment GDF15/TXNIP ratio for C, H, K. ‘Aggressive’ and ‘Non-aggressive’ defined as in M. (O) Post-treatment GDF15/TXNIP ratio divided by pre-treatment GDF15/TXNIP ratio for patient derived organoids grouped into primary tumours with and without extra-mural invasion. * p<0.05 using Sidak’s multiple comparisons test (C, H, K) ** p<0.01 *** p<0.001 using Mann Whitney (M, N) or unpaired t test (O). Western results shown are representative of three independent experiments.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Western Blot, Expressing, Derivative Assay, Microarray, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

doi: 10.1101/2023.08.10.552749

Figure Lengend Snippet: (A) A schematic model showing the process by which oxaliplatin-resistant CRC cells were generated. (B-C) IC50 values of oxaliplatin in oxaliplatin-resistant cells (OXAR) and their parental cells. DLD1 and DLD1-OXAR (B); HCT15 and HCT15-OXAR (C). (D) Bright field images of different organoids at different magnifications. (E-G) Receiver operating characteristic (ROC) curves showing area under the curve and p values for the use of GDF15/TXNIP ratio in predicting origin of cell line (E; primary [n=4] or secondary [n=2]), sensitivity to oxaliplatin (F; parental [n=3] or resistant [n=3]), aggression of tumour (G; non-aggressive [n=8] or non-aggressive [n=3]). (H-J) Post-treatment GDF15/TXNIP ratio divided by pre-treatment GDF15/TXNIP ratio for primary or secondary cell line source (H), parental or resistant cell line (I), or aggression of fresh primary tumour (J). ** p<0.01 using unpaired t test. H and J tested using Mann-Whitney.

Article Snippet: Human colon adenocarcinoma cell lines DLD1, DiFi, and SW48 were purchased from ATCC.

Techniques: Generated, MANN-WHITNEY

Journal: BMC Molecular and Cell Biology

Article Title: Role of tumor cell senescence in non-professional phagocytosis and cell-in-cell structure formation

doi: 10.1186/s12860-020-00326-6

Figure Lengend Snippet: a Immunofluorescence staining of the senescence markers HMGA2 and H3K9me3 in red without and after 6 days treatment with camptothecin (CPT) 120 nM and 200 nM for senescence induction in pancreas carcinoma cells BxPC-3. The cell nuclei were stained with DAPI (blue). Scale bar 10 μm. b Cell nucleus length and width of BxPC-3 cells after 6 days CPT treatment. c C 12 FDG β-galactosidase activity in untreated and 120 nM treated BxPC-3 cells on day 6. d Activity of the acidic β-galactosidase in pancreas carcinoma cells after CPT treatment on days 5, 6 and 7 using flow cytometry. The graphs represent the mean values from three independent experiments ± standard deviation, p -values: * < 0.05, ** < 0.01, *** < 0.001. e C 12 FDG β-galactosidase activity in 120 nM treated BxPC-3, SBLF-7 and SBLF-4 cells on day 6. f Flow cytometric determination of the amount of Annexin V−/7AAD- (living cells) compared to all others (dead cells) 7 days after 120 nM camptothecin treatment. p-values were determined from a two-tailed unpaired Mann Whitney U test: ** < 0.01, *** < 0.001

Article Snippet: Senescent cells were detected by anti-H3K9me3 antibodies and a fast-blue color reaction (Sigma-Aldrich, Deisenhofen, Germany).

Techniques: Immunofluorescence, Staining, Activity Assay, Flow Cytometry, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: BMC Molecular and Cell Biology

Article Title: Role of tumor cell senescence in non-professional phagocytosis and cell-in-cell structure formation

doi: 10.1186/s12860-020-00326-6

Figure Lengend Snippet: Representative microscopic images of typical cell-in-cell structures in phagocytosis experiments with ( a , d ) pancreas carcinoma cell line (BxPC-3) and fibroblast cell lines ( b , e ) (SBLF-7) and ( c , f ) (SBLF-4). Viable CTOG-stained (green) cell, which completely encloses a hyperthermia-damaged, CTFR-stained (red) cell ( a - c ). Senescent H3K9me3-stained (red) cells that completely enclose a hyperthermia-damaged CTOG-stained (green) cell ( d - f ). Cell nuclei were stained with DAPI (blue). Scale bars 10 μm (40x objective). In bar charts ( g ), ( h ) and ( j ) the cell-in-cell rates occurring in the respective cell lines on the left were shown for different combinations of senescent, living and heat-treated dead cells as indicated

Article Snippet: Senescent cells were detected by anti-H3K9me3 antibodies and a fast-blue color reaction (Sigma-Aldrich, Deisenhofen, Germany).

Techniques: Staining

Journal: BMC Molecular and Cell Biology

Article Title: Role of tumor cell senescence in non-professional phagocytosis and cell-in-cell structure formation

doi: 10.1186/s12860-020-00326-6

Figure Lengend Snippet: Kaplan Meier plots for overall survival, metastasis free survival and local recurrence-free survival for the cohort of patients ( a ) of which tissue micro arrays from pretherapeutic biopsies and ( b ) of post-RCT tumor resection were available. c Example of a micro array spot of rectal carcinoma tissue with a diameter of 2 mm that was immunohistochemically double stained by nuclear anti-H3K9me3 (blue, senescent) and anti-E-Cadherin (red membranous). Arrows indicating cell-in-cell events. d Individual cell-in-cell events. e Frequency of counted intraepithelial senescent cells/mm 2 of patients in pre-RCT biopsy and post-RCT central tumor, invasive tumor front and normal tissue. f Frequency of counted intraepithelial cell-in-cell phenomena/mm 2 in patients in pre-RCT biopsy and post-RCT central tumor, invasive tumor front and normal tissue area. Differences between groups were analyzed by unpaired Student’s t-test

Article Snippet: Senescent cells were detected by anti-H3K9me3 antibodies and a fast-blue color reaction (Sigma-Aldrich, Deisenhofen, Germany).

Techniques: Microarray, Staining

Journal: BMC Molecular and Cell Biology

Article Title: Role of tumor cell senescence in non-professional phagocytosis and cell-in-cell structure formation

doi: 10.1186/s12860-020-00326-6

Figure Lengend Snippet: a Part of a TMA immunohistochemical double stained for senescence by nuclear anti-H3K9me3 (blue) and membranous anti-E-Cadherin (red) in rectal cancer. Arrows indicating cell-in-cell events. Zoomed images of the tissue micro array, b a cell-in-cell event in which both involved cells are senescent, c a cell-in-cell event in which both involved cells are not senescent, d engulfed cell is senescent (asterisk marks the cell in the tissue micro array) and ( e ) a cell-in-cell event in which only the engulfing cell is senescent. f Distribution of senescence in the engulfing and in the engulfed cell in all observed cell-in-cell events. Differences were analyzed by the Student’s t-test

Article Snippet: Senescent cells were detected by anti-H3K9me3 antibodies and a fast-blue color reaction (Sigma-Aldrich, Deisenhofen, Germany).

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Microbiology

Article Title: An insert in the covS gene distinguishes a pharyngeal and a blood isolate of Streptococcus pyogenes found in the same individual

doi: 10.1099/mic.0.042614-0

Figure Lengend Snippet: Transcriptome profile analysis of pharyngeal and blood isolates. Microarrays were done on two separate microarray chips. Expression profiles to five GAS genomes were assessed: NC_002737 (M1), NC_003485 (M18), NC_004070 (M3), NC_006086 (M6) and NC_004606 (M3; not shown). Genes differing in expression by twofold and with a P value ≥0.05 between pharyngeal and blood isolates are depicted. Fold transcript differences between the pharyngeal and blood isolates for all known virulence genes and gene regulators are shown. Genes that were expressed at higher levels in the blood isolates are shown above 1.0; genes that were expressed at higher levels in the pharyngeal isolates are shown below 1.0.

Article Snippet: Total RNA from early stationary phases between the two isolates were compared using custom Nimblegen Systems microarray chips.

Techniques: Microarray, Expressing

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: The flowchart of experimental procedure. The study contained four procedural modules, including preparation of tissues, microarray analysis, bioinformatics analysis, and qRT-PCR

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques: Microarray, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: The sequences of lncRNA and mRNA primers used in the study

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques: Expressing, Amplification, Control

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: Differentially expressed lncRNAs and mRNAs in DBA/1 mice compared with C57BL/6 mice. The Volcano Plots of lncRNA ( a ) and mRNA ( b ) expression; Hierarchical clustering of differentially expressed lncRNA ( c ) and mRNA ( d ). ‘red’ indicates high relative expression, and ‘green’ indicates low relative expression. ‘C’ and ‘D’ respectively represent C57BL/6 and DBA/1 group (each group with four mice)

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques: Expressing

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: Differentially expressed antisense lncRNAs and nearby coding gene

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques:

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: a Fold change of significantly dysregulated lincRNA and their differentially expressed adjacent mRNAs for DBA/1mice vs. C57BL/6 mice; ( b ) The percentage of the length distribution of differentially expressed lincRNAs; ( c ) The percentage of the chromosome distribution of differentially expressed lincRNAs

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques:

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: Top 20 differentially expressed lincRNAs and adjacent mRNAs

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques:

Journal: BMC Genomics

Article Title: Gene expression profile for different susceptibilities to sound stimulation: a comparative study on brainstems between two inbred laboratory mouse strains

doi: 10.1186/s12864-022-09016-3

Figure Lengend Snippet: The qRT-PCR vadidation of differentially expressed lncRNAs and mRNAs between DBA/1 mice with C57BL/6. a The qRT-PCR results of up-regulated lncRNAs; ( b ) The qRT-PCR results of down-regulated lncRNAs; ( c ) The qRT-PCR results of differentially expressed mRNAs. By setting the expression value of target genes in C57BL/6 control group at 1, the expression level of which in DBA/1 mice group was the fold change relative to control group. Significant levels were indicated by * ( P < 0.05). The results of qRT-PCR were consistent with that in microarray analysis ( n = 4 animals/group)

Article Snippet: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD).

Techniques: Quantitative RT-PCR, Expressing, Control, Microarray

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A pGL3-Luc vector containing human KLF5 promoter (-4,000 to 0 bp) and pCDNA3.1-Flag-NRF2 were co-transfected into HEK293T cells with a ratio of 1:0.5 to 1:8 and luciferase activity was determined. B Analysis of NRF2 consensus motif enrichment in the KLF5 promoter (-4,000 to 0 bp) predicted by JASPAR database. Matched consensus motifs are shown in schematic. C ChIP-PCR analysis of the enrichment of NRF2 at the promoter region of KLF5 in A549 cells. D pGL3-Luc vector containing human SLC1A5 promoter (-500 to +10 bp) and pCDNA3.1-Flag-KLF5 were co-transfected into HEK293T cells with a ratio of 1:0.5 to 1:8 and luciferase activity was determined. E Analysis of KLF5 consensus motif enrichment in the SLC1A5 promoter predicted by JASPAR database. Matched consensus sequences are in bold. F ChIP-PCR analysis of the enrichment of KLF5 at the promoter region of SLC1A5 in A549 cells. G Q-PCR and immunoblot results of SLC1A5 in A549 cells treated with si NRF2 in the presence or absence of KLF5 overexpression. H Relative glutamine level in A549 cells following si NRF2 treatment in the presence or absence of KLF5 overexpression. I Colony formation of A549 cells treated with CsA or si NRF2 in the presence or absence of KLF5 overexpression. Quantitative results were shown in upper panel and representative colony image was presented in lower panel. J Correlation analysis of KLF5/NRF2 or KLF5/SLC1A5 gene expression in clinical NSCLC tumor samples ( n = 138 samples). The data are derived from public dataset (GSE8894) and analyzed in PrognoScan. The results of panels ( C , F , G ) are representative of three independent experiments. A , C , D , F , G – I represent mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: Antibody dilutions: NRF2 (Proteintech, 16396-1-AP; 1:1000 for WB; 2 μg for IP; 2 μg for ChIP; 1:100 for IHC), Histone-H3 (Proteintech, 17168-1-AP; 1:1000 for WB), PPIA (Proteintech, 10720-1-AP; 1:1000 for WB; 1:100 for IHC), Ubiquitin (Santa Cruz, sc-8017; 1:1000 for WB), SLC1A5 (Proteintech, 20350-1-AP; 1:1000 for WB), KLF5 (Proteintech, 21017-1-AP; 1:1000 for WB; 2 μg for ChIP), KEAP1 (Proteintech, 10503-2-AP; 1:1000 for WB), CRK (Proteintech, 16685-1-AP; 1:1000 for WB), TDP-43 (Proteintech, 10782-2-AP; 1:1000 for WB), HA (Proteintech, 51064-2-AP; 1:1000 for WB), Flag (Sigma-Aldrich, F3165; 1:1000 for WB; 2 μg for IP), GAPDH (Proteintech, 60004-1-Ig; 1:3000 for WB), IgG heavy chain (Proteintech, 16402-1-AP; 2 μg for IP).

Techniques: Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, Over Expression, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A Representative immunohistochemical analysis of PPIA and NRF2 in human LUAD tissue microarray containing lung tumor tissues and adjacent normal lung tissues. Scale bar = 50 μm. B Scatter plots showing a positive correlation of PPIA and NRF2 expression in IHC analysis of human NSCLC cancer tissues ( n = 69 samples). Linear regression with Pearson R and two-tailed P values are shown. C Kaplan-Meier survival curves of patients with NSCLC divided by high or low PPIA protein expression level according to IHC analysis ( n = 69 samples). D Kaplan-Meier survival curves of patients with NSCLC divided by high or low NRF2 protein expression level according to IHC analysis ( n = 69 samples). E – H Kaplan–Meier survival curves of patients with NSCLC based on PPIA , NRF2 , KLF5 and SLC1A5 gene expression level (for PPIA , n = 719 samples; for NRF2 , n = 672 samples; for KLF5 , n = 719 samples; for SLC1A5 , n = 719 samples). Data are integrated from Kaplan–Meier plotter ( http://kmplot.com/analysis/ ). Statistical significance for Kaplan–Meier survival curves ( C – H ) was calculated by Log-rank (Mantel-Cox) test. Source data are provided as a Source Data file.

Article Snippet: Antibody dilutions: NRF2 (Proteintech, 16396-1-AP; 1:1000 for WB; 2 μg for IP; 2 μg for ChIP; 1:100 for IHC), Histone-H3 (Proteintech, 17168-1-AP; 1:1000 for WB), PPIA (Proteintech, 10720-1-AP; 1:1000 for WB; 1:100 for IHC), Ubiquitin (Santa Cruz, sc-8017; 1:1000 for WB), SLC1A5 (Proteintech, 20350-1-AP; 1:1000 for WB), KLF5 (Proteintech, 21017-1-AP; 1:1000 for WB; 2 μg for ChIP), KEAP1 (Proteintech, 10503-2-AP; 1:1000 for WB), CRK (Proteintech, 16685-1-AP; 1:1000 for WB), TDP-43 (Proteintech, 10782-2-AP; 1:1000 for WB), HA (Proteintech, 51064-2-AP; 1:1000 for WB), Flag (Sigma-Aldrich, F3165; 1:1000 for WB; 2 μg for IP), GAPDH (Proteintech, 60004-1-Ig; 1:3000 for WB), IgG heavy chain (Proteintech, 16402-1-AP; 2 μg for IP).

Techniques: Immunohistochemical staining, Microarray, Expressing, Two Tailed Test, Gene Expression