Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, but not RALB, promotes metastatic growth in spontaneous and experimental models of TNBC lung metastasis. a Representative images (top) and quantitation (bottom) of lungs harvested from mice bearing MDA-MB-231 Ctrl ( n = 5), RALA-KO ( n = 6), or RALB-KO ( n = 6) tumors. Lungs were harvested when early removal criteria (ERC) was reached for each respective cohort. Scale bar is 100μm. b Representative H&E images and quantification of lung metastases arising in mice bearing orthotopic MVT1 shCtrl ( n = 6) or shRALA ( n = 6) mammary tumors. Lung metastasis were categorized as nodules, foci, or emboli. Representative images of each category are shown. Scale bars are 50μm except for the representative foci image which is 20μm. All mice were sacrificed when the shCtrl group met ERC. c Experimental metastasis assay. Representative IVIS images and quantification of total flux in the lungs of mice following tail vein inoculation of luciferase-tagged MDA-MB-231 shCtrl ( n = 15) or shRALA ( n = 10) cells over 24 days. e Extravasation and early colonization assay. Representative IVIS images and quantification of total flux in the lungs of mice following tail vein inoculation of luciferase-tagged MDA-MB-231 shCtrl ( n = 13) or shRALA ( n = 11) cells over 96 h. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: Quantitation Assay, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: BQU57 blocks TNBC growth in vitro and in vivo. a Western blot and quantification of GTP-bound RALB in MDA-MB-231 cells after BQU57 (50μM) or DMSO treatment for 24 h. b Quantification of MDA-MB-231 and MVT1 cell viability upon treatment with varying doses of BQU57 for 72 h. c Quantification of MDA-MB-231 and MVT1 growth in low adhesion (GILA) conditions upon treatment with varying doses of BQU57 for 5 days. d Quantification of MDA-MB-231 orthotopic mammary tumor volume over time [ n = 9 DMSO; n = 10 BQU57 treatment (50mg/kg by i.p. injection, M-F)]. e Representative H&E images (left) and quantification (right) of spontaneous lung metastases in MDA-MB-231 tumor bearing mice following treatment (arrows point to metastatic lesions). Scale bars = 500μm. f Quantification of subcutaneous TNBC PDX tumor volume over time. [ n = 9 DMSO; n = 9 BQU57 treatment (50mg/kg, M-F)]. g Quantification of MDA-MB-231 cell viability upon treatment with BQU57 (100μM) or DMSO in combination with various doses of paclitaxel for 72 h. h RALA expression, but not RALB expression, is predictive of BC patient response to chemotherapy. Data from ROC Plotter. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vitro, In Vivo, Western Blot, Injection, Expressing

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, not RALB, is pro-tumorigenic in preclinical in vivo models of TNBC utilizing CRISPR mediated knockout of RALA/B. a Western blots demonstrating RALA and RALB expression in MDA-MB-231 CRISPR control (Ctrl), RALA CRISPR knockout (RALA-KO), and RALB CRISPR knockout (RALB-KO) cells. b Quantification of RALA and RALB expression in MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells by ImageJ analysis ( n = 3). c Representative images of RALA and RALB immunofluorescence staining in MDA-MB-231 Ctrl, RALA-KO and RALB-KO cells (red = RALA or RALB, blue = DAPI; scale bars = 20μm). d Quantification of MDA-MB-231 Ctrl ( n = 10), RALA-KO ( n = 8), and RALB-KO ( n = 12) orthotopic mammary tumor growth. e Representative images and H-score quantification of Ki67 immunostaining in MDA-MB-231 CRISPR Ctrl, RALA-KO, and RALB-KO orthotopic mammary tumors (scale bar = 40μm). f Representative images and H-score quantification of cleaved caspase 3 (CC3) immunostaining in MDA-MB-231 Ctrl, RALA-KO, and RALB-KO orthotopic mammary tumors (scale bar = 20μm). Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vivo, CRISPR, Knock-Out, Western Blot, Expressing, Control, Immunofluorescence, Staining, Immunostaining

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA, not RALB, is pro-tumorigenic in preclinical in vivo models of TNBC utilizing short hairpin knockdown of RALA/B. a Western blots demonstrating RALA and RALB expression in MDA-MB-231 shRNA control (shCtrl), shRALA, and shRALB cells. b Comparison of MDA-MB-231 shCtrl ( n = 5) and shRALA ( n = 5) orthotopic mammary tumor growth. c Comparison of MDA-MB-231 shCtrl ( n = 7) and shRALB ( n = 8) orthotopic mammary tumor growth. d Representative Ki67 immunostaining images and H-score quantification of MDA-MB-231 shCtrl ( n = 4) and shRALA ( n = 4) orthotopic mammary tumors. e Representative cleaved caspase 3 (CC3) immunostaining images and H-score quantification of MDA-MB-231 shCtrl ( n = 4) and shRALA ( n = 4) orthotopic mammary tumors. f Quantification of Rala mRNA expression in MVT1 shCtrl and shRALA cells by qRT-PCR (left) and growth of orthotopic mammary tumors (right; n =6 per group). Inset shows western blot confirmation of RALA protein expression in MVT1 shCtrl and shRALA cells. Data are presented as mean ± SEM; (*), P < 0.05; scale bars = 20μm

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vivo, Knockdown, Western Blot, Expressing, shRNA, Control, Comparison, Immunostaining, Quantitative RT-PCR

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA supports, while RALB opposes, in vitro measures of aggressive cancer phenotypes in TNBC cell lines. a Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell migration over 6 h (scale bar = 100μm). b Representative images and quantification of MDA-MB-468 siRNA control (siCtrl), siRALA, and siRALB cell migration over 6 h (scale bar = 100μm). c Representative images and quantification of MVT1 shCtrl and shRALA cell migration over 6 h (scale bar = 200μm). d Representative images and quantification of MDA-MD-231 shCtrl and shRALA scratch assay over 6 h (scale bar = 500μm). e Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell invasion through matrigel coated transwell inserts over 24 h (scale bar = 200μm). f Representative images and quantification of MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cell invasion as three dimensional spheroids over 5 days (scale bar = 400μm). g Quantification of viability for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells ± siCtrl or siRALA/B transient knockdown over 72 h. h Quantification of viability for MDA-MB-468 siCtrl, siRALA and siRALB cells over 72 h. i Quantification of BrdU incorporation for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells over 72 h. j Quantification of Annexin V positivity for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells. k Quantification of growth in low adhesion (GILA) conditions for MDA-MB-231 Ctrl, RALA-KO, and RALB-KO cells ± siCtrl or siRALA/B transient knockdown over 5 days. h Quantification of growth in low adhesion (GILA) for MDA-MB-468 siCtrl, siRALA and siRALB cells over 5 days. Data are presented as mean ± SEM; (*), P < 0.05

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vitro, Migration, Control, Wound Healing Assay, Knockdown, BrdU Incorporation Assay

Journal: Breast Cancer Research : BCR

Article Title: The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer

doi: 10.1186/s13058-021-01438-3

Figure Lengend Snippet: RALA is elevated in TNBC and is prognostic of overall survival in ER-negative disease. a Western blots demonstrating RALA and RALB expression in a panel of BC cell lines denoted by molecular subtype. b RALA and RALB mRNA expression in a panel of 46 BC cell lines categorized according to molecular subtype. Data from the Broad Institute Cancer Cell Line Encyclopedia (CCLE), (*) P < 0.05. c Box and whisker plots of RALA and RALB expression in normal breast tissue ( n = 61), BC subtypes other than TNBC ( n = 300) , and TNBC ( n = 49). Data from TCGA Research Network. (*) P < 0.05. d Box and whisker plots of RALA and RALB expression in normal breast tissue ( n = 144), BC subtypes other than TNBC ( n = 1725), and TNBC ( n = 250). Data from METABRIC. (*) P < 0.05. e Left: Kaplan-Meier analysis segregates all BC patients by RALA (upper quartile vs lower three-quartiles) and RALB (spilt along the median) expression in METABRIC and TCGA datasets wherein high RALA is prognostic of worse disease specific survival (DSS) in the METABRIC ( P = 0.0079) and overall survival in the TCGA ( P = 0.0003) cohorts and low RALB is prognostic of worse DSS in the METABRIC cohort ( P = 0.0311), but is not prognostic in the TCGA cohort ( P = 0.9477). Right: Within the TNBC population of the METABRIC cohort, RALA (upper tenth percentile vs lower 90th percentail) is prognostic of worse DSS TNBC patients ( P = 0.0544) while RALB (spilt along the median) is not prognostic ( P = 0.9297). Significance determined by log-rank. f Top: Representative high and low RALA immunostaining of BC patient samples. Bottom: Kaplan-Meier analysis segregating all BC patients ( P = 0.0417) and TNBC patients ( P = 0.0440) by RALA H-score (upper tertile vs lower two tertiles) where high RALA is prognostic of worse overall survival (Scale bars = 60μm)

Article Snippet: Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h. Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Whisker Assay, Immunostaining

Journal: Advanced Science

Article Title: Inhibition of CARM1‐Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202303484

Figure Lengend Snippet: CARM1 was negatively associated with ferroptosis. a) HE staining of clinical specimens of colon cancer. Scale bar, 20 µm. b) Twenty‐five pairs of colorectal cancer (CRC) tissues and adjacent tissues were digested into single‐cell suspensions, and lipid ROS production was assayed via flow cytometry by using C11‐BODIPY after RSL3 treatment for 4 h ( n = 25). c) Malondialdehyde (MDA) levels were detected by using a lipid peroxidation MDA assay kit in single‐cell suspensions treated with RSL3 for 4 h from 25 pairs of CRC tissues and adjacent tissues ( n = 25). d) Heatmap of RNA‐seq using six patient tumors with different lipid ROS levels showing changes in gene expression, including CARM1. e) Quantitative real‐time PCR (qPCR) analysis of CARM1 mRNA levels in tumors from 10 patients. f) Western blot analysis of CARM1 in the same tissues as (e). g) Cell viability was assayed in vector‐ and CARM1‐overexpressing LoVo and HCT116 cells treated with the indicated doses of RSL3 and erastin for 24 h. h) Scatter plot of the immunohistochemistry (IHC) staining score for CARM1, lipid ROS, and MDA levels in CRC tissues ( n = 25). All p values and R values were calculated with Spearman's r test. i) Representative results of immunohistochemical staining for CARM1 from 25 clinical CRC patients. Scale bars, 20 µm. j,k) Lipid ROS (left) and MDA (right) levels were compared in CARM1 high (CARM1 IHC score ≥ 6) and CARM1 low (CARM1 IHC score<6) groups. The data shown represent the mean ± SD. In (b) and (c), comparisons were made by using paired Student's t ‐test, and in (j) and (k), comparisons were made by using the two‐tailed, unpaired Student's t ‐test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The colon cancer cell lines LoVo, HCT116, and MC38, as well as the human embryonic kidney cell line HEK293T, were obtained from the American Type Culture Collection.

Techniques: Staining, Flow Cytometry, Multiple Displacement Amplification, RNA Sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Immunohistochemistry, Immunohistochemical staining, Two Tailed Test

Journal: Advanced Science

Article Title: Inhibition of CARM1‐Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202303484

Figure Lengend Snippet: CARM1 directly interacts with and decreases ACSL4 protein levels in colon cancer cells. a) Mass spectrometry analysis identified ACSL4 in the binding protein pool of CARM1. b) Immunoprecipitation (IP) analyses were performed to examine the endogenous interaction between CARM1 and ACSL4 by using antibodies against CARM1 and ACSL4 in LoVo cells. c) IP analyses were performed to examine the exogenous interaction between CARM1 and ACSL4 by using antibodies against Flag and HA, respectively, in HEK293T cells. d) In vitro GST pull‐down assay to verify the binding of CARM1 and ACSL4. e) Immunofluorescence staining was performed to observe the colocalization of CARM1 (green) and ACSL4 (red) in LoVo and HCT116 cells. The nucleus is labeled via DAPI (blue). Scale bar, 20 µm. f) Western blot analysis of the indicated LoVo cells. Protein levels of CARM1, ACSL4, and H3R17me2a were assayed. g) Quantitative real‐time PCR (qPCR) analysis of CARM1 and ACSL4 mRNA levels in the indicated LoVo cells ( n = 3 independent experiments). h) Western blot analysis of the indicated LoVo cells. Protein levels of CARM1, ACSL4, and H3R17me2a were assayed. i) Western blot analysis of vector‐ and CARM1‐overexpressing LoVo cells treated with 50 µg mL −1 cycloheximide for the indicated times. Quantitative analysis was conducted on ACSL4 levels at the indicated time points. j) IP with an anti‐Flag antibody and Western blotting with an anti‐Myc antibody were performed to detect the ubiquitination level of ACSL4. k) CARM1‐knockdown cells were transfected with the indicated plasmid and treated with MG132 (10 × 10 −6 m ) for 8 h. IP with an anti‐ACSL4 antibody and Western blot analysis of the ubiquitination of endogenous ACSL4 were performed. The data shown represent the mean ± SD. Comparisons were made by using one‐way ANOVA with Tukey's test; *** p < 0.001; n.s., no significant difference.

Article Snippet: The colon cancer cell lines LoVo, HCT116, and MC38, as well as the human embryonic kidney cell line HEK293T, were obtained from the American Type Culture Collection.

Techniques: Mass Spectrometry, Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay, Immunofluorescence, Staining, Labeling, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Ubiquitin Proteomics, Knockdown, Transfection

Journal: Advanced Science

Article Title: Inhibition of CARM1‐Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202303484

Figure Lengend Snippet: RNF25 knockdown inhibits CARM1‐induced ferroptosis resistance. a) Western blot analysis of LoVo and HCT116 cells transfected with the indicated plasmid and siRNAs. Protein levels of CARM1, RNF25, ACSL4 and ACSL4 R339me2a were assayed. b) Western blot analysis of vector‐ and RNF25‐overexpressing LoVo and HCT116 cells treated with DMSO or 10 × 10 −9 m EZM2302 for 24 h. Protein levels of RNF25, ACSL4, and H3R17me2a were assayed. c) HEK293T cells transfected with the indicated plasmids and treated with or without 10 × 10 −9 m EZM2302 for 24 h. Immunoprecipitation (IP) with an anti‐Flag antibody and Western blotting with an anti‐Myc antibody were performed to detect the ubiquitination level of ACSL4. d) Cell viability was assayed in the indicated LoVo and HCT116 cells as (a) treated with 2. 5 × 10 −6 m RSL3 for 12 h ( n = 5 independent experiments). e,f) Malondialdehyde (MDA) levels and relative lipid ROS were assayed in the indicated LoVo and HCT116 cells treated with 2. 5 × 10 −6 m RSL3 for 12 h ( n = 3 independent experiments). g) Mitochondrial membrane potential was detected for the same cells as (e) by using fluorescence staining of mitochondria with JC‐1 dye ( n = 3 independent experiments). h) Schematic diagram of our hypothesis about this project. The data shown represent the mean ± SD. Comparisons were made by using one‐way ANOVA with Tukey's test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The colon cancer cell lines LoVo, HCT116, and MC38, as well as the human embryonic kidney cell line HEK293T, were obtained from the American Type Culture Collection.

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Fluorescence, Staining

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Knockdown of SChLAP1 inhibits the growth of triple negative breast cancer cells. (A) MDA-MB-231 and SKBR3 cells were transfected with control-siRNA or SChLAP1-siRNA. The knockdown of SChLAP1 was confirmed via reverse transcription-quantitative PCR analysis. A Cell Counting Kit-8 assay was performed to determine the viability of (B) MDA-MB-231 and (C) SKBR3 cells after transfection with siRNA-SChLAP1. (D) Colony formation ability was suppressed in MDA-MB-231 and SKBR3 cells upon knockdown of SChLAP1 (magnification, ×40). (E) Knockdown of SChLAP1 significantly promoted the apoptosis of both MDA-MB-231 and SKBR3 cells compared with cells expressing a control vector. ***P<0.001 vs. siRNA-Control. OD, optical density; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Expressing, Plasmid Preparation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: SChLAP1 acts as a sponge of miR-524-5p. (A) Predicted binding site of miR-524-5p on the sequence of SChLAP1. Luciferase activity of (B) MDA-MB-231 and (C) SKBR3 cells co-transfected with miR-524-5p mimics and WT-SChLAP1 was significantly decreased, while the activity of cells expressing miR-524-5p and Mut-SChLAP1 was not obviously affected. (D) Knockdown of SChLAP1 increased the expression of miR-524-5p in MDA-MB-231 and SKBR3 cells. (E) Expression of miR-524-5p in TNBC tissues was significantly downregulated compared with that of adjacent non-cancerous tissues. (F) Expression level of miR-524-5p was negatively correlated with SChLAP1 expression in TNBC tissues. ***P<0.001 vs. corresponding control. NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Transfection, Expressing, Knockdown, Control, Negative Control, Mutagenesis, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: HMGA2 is a target of miR-524-5p in triple negative breast cancer. (A) MDA-MB-231 and SKBR3 cells were transfected with miR-524-5p mimic and mimic-NC, the expression of miR-524-5p was validated via reverse transcription-quantitative PCR. Cell Counting Kit-8 assay showed the decreased viability of (B) MDA-MB-231 and (C) SKBR3 cells induced by miR-524-5p overexpression. (D) Predicted binding sites of miR-524-5p in the 3′-UTR of HMGA2. Relative luciferase activities in (E) MB-231 and (F) SKBR3 cells co-transfected with miR-524-5p mimics or mimic-NC and luciferase reporter plasmid containing WT or Mut 3′-UTR of HMGA2. Overexpression of miR-524-5p decreased the (G) mRNA and (H) protein expression levels of HMGA2 in TNBC cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using ImageJ software. **P<0.01. ***P<0.001 vs. mimic-NC. UTR, untranslated region; NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; HMGA2, High Mobility Group AT-Hook 2; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Binding Assay, Luciferase, Plasmid Preparation, Software, Negative Control, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Reintroduction of HMGA2 reverses SChLAP1 knockdown-induced viability defects in TNBC cells. Knockdown of SChLAP1 decreased both the (A) mRNA and (B) protein expression levels of HMGA2 in MDA-MB-231 and SKBR3 cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using the ImageJ software. (C) Expression of Flag-tagged HMGA2 was detected via western blotting with anti-Flag antibody. Overexpression of HMGA2 significantly attenuated the effect of SChLAP1 knockdown on the viability of (D) MDA-MB-231 and (E) SKBR3 cells. (F) Co-transfection of HMGA2 promoted the colony formation of TNBC cells compared with cells expressing siRNA-SChLAP1 (magnification, ×40). ***P<0.001 vs. control group. HMGA2, High Mobility Group AT-Hook 2; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Software, Western Blot, Over Expression, Cotransfection, Control, Small Interfering RNA

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: Ferroptosis promotes PRV replication. ( A ) Cells were treated with different concentrations of Fer-1 (40 or 80 µM) or vehicle (DMSO) for 24 h. Cell viability of N2a cells was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. ( B ) N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 (80 µM) or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. ( C ) At 24 hpi, cells and supernatants were harvested, and virus titers were determined in Vero cells using a plaque assay. ( D ) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. ( E ) N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection or mock infection and additional treatment of Fer-1 or vehicle. At 6 or 12 hpi, apoptosis was assessed using Annexin V-FITC/PI staining and analyzed by fluorescence-activated cell sorting (FACS). The values in each panel represent the percentage of viable cells. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: CCK-8 Assay, Infection, Virus, Plaque Assay, Western Blot, Staining, Fluorescence, FACS

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: PRV replication depends on extracellular iron concentration. N2a cells were treated with either 15 µM FAC, 50 µM DFOM, or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of FAC/DFOM or vehicle. At 24 hpi, the following assays were performed: ( A, G ) Cell viability was determined using a CCK-8 assay, with vehicle-treated cells defined as 100%. ( B, H ) MDA concentrations in cell lysates were determined using MDA assay. ( C, I ) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. ( D, J ) ROS levels in cells were determined using a ROS assay. ( E, K ) Cells and supernatants were harvested, and virus titers were determined in Vero cells using a plaque assay. ( F, L ) Cell lysates were analyzed by Western blot for gB and β-actin. The protein levels were quantified using ImageJ and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Concentration Assay, Infection, CCK-8 Assay, Multiple Displacement Amplification, Colorimetric Assay, ROS Assay, Virus, Plaque Assay, Western Blot

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: PRV infection induces ferroptosis through disruption of iron metabolism. ( A ) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for TfR1, FTH1, VP16, and β-actin. TfR1-knockdown ( B ), FTH1-overexpression ( H ), and wild-type N2a cells were treated with Fer-1 (80 µM) or the vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. TfR1-knockdown, FTH1-overexpression and wild-type N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. MDA concentrations in TfR1-knockdown ( C ) and FTH1-overexpression ( I ) cell lysates were determined. Ferrous iron concentrations in TfR1-knockdown ( D ) and FTH1-overexpression ( J ) cell lysates were determined. ROS levels in TfR1-knockdown ( E ) and FTH1-overexpression ( K ) cells were determined. Cells and supernatants were harvested, and total virus titers in TfR1-knockdown ( F ) and FTH1-overexpression ( L ) cells were determined in Vero cells using a plaque assay. Cell lysates of TfR1-knockdown ( G ) and FTH1-overexpression ( M ) were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Infection, Disruption, Western Blot, Knockdown, Over Expression, CCK-8 Assay, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: HIF-1β promotes TfR1 mRNA expression upon PRV infection. ( A ) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. RNA was extracted for RT-PCR analysis of TfR1 and RNA18s rRNA, and cell lysates were analyzed by Western blot for TfR1 and β-actin. ( B ) N2a cells were mock- or PRV-infected (MOI = 1) at various times. Cell lysates were analyzed by western blot for HIF-1α, HIF-1β, IRP1, IRP2, TfR1, VP16, and β-actin. HIF-1α-knockdown ( C ), HIF-1β-knockdown ( D ), IRP1-knockdown ( E ), IRP2-knockdown ( F ), and Wild-type N2a cells were harvested for RNA extraction and RT-PCR analysis of TfR1 and RNA18s rRNA, and cell lysates were analyzed by western blot for TFR1 and β-actin. ( G ) HIF-1β-knockdown cells and wild-type N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 (80 µM) or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. Wild-type and HIF-1β-knockdown N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. ( H ) MDA concentrations in cell lysates were determined using MDA assay. ( I ) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. ( J ) ROS levels were determined using a ROS assay. ( K ) Cells and supernatants were harvested, and total virus titers were determined in Vero cells using a plaque assay. ( L ) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, RNA Extraction, CCK-8 Assay, Multiple Displacement Amplification, Colorimetric Assay, ROS Assay, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: Rab11a promotes endosomal trafficking of TfR1 upon PRV infection. N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. ( A ) Cell lysates were analyzed by western blot for Rab11a, TfR1, VP16, and β-actin. ( B ) Cell lysates were co-immunoprecipitated (co-IP) with anti-TfR1 antibody and then subjected to western blot analysis for Rab11a, TfR1, VP16, and β-actin. The grayscale intensity in the mock-infected group was set at 1.00 in the co-IP analysis. ( C ) N2a cells were mock-infected and infected with PRV at an MOI = 1 for 6 h or 12 h. Cells were stained for TfR1, Rab11a, and DAPI and performed using the Duolink In Situ Detection Reagents Green. Then observed by confocal microscopy. Scale bar, 10 µm. ( D ) Rab11a-knockdown and wild-type N2a cells were mock- or PRV-GFP-infected (MOI = 1) for 12 h. Cells were stained for TfR1and DAPI and observed by confocal microscopy. Scale bar, 10 µm. ( E ) Rab11a-knockdown cells and wild-type N2a cells were treated with Fer-1 (80 µM) or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. Wild-type and Rab11a-knockdown N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. ( F ) MDA concentrations in cell lysates were determined using MDA assay. ( G ) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. ( H ) ROS levels were determined using a ROS assay. ( I ) Cells and supernatants were harvested, and total virus titers were determined in Vero cells using a plaque assay. ( J ) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Infection, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, In Situ, Confocal Microscopy, Knockdown, CCK-8 Assay, Multiple Displacement Amplification, Colorimetric Assay, ROS Assay, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

doi: 10.1128/jvi.00974-25

Figure Lengend Snippet: PRV induces ferritinophagy. ( A ) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for NCOA4, TAX1BP1, FTH1, VP16, and β-actin. ( B ) N2a cells were mock- or PRV-infected (MOI = 1) for 2 h and then treated with 5 mM 3-MA or the vehicle. At 12 hpi, cell lysates were analyzed by western blot for NCOA4, TAX1BP1, FTH1, LC3, VP16, and β-actin. ( C ) N2a cells were transfected with FLAG-FTH1 (1 µg) for 24 h, and then mock- or PRV-GFP-infected (MOI = 1). At 12 hpi, cells were stained for FTH1, LAMP1, and DAPI and observed by confocal microscopy. Scale bar, 10 µm. ( D ) N2a cells were mock- or PRV-infected (MOI = 1) for 2 h and then treated with 5 mM 3-MA or the vehicle. At 12 hpi, cell lysates were precipitated (co-IP) with anti-NCOA4 antibody and analyzed by western blot analysis for TAX1BP1, FTH1, NCOA4, VP16, and β-actin. The grayscale intensity was set at 1.00 for the mock-infected group. ( E ) NCOA4-knockdown and wild-type N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for NCOA4, FTH1, LC3, VP16, and β-actin. ( F ) TAX1BP1-knockdown and wild-type N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for TAX1BP1, FTH1, LC3, VP16, and β-actin. NCOA4-knockdown, TAX1BP1-knockdown, and wild-type N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. NCOA4-knockdown ( G ) and TAX1BP1-knockdown ( M ) cells and wild-type N2a cells were treated with Fer-1 (80 µM) or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. MDA concentrations in NCOA4-knockdown ( H ) and TAX1BP1-knockdown ( N ) cell lysates were determined using MDA assay. Ferrous iron concentrations in NCOA4-knockdown ( I ) and TAX1BP1-knockdown ( O ) cell lysates were determined using a ferrous iron colorimetric assay. ROS levels in NCOA4-knockdown ( J ) and TAX1BP1-knockdown ( P ) cells were determined using a ROS assay. Cells and supernatants were harvested, and total virus titers of NCOA4-knockdown ( K ) and TAX1BP1-knockdown ( Q ) cells were determined in Vero cells using a plaque assay. Cell lysates of NCOA4-knockdown ( L ) and TAX1BP1-knockdown ( R ) cells were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Article Snippet: Human embryonic kidney-293T (HEK-293T, CRL-11268), Porcine kidney cell 15 (PK-15; CCL-33), Vero (CCL-81), SH-SY5Y (CRL-2266), and Neuro-2a (N2a; CCL-131) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Infection, Western Blot, Transfection, Staining, Confocal Microscopy, Co-Immunoprecipitation Assay, Knockdown, CCK-8 Assay, Multiple Displacement Amplification, Colorimetric Assay, ROS Assay, Virus, Plaque Assay