Journal: Science Advances

Article Title: CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial cells

doi: 10.1126/sciadv.abo6566

Figure Lengend Snippet: ( A ) Gene editing strategy to generate inducible CRISPRi iPSC/ESC lines with an NKX2-1–GFP and/or SFTPC-tdTomato reporter. ( B ) Schematic representation of dox-inducible dCas9-KRAB expression and lentiviral-delivered gRNA targeted to the transcriptional start site (TSS) of genes of interest (GOI) to mediate knockdown. ( C ) Effector genes at COPD genome-wide significant loci were identified using gene expression, methylation status, coding associations, deoxyribonuclease (DNase) hypersensitivity, chromatin interactions, and/or similarity in gene sets, as previously described . Genes were further filtered on the basis of expression during lung-directed differentiation of iPSCs, expression in differentiated iAT2s, and expression changes during human lung development. Expression levels of the final selected genes, FAM13A, DSP, TGFB2, MFAP2, RBMS3, SOX4, SOX9, HHIP, and ADGRG6, are shown in iPSCs, iPSC-lung progenitors, early (16 to 17.5 weeks of gestation) and late (20 to 21 weeks of gestation) human fetal lung (HFL), iAT2s grown as 3D alveolospheres or at air-liquid interface (ALI), primary pediatric (13-month-old male donor) and adult (32-year-old male donor) AT2s grown in CK + DCI with MRC-5 cells, and freshly isolated primary adult AT2s .

Article Snippet: TaqMan (Thermo Fisher Scientific) probes used were as follows: SFTPC ( Hs00161628_m1), SFTPA1 (Hs00831305_s1), SFTPA2 (Hs04195463_g1), SLPI (Hs00268204_m1), NKX2-1 (Hs00968940_m1), DSP (Hs00950591_m1), FAM13A (Hs01596554_m1), TGFB2 (Hs00234244_m1), RBMS3 (Hs01104892_m1), MFAP2 (Hs01027737_m1), SOX4 (Hs04987498_s1), SOX9 (Hs00165814_m1), ADGRG6 (Hs01089210_m1), HHIP (Hs01011009_m1), MKI67 (Hs04260396_g1), CDH1 (Hs01023895_m1), CLDN4 (Hs00533616_s1), CLDN1 (Hs00221623_m1), TJP1 (Hs01551876_m1), TJP3 (Hs00274276_m1), KRAS (Hs00364284_g1), COL1A1 (Hs00164004_m1), COL2A1 (Hs00264051_m1), and FN1 (Hs01549976_m1).

Techniques: Expressing, Knockdown, Genome Wide, Gene Expression, Methylation, Isolation

Journal: Science Advances

Article Title: CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial cells

doi: 10.1126/sciadv.abo6566

Figure Lengend Snippet: Guide RNA (gRNA) sequences used in this study.

Article Snippet: TaqMan (Thermo Fisher Scientific) probes used were as follows: SFTPC ( Hs00161628_m1), SFTPA1 (Hs00831305_s1), SFTPA2 (Hs04195463_g1), SLPI (Hs00268204_m1), NKX2-1 (Hs00968940_m1), DSP (Hs00950591_m1), FAM13A (Hs01596554_m1), TGFB2 (Hs00234244_m1), RBMS3 (Hs01104892_m1), MFAP2 (Hs01027737_m1), SOX4 (Hs04987498_s1), SOX9 (Hs00165814_m1), ADGRG6 (Hs01089210_m1), HHIP (Hs01011009_m1), MKI67 (Hs04260396_g1), CDH1 (Hs01023895_m1), CLDN4 (Hs00533616_s1), CLDN1 (Hs00221623_m1), TJP1 (Hs01551876_m1), TJP3 (Hs00274276_m1), KRAS (Hs00364284_g1), COL1A1 (Hs00164004_m1), COL2A1 (Hs00264051_m1), and FN1 (Hs01549976_m1).

Techniques:

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A-D: Representative images of different genotypes of harvested mouse lung sections following necropsy analyses stained with hematoxylin and eosin (H&E) 13 weeks post initiation with 5 × 104 pfu lenti-CRE. CRISPR/CAS9-mediated genome editing was used in panels A, C, D to edit Rbms3 in vivo. Genotype and average tumor burden calculation of each experimental group was: A: sgNT-CRE virus in BrafCAT/+; H11LSL-CAS9 (BC) mice: 8.5%. B: sgRbms3-CRE virus in BrafCAT/+ (B) mice: 7.7%. C: sgRbms3-CRE virus in BC mice: 38.8%. D: sgRbms3-CRE virus in H11LSL-CAS9/+ (C) mice: 0%. Black bar in bottom left of each panel represents a 1000-micron scale bar. E: Quantification of individual tumor burden from genotypes in panel A compared to panel C. Tumor bearing lungs from panel B were identical to panel A. A paired T-test was used to determine statistical significance; p < 0.01.F: Quantification of tumor diameter was performed in microns using 25 individual tumors from genotypes in panel A compared to panel C using the 3D Histech MIDI Slide Scanner QuantCenter. Comprehensive analyses was conducted with over 200 lung tumors. N=50 mice individual or (biological replicates). N = 2 experimental replicates were performed comparing the indicated genotypes in A and C. Individual values are graphed, the black bar represents the mean, and the error bars represent SEM. A paired T-test was used to determine statistical significance; p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1).

Techniques: Staining, CRISPR, In Vivo, Virus

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A-C: Images of harvested mouse lung sections following necropsy analyses stained with hematoxylin and eosin (H&E) 11 weeks post initiation with 1 × 105 pfu lenti-CRE, followed by continuous administration of doxycycline chow to induce EGFRL858R expression. CRISPR/CAS9-mediated genome editing was used in panels B and C to edit Rbms3 in vivo. Genotype of each experimental group was: A: sgRbms3-CRE virus in SPC::CRE-ERT2/+; Rosa26CAGs-LSL-rTTa3; EGFRL858R (SRE) mice. B: sgNT-CRE virus in or SPC::CRE-ERT2/+; Rosa26CAGs-LSL-rTTa3; EGFRL858R; H11LSL-CAS9/+ (SREC) mice. C: sgRbms3-CRE virus in SREC mice. Black bar in bottom left of each panel represents a 1000-micron scale bar. D: Quantification of tumor burden from genotypes in panel B compared to panel C. N=5 mice per group. The mean is graphed, and error bars represent SEM. Statistical analysis was conducted using a paired T test; **** = p < 0.0001. E: Representative images of immunohistochemistry on FFPE lung tissue sections from SREC mice initiated with either sgNT- or sgRbms3-CRE and stained with pEGFR (pY1068) or pERK (pT202; Y204) shown at 20x magnification. Scale bar shown in black at the bottom left corner of each image represent 50 microns.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1).

Techniques: Staining, Expressing, CRISPR, In Vivo, Virus, Immunohistochemistry

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A: Oncoprint of statistically significant drivers in BRAFV600E-driven lung tumors detected using GKC analysis, using SB common integration regions (CIRs), and Truncal SB Driver Analysis, using unique, directional SB insertions at TA-dinucleotides.B: SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) and within CIRs (blue lines) for Foxp1 (3 transcripts of the genes are shown).C: SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) and within CIRs (blue lines) for Rbms3 (6 transcripts of the candidate gene are shown).

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1).

Techniques:

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: Trunk CIS genes involved in lung adenocarcinoma progression of BRAF V600E -initiated tumors. CIS genes containing 5 or more sequence read counts per tumor from 3 or more tumors and have corrected p < 0.05 by gCIS analysis.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1).

Techniques: Sequencing

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A: Representative images are shown of qualitative analyses of phase contrast images of organoids established following tumor dissociation from BC mice at 7 days post-initiation of organoids. White scale bar indicates 400 microns, and was taken with 10x magnification. N=12 technical replicates with 3 biological replicates leveraging pooled lung lobes from N=8 mice. B: Quantification of organoid diameters at 7 days post-initiation of organoids from lungs of the indicated mouse genotypes described in (A). C: qRT-PCR analysis of Rbms3 mRNA expression in organoids derived from BC mice labeled by the lentivirus they were initiated with. Transient over-expression of wild-type Rbms3 was used as a positive control for gene expression. Mean is graphed and error bars represent SEM. Statistical analysis was conducted using a paired T test; * = p < 0.05; **** = p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Labeling, Over Expression, Positive Control, Gene Expression

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A-D: Representative images of different genotypes of harvested mouse lung sections following necropsy analyses stained with hematoxylin and eosin (H&E) 13 weeks post initiation with 5 × 104 pfu lenti-CRE. CRISPR/CAS9-mediated genome editing was used in panels A, C, D to edit Rbms3 in vivo. Genotype and average tumor burden calculation of each experimental group was: A: sgNT-CRE virus in BrafCAT/+; H11LSL-CAS9 (BC) mice: 8.5%. B: sgRbms3-CRE virus in BrafCAT/+ (B) mice: 7.7%. C: sgRbms3-CRE virus in BC mice: 38.8%. D: sgRbms3-CRE virus in H11LSL-CAS9/+ (C) mice: 0%. Black bar in bottom left of each panel represents a 1000-micron scale bar. E: Quantification of individual tumor burden from genotypes in panel A compared to panel C. Tumor bearing lungs from panel B were identical to panel A. A paired T-test was used to determine statistical significance; p < 0.01.F: Quantification of tumor diameter was performed in microns using 25 individual tumors from genotypes in panel A compared to panel C using the 3D Histech MIDI Slide Scanner QuantCenter. Comprehensive analyses was conducted with over 200 lung tumors. N=50 mice individual or (biological replicates). N = 2 experimental replicates were performed comparing the indicated genotypes in A and C. Individual values are graphed, the black bar represents the mean, and the error bars represent SEM. A paired T-test was used to determine statistical significance; p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1). . cBioPortal analysis of human lung cancer databases Point mutations were defined as single base-pair alterations and copy number alterations were defined with copy number values less than or equal to −1, consistent with TCGA standards.

Techniques: Staining, CRISPR, In Vivo, Virus

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A-C: Images of harvested mouse lung sections following necropsy analyses stained with hematoxylin and eosin (H&E) 11 weeks post initiation with 1 × 105 pfu lenti-CRE, followed by continuous administration of doxycycline chow to induce EGFRL858R expression. CRISPR/CAS9-mediated genome editing was used in panels B and C to edit Rbms3 in vivo. Genotype of each experimental group was: A: sgRbms3-CRE virus in SPC::CRE-ERT2/+; Rosa26CAGs-LSL-rTTa3; EGFRL858R (SRE) mice. B: sgNT-CRE virus in or SPC::CRE-ERT2/+; Rosa26CAGs-LSL-rTTa3; EGFRL858R; H11LSL-CAS9/+ (SREC) mice. C: sgRbms3-CRE virus in SREC mice. Black bar in bottom left of each panel represents a 1000-micron scale bar. D: Quantification of tumor burden from genotypes in panel B compared to panel C. N=5 mice per group. The mean is graphed, and error bars represent SEM. Statistical analysis was conducted using a paired T test; **** = p < 0.0001. E: Representative images of immunohistochemistry on FFPE lung tissue sections from SREC mice initiated with either sgNT- or sgRbms3-CRE and stained with pEGFR (pY1068) or pERK (pT202; Y204) shown at 20x magnification. Scale bar shown in black at the bottom left corner of each image represent 50 microns.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1). . cBioPortal analysis of human lung cancer databases Point mutations were defined as single base-pair alterations and copy number alterations were defined with copy number values less than or equal to −1, consistent with TCGA standards.

Techniques: Staining, Expressing, CRISPR, In Vivo, Virus, Immunohistochemistry

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A: Oncoprint of statistically significant drivers in BRAFV600E-driven lung tumors detected using GKC analysis, using SB common integration regions (CIRs), and Truncal SB Driver Analysis, using unique, directional SB insertions at TA-dinucleotides.B: SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) and within CIRs (blue lines) for Foxp1 (3 transcripts of the genes are shown).C: SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) and within CIRs (blue lines) for Rbms3 (6 transcripts of the candidate gene are shown).

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1). . cBioPortal analysis of human lung cancer databases Point mutations were defined as single base-pair alterations and copy number alterations were defined with copy number values less than or equal to −1, consistent with TCGA standards.

Techniques:

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: Trunk CIS genes involved in lung adenocarcinoma progression of BRAF V600E -initiated tumors. CIS genes containing 5 or more sequence read counts per tumor from 3 or more tumors and have corrected p < 0.05 by gCIS analysis.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1). . cBioPortal analysis of human lung cancer databases Point mutations were defined as single base-pair alterations and copy number alterations were defined with copy number values less than or equal to −1, consistent with TCGA standards.

Techniques: Sequencing

Journal: Cancer research

Article Title: Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1158/0008-5472.CAN-21-3214

Figure Lengend Snippet: A: Representative images are shown of qualitative analyses of phase contrast images of organoids established following tumor dissociation from BC mice at 7 days post-initiation of organoids. White scale bar indicates 400 microns, and was taken with 10x magnification. N=12 technical replicates with 3 biological replicates leveraging pooled lung lobes from N=8 mice. B: Quantification of organoid diameters at 7 days post-initiation of organoids from lungs of the indicated mouse genotypes described in (A). C: qRT-PCR analysis of Rbms3 mRNA expression in organoids derived from BC mice labeled by the lentivirus they were initiated with. Transient over-expression of wild-type Rbms3 was used as a positive control for gene expression. Mean is graphed and error bars represent SEM. Statistical analysis was conducted using a paired T test; * = p < 0.05; **** = p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofisher) and the following 20x probes: Ppia (Mm03302254_g1 and Mm02342429_g1) as a housekeeping gene for normalization, Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1), Axin2 (Mm00443610_m1), Lgr5 (Mm00438890_m1), Ccnd1 (Mm00432359_m1), C-Myc (Mm00487804_m1), and Ctnnb1 (Mm00483029_g1). . cBioPortal analysis of human lung cancer databases Point mutations were defined as single base-pair alterations and copy number alterations were defined with copy number values less than or equal to −1, consistent with TCGA standards.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Labeling, Over Expression, Positive Control, Gene Expression

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: (A) Oncoprint of statistically significant drivers in the 28 SB driver genes occurring in oncogenic Braf lung tumors detected using SB Trunk Drvier analysis, from SB common integration regions (CIRs) and unique, directional SB insertions at TA-dinucleotides. (B) SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) SB insertions, and within CIRs (blue lines), for gene Foxp1 (3 transcripts of the genes are shown). (c) SB insertions at TA-dinucleotides with sense (red arrowhead) and anti-sense (blue arrowheads) SB insertions, and within CIRs (blue lines), for genes Rbms3 (6 transcripts of the candidate gene are shown).

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques:

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: ( A ) – ( D ) Panels of 4 genotypes of harvested mouse lung sections following necropsy analyses stained with hematoxylin and eosin (H&E) 13 weeks post initiation with 5 x 10 4 pfu lenti-CRE. CRISPR/CAS9-mediated genome editing was used in panels A , C , D to edit Rbms3 in vivo . Genotype and tumor burden calculation of each experimental group was ( A ) sgNT-CRE virus in BRAF CAT/+; H11 LSL-CAS9 : 8.5% . ( B ) sgRbms3-CRE virus in BRAF CAT/+ : 7.7% . ( C ) sgRbms3-CRE virus in BRAF CAT/+; H11 LSL-CAS9/+ : 38.8% . ( D ) sgRbms3-CRE virus in H11 LSL-CAS9/+ : <1% . Black bar in bottom left of each panel represents a 1000 micron scale bar. ( E ). Quantification of tumor burden from genotypes in panel A compared to panel C . Tumor bearing lungs from panel B were identical to panel A . A paired T-test was used to determine statistical significance; p < 0.01. ( F ) Quantification of tumor diameter was performed in microns using 25 tumors from genotypes in panel A compared to panel C using the 3D Histech MIDI Slide Scanner QuantCenter. Comprehensive analyses was conducted with over 200 lung lobes. N = 50 mice individual or (biological replicates). N = 2 experimental or (technical replicates) were performed comparing the indicated genotypes in (A) and (C) . A paired T-test was used to determine statistical significance; p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Staining, CRISPR, In Vivo, Virus

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: Validation of Rbms3 loss in tumors initiated with sgRbms3-CRE lentivirus. ( A ) A PCR of Rbms3 genomic locus is 850 bp, and contains each sgRNA used against the mouse Rbms3 gene. Digestion of wild-type or mutant duplxed DNA with Surveyor enzyme reveals Rbms3 locus is edited in large tumor DNA via CRISPR/CAS9-mediated editing: indicated by mismatch cleavage fragments below the 850 bp band in lane 10. L represents ladder. Numbers represent lanes. C is technical control (lane 1), S represents addition of Surveyor enzyme (lane 2), N is negative control (lane 11), P is positive or Wild-type Rbms3 gDNA (lanes 3 and 4), sgRbms3 tumor gDNA (lanes 5-8)and D represents wild-type and sgRbms3 tumor gDNA duplexed with the Surveyor enzyme. ( B ) qRT-PCR indicates significantly lower Rbms3 gene expression in 4 of 6 tumors excised from lung sections of mice initiated with sgRbms3 virus compared to those initiated with sgNT virus by a paired T-test. * = p < 0.05; **= p < 0.01; **** = p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Biomarker Discovery, Mutagenesis, CRISPR, Control, Negative Control, Quantitative RT-PCR, Gene Expression, Virus

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: Histological analyses of the progression of grade of tumor bearing mouse lungs expressing BRAF V600E with or without Rbms3 . (A-I) High magnification images of formalin-fixed paraffin-embedden (FFPE) sections of mouse lungs stained with hematoxylin and eosin (H&E). (A-B) Tumor bearing lungs from BRAF CAT/+, H11 LSL-CAS9/+ mice initiated with sgNT-CRE virus harvested at 13 weeks post-initiation using (A) 40x or (B) 400X magnification. Shown are typical BRAF V600E only papillary adenomas with well-circumscribed borders. (C-D) Tumor bearing lungs from BRAF CAT/+, H11 LSL-CAS9/+ mice 13 weeks post-initiation with sgRbms3-CRE virus harvested at 13 weeks using (C) 40x or (D) 400X magnification. Shown are an increased number of papillary adenomas with poorly circumscribed borders, discohesive neoplastic cells, and small, avascular nests free-floating in air spaces. Blue arrows indicate micropapillary tufts. (E-F) Largest tumor bearing lungs from BRAF CAT/+, H11 LSL-CAS9/+ mice 13 weeks post-initiation with sgRbms3-CRE virus harvested at 13 weeks using (E) 40x or (F) 400X. Shown here are higher grade adenocarcinoma in a larger tumor with blue arrows indicating complex papillary and micropapillary architecture. The large tumor shown here was validated to have edited Rbms3 at the DNA and RNA level in . Scale bars are shown in black in the bottom left corner and are 200 microns ( A , C , E ) and 20 microns ( B , D , F ), respectively.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Expressing, Staining, Virus

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: ( A ) Representative images are shown of qualitative analyses of phase contrast images of organoids established following tumor dissociation from the indicated mouse genotypes at 7 days post-initiation of organoids. White scale bar indicates 400 microns, and was taken with 10x magnification. N = 2 experimental replicates with 3 biological replicates leveraging multiple unique mouse lung lobes. ( B ). qRT-PCT analysis of Rbms3 gene expression in organoids derived from BRAF CAT/+; H11 LSL-CAS9 mice labeled by the lentivirus they were initiated with. Transient over-expression of wild-type Rbms3 in the lentiviral PCDH expression backbone was used here as a positive control. Statistical analysis was conducted using a paired T test; * = p < 0.05; **** = p < 0.0001.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Gene Expression, Derivative Assay, Labeling, Over Expression, Expressing, Positive Control

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: Immunohistochemistry of c-MYC protein expression in BRAF V600E + mouse lung tumors with and without RBMS3. ( A ) Panels of c-MYC immunohistochemistry in BRAF CAT/+; H11 LSL-CAS9 mice initiated with sgNT-CRE at 10, 40, and 100x magnifications. ( B ) Panels of c-MYC immunohistochemistry in BRAF CAT/+; H11 LSL-CAS9 mice initiated with sgRbms3-CRE at 10, 40, and 100x magnifications. Scale bars are indicated in the bottom left corner of each image.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: ( A ) Panels of ß-Catenin immunofluorescent staining analyses in the indicated genotypes of tumor bearing FFPE mouse lung sections shown at 2x, 5x, and 40 magnifications. Scale bars are shown in white in the bottom left corner of each image. ( B ) Median fluorescence intensity quantitation using cellprofiler software. Quantitation revealed a trend of increased median staining intensity in lungs without Rbms3 , but no statistically significant difference was seen.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Staining, Fluorescence, Quantitation Assay, Software

Journal: bioRxiv

Article Title: Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAF V600E -Driven Lung Tumorigenesis

doi: 10.1101/2022.02.28.482366

Figure Lengend Snippet: Rbms3 is lost frequently in human non-small cell lung cancer patients. ( A ) Schematic depicting genes located on chromosome 3p24.1 (not drawn to scale). ( B ) Quantitation of cBioportal analyses of TCGA human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients with identified relative copy number alteration frequences of chromosome 3p either by gain (pink) or deletion (blue). (C) and (D) Copy number alteration heat map depicting chromosome 3 gain/loss in red/blue (respectively) on both 3p and 3q in (C) lung adenocarcinoma (LUAD) or (D) lung squamous cell carcinoma (LUSC) patients, respectively. Each row corresponds to a patient. (E) Kaplain-meier survival curve of patient cohorts that do (orange) or do not (blue) have deletion of chromosome 3p. Deletion was defined to be a copy number threshold of −1 within cBioPortal data. Decreased survival is observed in patients that have lost chromosome 3p, but this was not found to be statistically significant.

Article Snippet: SSOAdvanced Universal Probes Supermix (Bio-Rad; catalog # 1725280) was used also according to the manufacter’s protocol. qRT-PCR was performed using Taqman Gene Expression assays (Applied Biosystems; Thermofiosher) and the following 20x probes: Ppia (mm03302254_g1) as a housekeeping gene for normalization, and Rbms3 (mm01350499_m1; mm00618362_m1; mm01350496_m1).

Techniques: Quantitation Assay

Journal: medRxiv

Article Title: Proteo-genomics of soluble TREM2 in cerebrospinal fluid provides novel insights and identifies novel modulators for Alzheimer’s disease

doi: 10.1101/2023.06.14.23291409

Figure Lengend Snippet: A) Our study included two stages of analyses: first stage GWAS analyses using 3350 European samples from eight cohorts and 12,621,222 autosomal genotypic variants, and second stage multi-ethnic fine mapping using 250 non-European (non-EUR) samples from eight cohorts and 8,909,120 autosomal genotypic variants. CSF sTREM2 was measured by SomaScan or MSD. In the first-stage GWAS analyses, using an additive linear model adjusting for age at CSF draw, sex, genotype platform/cohorts, and 10 PCs, we identified 4 loci associated with CSF sTREM2 levels: chromosome 3 RBMS3-TGFBR2 (novel), chromosome 6 TREM2 (novel), chromosome 11 MS4A (known), and chromosome 19 APOE (novel) as shown in Manhantan plot and locus zoom plots. For these 4 loci, we then conducted post-GWAS analyses. First, we used multi-ethnic fine mapping to detect the true causal variants underlying each locus. For each of the four loci, we then performed stepwise conditional analyses to identify the independent genotypic variants. To identify the functional genes underlying three novel loci, we performed colocalization analyses of each locus with the AD GWAS, GTEx eQTL, and MetaBrain eQTL. The regulatory role of these loci were annotated with the brain cell type-specific enhancer-promoter interaction map. For chromosome 3 RBMS3-TGFBR2 locus, in vitro functional validation using overexpression of TGFBR2 and RBMS3 in human primary macrophages was conducted. The overall genetic architecture overlapped between AD PRS and CSF sTREM2 was estimated using multivariate linear regression. Finally to determine whether CSF sTREM2 is causal for AD, two-sample Mendelian randomization was analyzed using CSF sTREM2 GWAS as exposure and the latest AD GWAS as outcome. B) Manhantan plots of GWAS for cerebrospinal fluid (CSF) soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in European individuals (EURs). P values are two-sided raw P values estimated from a linear additive model. The blue solid horizontal line denotes the genome-wide significance level (P = 5 × 10 -8 ), and the red solid horizontal line represents the suggestive significance level (P = 1 × 10 - 6 ). X-axis depicts genomic coordinates by chromosome number and y-axis denotes the negative log10-transformed P value for each genetic variant. C) LocusZoom plot of GWAS of CSF sTREM2 at chromosome 3, 6, 11, and 19. The X-axis depicts genomic coordinates and the y-axis denotes the negative log10-transformed P value for each genetic variant.

Article Snippet: The following primary antibodies and dilutions were used: recombinant anti-TGF beta Receptor II antibody [EPR14673] (1:1000, ab184948, Abcam, Waltham, Boston, MA, USA), anti-RBMS3 (1:1000, NBP1-89497, Novus biologicals, Englewood, CO, USA), anti-TREM2 (AF1828, R&D Systems, Minneapolis, MN, USA), anti-GAPDH (1:4000, MA5-15738, Thermo Scientific) and anti β-actin (1:5000, MilliporeSigma, St. Louis, USA).

Techniques: Functional Assay, In Vitro, Over Expression, Genome Wide, Transformation Assay, Variant Assay

Journal: medRxiv

Article Title: Proteo-genomics of soluble TREM2 in cerebrospinal fluid provides novel insights and identifies novel modulators for Alzheimer’s disease

doi: 10.1101/2023.06.14.23291409

Figure Lengend Snippet: A) Forest plots of effect size estimated by cohort for rs73823326. The effect allele is T for rs73823326. Heterogeneity P is 7.53 × 10 -1 for rs73823326. B) Violin plots of CSF sTREM2 Z Score Residuals by genotypes of rs73823326. C) UCSC genome browser visualization of Microglia and Neurons specific assay for transposase-accessible chromatin with sequencing ( ATAC-seq), H3K27ac Chromatin immunoprecipitation followed by sequencing (ChiP-seq), H3K4me3 ChiP-seq and proximity ligation-assisted ChIP-Seq (PLAC-seq) loops at the chr 3 RBMS3 – TGFBR2 locus. Chromatin loops linking the promoter of TGFBR2 to active gene-regulatory region close to rs73823314 (LD r2=1 with rs73823326 and P=2.54 x 10 -8 ) is specific in microglia. D) Quantification of intracellular TGFBR2 (left panel), TREM2 (middle panel) and extracellular sTREM2 protein levels in PBMC-derived macrophages upon TGFBR2 overexpression. E) Quantification of intracellular RBMS3 (left panel), TREM2 (middle panel) and extracellular sTREM2 protein levels in PBMC-derived macrophages upon RBMS3 overexpression. n = 15 from 4 independent experiments. F) Quantification of intracellular TGFBR2 (left panel), TREM2 (middle panel) and extracellular sTREM2 (right panel) protein levels in PBMC-derived macrophages upon TGFBR2 knockdown. n = 9 from 3 independent experiments. ns: not significant, ** p < 0.01, **** p < 0.0001. Results are shown in mean ± SEM.

Article Snippet: The following primary antibodies and dilutions were used: recombinant anti-TGF beta Receptor II antibody [EPR14673] (1:1000, ab184948, Abcam, Waltham, Boston, MA, USA), anti-RBMS3 (1:1000, NBP1-89497, Novus biologicals, Englewood, CO, USA), anti-TREM2 (AF1828, R&D Systems, Minneapolis, MN, USA), anti-GAPDH (1:4000, MA5-15738, Thermo Scientific) and anti β-actin (1:5000, MilliporeSigma, St. Louis, USA).

Techniques: Sequencing, Chromatin Immunoprecipitation, ChIP-sequencing, Ligation, Derivative Assay, Over Expression, Knockdown

Journal: medRxiv

Article Title: Proteo-genomics of soluble TREM2 in cerebrospinal fluid provides novel insights and identifies novel modulators for Alzheimer’s disease

doi: 10.1101/2023.06.14.23291409

Figure Lengend Snippet: A) Experimental design for PBMC-derived macrophages. B) Intracellular TGFBR2 and RBMS3 protein levels and C) TREM2 levels upon TGFBR2 and RBMS3 overexpression. D) Representative western blot upon TGFBR2 knock down. Quantification of intracellular E) TGFBR2, F) intracellular TREM2 and G) extracellular sTREM2 levels upon TGFBR2 knockdown using TGFBR2_D shRNA. n = 9 from 3 independent experiments. ns: not significant, ** p < 0.01. Results are shown in mean ± SEM.

Article Snippet: The following primary antibodies and dilutions were used: recombinant anti-TGF beta Receptor II antibody [EPR14673] (1:1000, ab184948, Abcam, Waltham, Boston, MA, USA), anti-RBMS3 (1:1000, NBP1-89497, Novus biologicals, Englewood, CO, USA), anti-TREM2 (AF1828, R&D Systems, Minneapolis, MN, USA), anti-GAPDH (1:4000, MA5-15738, Thermo Scientific) and anti β-actin (1:5000, MilliporeSigma, St. Louis, USA).

Techniques: Derivative Assay, Over Expression, Western Blot, Knockdown, shRNA

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Primer sequences.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques:

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Antibody information used in the experiment.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques:

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The poor prognosis of OC patients is linked to down-regulated RBMS3 expression in OC tissues. (A) RBMS3 mRNA expression levels from the GEPIA database in both normal tissue and OC. Grey (n = 88) normal samples and red (n = 426) tumor samples. (B, C)Data on OS and PFS for patients with OC were collected with the assistant of the Kaplan-Meier plotter ( https://kmplot.com/analysis/ ). (D) qRT-PCR was used to measure the mRNA levels of RBMS3 in 29 instances of EOC and 23 cases of normal ovarian tissue. (E, F) Western blot analysis reveals the RBMS3 protein expression level in eight pairs of EOC tissues. (G) Typical IHC staining pictures of RBMS3 in normal tissue (Normal) and OC (Tumor). The scale bars are 50 μm and 20 μm respectively. (H, I) Kaplan-Meier study of the effects of 60 EOC patients' RBMS3 expression on OS and PFS. * P < 0.05, ** P < 0.01, *** P < 0.001 and**** P < 0.0001.The uncropped versions of E is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Expression of RBMS3 in EOC and normal tissue.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relation between clinical parameters and the expression of RBMS3.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The overexpression and suppression of the RBMS3 gene can control the capabilities to migrate, invade, and reproduce of OC cells. (A, B) Stable ID8 cell lines that overexpress and knockdown RBMS3 were created using the lentiviral transfection approach. The fluorescence plot illustrates representative images of stably transfected cells in both dark and bright settings (Microscopic magnification: 20 × ). (C) Comparative mRNA expression levels of RBMS3 in lentiviral-transfected ID8 cells. (D) Western Blot analysis verified that ID8 cells had overexpressed and knocked down RBMS3 genes. (E, F) Plate cloning and CCK-8 were used to determine cell growth. (G, H) Experiments on wound healing demonstrate how RBMS3 affects the capacity for cell migration (Microscopic magnification: 4 × ). (I, J) The Transwell technique for identifying the capacity for the capabilities in invading and migrating of cells (Microscopic magnification: 10 × ). The scale bar is 100 μm * P < 0.05, ** P < 0.01, and *** P < 0.001. The uncropped versions of D is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Over Expression, Control, Knockdown, Transfection, Fluorescence, Stable Transfection, Expressing, Western Blot, Cloning, CCK-8 Assay, Migration

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The RBMS3 gene induces cell cycle arrest and apoptosis, potentially preventing the progression of OC cells. (A, B) Histograms of the flow cytometry data and flow cytometry detection of the cell cycle distribution of ID8 cells before and after RBMS3 gene overexpression and knockdown. (C, D) Using flow cytometry, the apoptotic rate of ID8 cells with RBMS3 lentivirus overexpression and knockdown was examined. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Flow Cytometry, Over Expression, Knockdown

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Immune cell infiltration and RBMS3 expression are correlated in OC tissues. (A) TISIDB database data shows the connection between RBMS3 expression and 28 tumor-infiltrating immune cells in human cancer (red indicates positive infiltration, blue indicates negative). (B) The degree of immune cell infiltration in OC tissues is correlated with the expression of RBMS3. (C) An IHC staining diagram showing the relationship between immune cell-related markers and RBMS3 expression (magnification: 400 × ). The scale bar is 20 μm.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunohistochemistry

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relationship between high and low RBMS3 expression in ID8 cells and immune cell markers. (A,B) RBMS3 expression in ID8 cells in both groups. (C,D) Immunofluorescence imagesof INOS, ARG1, FOXP3 and CD11b in cells of Lv-Vector and Lv-RBMS3 groups (200 × and 400 × ). The scale bars are 100 μm and 50 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunofluorescence, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Impact of overexpressing the RBMS3 gene on in vivo angiogenesis and tumor growth. (A) Diagram of mouse subcutaneous tumor growth. (B) Tumor weight 28 days after inoculating cells. (C) Mouse weight growth curve. (D) Measure the subcutaneous tumor volume, tumor volume = 0.5 × largest dimension × shortest dimension 2 . (E, F) Immunohistochemistry (IHC) staining of Ki-67 and CD31 in transplanted tumor tissue Lv-Vector and Lv-RBMS3 groups (200 × and 400 × ). The scale bars are 50 μm and 20 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: In Vivo, Immunohistochemistry, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relationship between high and low expression of RBMS3 in tumor tissues and immune cell markers. (A,B) IHC staining detects the expression of RBMS3 in mouse tumor tissues. (C,D) Immunofluorescence detection images of INOS, ARG1, FOXP3 and CD11b in the Lv-Vector and Lv-RBMS3 groups of transplanted tumor tissues (200 × and 400 × ). The scale bars are 50 μm and 20 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Effects of RBMS3 on the Wnt/β-catenin signalling pathway in ovarian cancer cells. (A, B) Up-regulation of RBMS3 regulates the Wnt/β-catenin signalling pathway in ID8 cells. (C) After overexpression of RBMS3 and activation of the Wnt/β-catenin signalling pathway, the migratory activity of ID8 cells was detected by transwell. (D) After overexpression of RBMS3 and activation of the Wnt/β-catenin signalling pathway, the invasive activity of ID8 cells was detected by transwell. The scale bars is 100 μm ** P < 0.01, *** P < 0.001 and **** P < 0.0001. The uncropped versions of A is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Over Expression, Activation Assay, Activity Assay

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Correlation between RBMS3 Expression and Clinical Pathological Features of Lung Cancer by IHC

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Expression of RBMS3 and LKB1 in lung cancer via IHC. (A) Representative images of RBMS3 and LKB1 staining in tumors and normal tissues. The right panel provides an enlarged view of the specific features highlighted in the rectangular image presented in the left panel. (Scale bar, 100 μm, 250 μm). Statistical analysis of the expression of RBMS3 (B) and LKB1 (C) proteins in lung cancer. (D) Correlation between RBMS3 and LKB1 in lung cancer patients. (*p<0.05).

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing, Staining

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: The LKB1/AMPK axis promotes the expression of RBMS3. (A) Correlation between RBMS3 and LKB1 in lung cancer from the TCGA database. (B) Western blotting to detect the change in RBMS3 expression after LKB1 knock-out. (C) A549-NC, A549-LKB1, A549-LKB1-AMPK-β-KO cells treated with phenformin (1mM) for 8 hours and cell extracts immunoblotted with antibodies as indicated. (D.E) A549-NC, A549-LKB1, A549-LKB1-AMPK-β-KO cells treated with AICAR (1mM) and metformin (5mM) for 8 hours. qPCR was used to detect RBMS3 expression. (F) A549-LKB1 and (I) H1975 cell lines were subjected to a gradient treatment of metformin for 8 hours, followed by immunoblotting analysis using the specified antibodies. The presented data represents a representative blot from three independent experiments. (G.H) The Western blots of (F) A549-LKB1 was scanned, and the resulting data was presented as densitometric unit ratios. (J.K) The Western blots of (I) H1975 was scanned, and the resulting data was presented as densitometric unit ratios. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing, Western Blot, Knock-Out

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Expression of RBMS3 in lung cancer and its subgroups. (A) RBMS3 expression profiles across 33 types of cancers from the TCGA and GTEx databases. (B) Differences in RBMS3 expression within paired LUAD samples from the TCGA database. (C) Comparison of RBMS3 expression between cancerous and adjacent normal tissue in lung adenocarcinoma from the TCGA and GTEx databases. The Wilcoxon rank-sum test was used to assess the association between RBMS3 expression and various clinicopathological parameters, including gender (D), age (E), smoking history (F), mortality (G), clinical T stage (H), clinical N stage (I), clinical M stage (J), and clinical stage (K). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing, Comparison

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Correlation between RBMS3 Expression and Clinical Pathological Features of Lung Cancer in TCGA Database

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Diagnostic and prognostic value of RBMS3 in lung cancer. (A) ROC curves of RBMS3 protein expression in lung cancer tissue from hospitalized patients. (B) ROC curves of RBMS3 mRNA expression in lung cancer tissue cohort from the TCGA database. (C) Kaplan-Meier curves for overall survival in lung cancer for all cases from hospitalized patients. (D) Kaplan-Meier curves for overall survival in lung cancer for all cases from the TCGA database. (E) M0. (F) N1/N2. (G) T3/T4.

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Diagnostic Assay, Expressing

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: Cox Regression Analysis of Clinical Prognosis in Lung Cancer Patients

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques:

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: RBMS3 suppresses lung cancer cell proliferation and metastasis. (A) Up-regulation of RBMS3 expression in A549 cells. (B) Knockout of RBMS3 expression in H1975 cells. (C) CCK8 assay to evaluate the effect of RBMS3 on lung cancer cell proliferation. (D) Colony formation assay to investigate the effect of RBMS3 on lung cancer cell growth. (E.F) Scratch-wound assay to assess the effect of RBMS3 on lung cancer cell migration. (G.H) Transwell assay to test the effect of RBMS3 on cell migration and invasion ability. G) Migration assay, H) Invasion assay. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Expressing, Knock-Out, CCK-8 Assay, Colony Assay, Scratch Wound Assay Assay, Migration, Transwell Assay, Invasion Assay

Journal: Journal of Cancer

Article Title: RBMS3, a downstream target of AMPK, Exerts Inhibitory Effects on Invasion and Metastasis of Lung Cancer

doi: 10.7150/jca.86572

Figure Lengend Snippet: RBMS3 mediates the function of AMPK in inhibiting lung cancer cell invasion and metastasis. (A) Western blot analysis of RBMS3 expression in A549 cells co-transfected with RBMS3 overexpression plasmid, compound C, and corresponding controls. (B) Western blot analysis of RBMS3 expression in H1975 cells co-transfected with sgRNA-RBMS3, Metformin, and corresponding controls. (C, D) Cell scratch assays demonstrated that RBMS3 inhibited lung cancer cell migration and that RBMS3 mediated the function of AMPK in lung cancer cell migration. (E, F) Transwell migration and invasion assays revealed that RBMS3 inhibited the migration and invasion of lung cancer cells, and that RBMS3 mediated the effect of AMPK on the migration and invasion of lung cancer cells. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The over-expression lentivirus of RBMS3 is acquired from the Genechem and then infects the A549 cell line.

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Migration