Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-PAI-1 (clone C-9) , Santa Cruz Biotechnology , Cat# sc-5297; RRID: AB_628154.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: Life

Article Title: PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells

doi: 10.3390/life14081040

Figure Lengend Snippet: Real-time PCR primers used in experiments.

Article Snippet: Primary antibodies—Mouse monoclonal PAI1 (sc-5297, Santa Cruz [Santa Cruz, CA, USA]) and Mouse anti-α-Tubulin (Sigma [Burlington, MA, USA])—were diluted to 1/1000 in 5% skimmed milk in TBST and incubated overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Life

Article Title: PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells

doi: 10.3390/life14081040

Figure Lengend Snippet: Acquired trastuzumab resistance increases PAI1 gene expression and migration markers in parental and resistant SKBR3 and HCC1954 cell lines. ( A ) Approximately a 25-fold increase was observed in HCC1954, and a 40-fold increase was observed in SKBR3-resistant cell lines. ( B ) Col4a1 , Fibronectin , ICAM1 , Timp2 , Vimentin gene expressions were significantly increased in SKBR3 and HCC1954 cell lines resistant cell lines. P: parental, R: resistant. A two-tailed Student’s t -test was used. * p ≤ 0.05, n = 3 ± SD.

Article Snippet: Primary antibodies—Mouse monoclonal PAI1 (sc-5297, Santa Cruz [Santa Cruz, CA, USA]) and Mouse anti-α-Tubulin (Sigma [Burlington, MA, USA])—were diluted to 1/1000 in 5% skimmed milk in TBST and incubated overnight at 4 °C.

Techniques: Gene Expression, Migration, Two Tailed Test

Journal: Life

Article Title: PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells

doi: 10.3390/life14081040

Figure Lengend Snippet: PAI1 regulates migration markers in HCC1954 and SKBR3 parental and resistant cells: ( A ) Overexpression of PAI1 in SKBR3 and ( B ) HCC1954 parental and resistant cells increased migration marker gene expressions. ( C ) Silence of PAI1 in SKBR3 and ( D ) HCC1954 parental and resistant cells decreased migration marker gene expressions. ( E ) Silence and overexpression of PAI1 in HCC1954 and SKBR3 cells. P: parental, R: resistant. Two-way ANOVA variation test and Tukey post hoc test were used. * p ≤ 0.05, n = 3 ± SD.

Article Snippet: Primary antibodies—Mouse monoclonal PAI1 (sc-5297, Santa Cruz [Santa Cruz, CA, USA]) and Mouse anti-α-Tubulin (Sigma [Burlington, MA, USA])—were diluted to 1/1000 in 5% skimmed milk in TBST and incubated overnight at 4 °C.

Techniques: Migration, Over Expression, Marker

Journal: Life

Article Title: PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells

doi: 10.3390/life14081040

Figure Lengend Snippet: PAI1 regulates cell morphology in parental and resistant SKBR3 and HCC1954 cell lines. Immunofluorescence staining of parental and resistant SKBR3 and HCC1954 cell lines, DAPI indicates nucleus, green is β-Actin. Immunofluorescence of ( A ) parental cells showed epithelial and ( B ) resistant cells showed mesenchymal morphology in SKBR3 and HCC1954. ( C ) Overexpression of PAI1 in parental cells and ( D ) resistant cells showed mesenchymal morphology in SKBR3 and HCC1954. ( E ) Silencing of PAI1 in parental and ( F ) resistant cells showed epithelial morphology. Images are representative of three independent experiments. Scale bar 10 μm.

Article Snippet: Primary antibodies—Mouse monoclonal PAI1 (sc-5297, Santa Cruz [Santa Cruz, CA, USA]) and Mouse anti-α-Tubulin (Sigma [Burlington, MA, USA])—were diluted to 1/1000 in 5% skimmed milk in TBST and incubated overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Over Expression

Journal: Life

Article Title: PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells

doi: 10.3390/life14081040

Figure Lengend Snippet: Trastuzumab+cilengitide combination decreased PAI1 expression in parental and resistant SKBR3 and HCC1954 cell lines in both the short term and the long term. Trastuzumab+aleplasinin therapy decreased PAI1 expression on day 2 and continued to decrease on day 4, day 10, and day 15 in ( A ) parental SKBR3, ( B ) resistant SKBR3, ( C ) parental HCC1954, and ( D ) resistant HCC1954 cells treated with DMSO, trastuzumab, aleplasinin as monotherapies, and trastuzumab+aleplasinin as combination therapy. Two-way ANOVA variation test and Tukey post hoc test were used. * p ≤ 0.05, n = 3 ± SD.

Article Snippet: Primary antibodies—Mouse monoclonal PAI1 (sc-5297, Santa Cruz [Santa Cruz, CA, USA]) and Mouse anti-α-Tubulin (Sigma [Burlington, MA, USA])—were diluted to 1/1000 in 5% skimmed milk in TBST and incubated overnight at 4 °C.

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: RISC complex-associated mRNAs in TGF-β-treated cells for 4 h.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Control

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: A Location of AGO2-mRNA tags in the genome of control NMuMG cells and cells treated for 4, and 48 h with TGF-β. B Differential association of transcripts to AGO2 after 4 h of TGF-β treatment [Log 2 (Fold Change 4 h of TGF-β versus Control)], respect to transcript mean expression. The Serpine1 point was highlighted in red. C Distribution of the AGO2 34 crosslinking sites along the Serpine1 mRNA. 3´UTR, 5´UTR, coding region and exons of the Serpine1 mRNA are indicated.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Control, Expressing

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: Crosslinking sites of RISC complex-associated mRNAs in control cells and TGF-β-treated cells for 4 h.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Control

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: Relative levels of mRNA Serpine1 ( A ) and protein SERPINE1 ( B ) in cells treated with TGF-β at the indicated times. A representative immunoblotting of SERPINE1 is shown. Quantification of Serpine1 and SERPINE1 in three independent experiments is shown. Error bars represent S.D. *** p < 0.001, ** p < 0.01 by two-tailed Student´s t test. Protein-loading normalization was performed by measuring total protein directly on the membrane using the criterion stain-free gel imaging system.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Western Blot, Two Tailed Test, Membrane, Staining, Imaging

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: A Diagram of the complete Serpine1 cDNA (Serpine1wt) and the cDNA with the start codon and three additional ATG codons mutated (Serpine1ATG*). ORF, open reading frame. Xs represent mutations of the ATG codons in the Serpine1 ORF. B Real-time PCR analysis of Serpine1 levels in cells infected at MOI 2 with pHRSIN-DUAL lentivirus control (RelControl), carrying Serpine1wt or Serpine1ATG*. Experiments were repeated fourfold in triplicate samples; error bars represent S.D. **** P < 0.0001 by two-tailed Student´s t test. C Immunoblotting of SERPINE1 in RelControl, Serpine1ATG* and Serpine1wt cells. Protein-loading normalization was performed by measuring total protein directly on the membrane using the criterion stain-free gel imaging system. Experiments were repeated threefold; error bars represent S.D. *** P < 0.001 by two-tailed Student´s t test; ns not significant. D The migratory capacity of RelControl, Serpine1ATG*, and Serpine1wt cells was tested in wound-healing assays. Cells were imaged at 10-min intervals for 24 h. The frames of the movie at 0, 5, and 9 h are shown. E The frames at 5 and 9 h were used to estimate the percentage of surface covered by the cells. Values represent the average (%) of wound closure. Error bars represent S.D. ** P < 0.01 by two-tailed Student’s t test. F Representative images of invasion of RelControl, Serpine1ATG*, and Serpine1wt cells were analyzed in transwell invasion assays. After 24 h, cells were fixed and stained with crystal violet. Invasive cells were solubilized with 1% SDS at room temperature for 30 min and quantified at O.D. 590 nm. G Histograms represent the mean from four independent invasion assays. Error bars represent S.D. ** P < 0.01, * P < 0.05 by two-tailed Student’s t test. H The percentage of sub-G1 cells was determined by flow cytometry. Results are the averages of five independent experiments and were analyzed by one-way ANOVA, followed by the Bonferroni post-test for significance versus control cells. Error bars represent S.D. *** P < 0.001. I Proton efflux rate derived from glycolysis (glycoPER) of RelControl, Serpine1ATG*, and Serpine1wt cells. Rot/AA rotenone/antimycin, 2-DG 2-deoxy-D-glucose. GlycoPER is expressed as pmol/min and was corrected using total µg of protein. Data from three independent experiments. Error bars represent S.D.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Real-time Polymerase Chain Reaction, Infection, Control, Two Tailed Test, Western Blot, Membrane, Staining, Imaging, Flow Cytometry, Derivative Assay

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: Location of selected miRNAs recognition elements in Serpine1 mRNA.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques:

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: A Heatmap of upregulated proteins detected at significantly different levels (false discovery rate: <0.05) in Serpine1ATG cells versus RelControl cells. B Representative immunoblotting of TRA2B, ADH7 and PGM1 proteins in RelControl and Serpine1ATG* cells. Protein-loading normalization was performed by measuring total protein directly on the membrane using the criterion stain-free gel imaging system. C Quantification of TRA2B, ADH7, and PGM1 in three independent experiments. Error bars represent S.D. ** P < 0.01, * P < 0.05 by two-tailed Student’s t test. D Tra2b, Adh7 , and Pgm1 mRNAs were similarly expressed by RelControl and Serpine1ATG* cells. Expression was normalized to Hprt mRNA. Experiments were performed independently three times; error bars represent S.D. ns not significant. E Relative levels of Tra2b mRNA in cells treated with TGF-β at the indicated times. F Representative immunoblotting of TRA2B in control and Serpine1 -depleted cells treated with TGF-β at the indicated times. G Quantification of TRA2B in two independent experiments. Error bars represent S.D. ** P < 0.01, * P < 0.05, ns not significant by two-tailed Student’s t test. H Tra2b, Adh7 , and Pgm1 mRNAs were similarly expressed in Serpine1 -depleted control cells and Serpine1 -depleted cells expressing Serpine1ATG * mRNA. Expression was normalized to Hprt mRNA; error bars represent S.D. ns not significant. I immunoblotting of TRA2B, ADH7 and PGM1 proteins in Serpine1 -depleted control cells and Serpine1 -depleted cells expressing Serpine1ATG * mRNA. Protein-loading normalization was performed by measuring total protein directly on the membrane using the criterion stain-free gel imaging system. J Quantification of TRA2B, ADH7, and PGM1 proteins in two independent experiments. Error bars represent S.D. ** P < 0.01, * P < 0.05 by two-tailed Student’s t test.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Western Blot, Membrane, Staining, Imaging, Two Tailed Test, Expressing, Control

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: A Diagram of Serpine1 constructs containing the Serpine1 cDNA with selected miRNAs sites in the coding region mutated ( Serpine1ATG*Ex*) ; the cDNA with selected miRNAs sites in the 3’UTR mutated ( Serpine1ATG*3’UTR*) ; the cDNA with selected miRNAs sites in both coding region and 3’UTR mutated (Serpine1ATG*All*) ; and the cDNA with all three miR-130b-5p sites mutated (Serpine1ATG*130b-5p*) . ORF, open reading frame, Xs represent mutations of the ATG codons, *represent mutations of the selected miRNAs sites in the Serpine1 cDNA. B Quantification and representative immunoblotting of TRA2B protein in RelControl, Serpine1ATG*, Serpine1ATG*Ex*, Serpine1ATG*3’UTR* and Serpine1ATG*All* cells. C Quantification and representative immunoblotting of TRA2B protein in RelControl, Serpine1ATG* and Serpine1ATG*130b-5p* cells. D Cartoon illustrating the splice junctions of transcripts differentially spliced between RelControl and Serpine1ATG* or Tra2b cells. Each gene is diagrammed by vertical blue bars (exons) and thin horizontal lines (introns) with black arrows representing specific RT-PCR primers and red arrows start transcription sites. E Regulated splicing in RelControl and Serpine1ATG* cells was analyzed by RT-qPCR, and the rate of isoforms was indicated. F Volcano plot for differentially expressed genes in RelControl versus Serpine1ATG*- overexpression cells and RelControl versus Tra2b -overexpression cells. Genes upregulated and downregulated with more than 2-fold change with a P -value < 0.05 are depicted in green and red boxes respectively. G Venn diagram showing the intersection of genes downregulated in Serpine1ATG*- and Tra2b -overexpression cells. The probability of overlapping based on hypergeometric distribution is provided. (H) Relative mRNA quantification of indicated genes by qPCR in RelControl, Serpine1ATG*, and Tra2b DsiRNA-Serpine1ATG* cells. For each mRNA, results are presented relative to the average value of Hprt reference gene. Error bars represent S.D. ** P < 0.01, * P < 0.05, ns not significant by two-tailed Student’s t test. I Gene set enrichment analysis of downregulated genes in Serpine1ATG*- and Tra2b -overexpression cells.

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Construct, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

doi: 10.1038/s41420-024-01886-8

Figure Lengend Snippet: A , B Expression of SERPINE1 mRNA and SERPINE1 protein in COAD samples and adjacent normal samples from TCGA and CPTAC databases respectively. C The Spearman correlation coefficients in the analysis of the expression levels of SERPINE1 mRNA and the infiltration levels of CD8+ T cells, CD4 + T cells, B cells, macrophages and neutrophils in patients with COAD ( n = 458). D , E Linear correlation analysis between SERPINE1 and CD8A mRNAs and SERPINE1 and CD3A mRNAs, respectively, in COAD surgical samples. F Representative images of COAD samples showing low, medium, and high signal for SERPINE1 mRNA, detected by in situ hybridization (ISH) and the corresponding immunofluorescence signal (IFC) using anti-CD8A antibody (red) and DAPI (blue).

Article Snippet: The following antibodies were used in our study: anti-TRA2B (Santa Cruz; sc166829), anti-PGM1 (Invitrogen; PA5-55008), anti-ADH7 (Invitrogen; PA5-97437), anti-mouse Serpine1 (R&D Systems; AF3828), anti-human Serpine1 (ThermoFisher, #66261-1-IG), anti-CD8A (Sigma-Aldrich, HPA037756), anti-AGO2 (Wako Chemicals; clone 2D4, # 018-22021).

Techniques: Expressing, In Situ Hybridization, Immunofluorescence