Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: HEK293T and DF1 cells were either: non-infected (MOCK), treated with DOPS-SUV (DOPS, positive control), infected with FPV or with WSN influenza A strains. All cells were expressing the FRET-sensor MCS+ and emission spectrum images (22 spectral channels from 499 nm to 695 nm) were acquired 16 hpi using 488 nm excitation. (A) Average normalized emission spectra of all the selected regions of interest (ROI) at the equatorial plane of HEK293T and DF1 cells expressing MCS+, following the indicated treatment. Data are represented as mean ± SD from 50-55 HEK293T cells and 21-33 DF1 cells. (B) Representative ratiometric FRET images (RG ratio, pseudo-colored as indicated by the color scale) of HEK293T and DF1 cells expressing MCS+. White rectangles represent examples of ROIs at the PM selected for FRET quantification. Scale bars represent 10 µm. (C) RG ratio derived from the average intensity spectra of each cell type for the indicated treatment. Data from two separate experiments were pooled, plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (**** p < 0.0001). Each data point represents the average value measured for a ROI at the PM in one cell (Table S2).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Infection, Positive Control, Expressing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: HEK293T and DF1 cells were either: non-infected (MOCK), treated with methyl-β-cyclodextrin (MbCD), infected with FPV or with WSN influenza A strains. All cells were labelled with the solvatochromic probes Laurdan (A-C) and Di-4-ANEPPDHQ (D-F) , and then imaged 16 hpi. Averaged normalized fluorescence emission spectra of all selected regions of interest (ROI) at the equatorial plane of HEK293T and DF1 cells stained with Laurdan (A) or Di-4-ANEPPDHQ (D) , for the indicated treatment. Data are represented as mean ± SD of 52-110 cells stained with Laurdan and 36-127 cells stained with Di-4-ANEPPDHQ (Table S3 and S4). Representative ratiometric GP images (GP index, pseudo-colored as indicated by the color scale) of HEK293T and DF1 cells stained with Laurdan (B) or Di-4-ANEPPDHQ (E) . White lines represent examples of ROIs at the PM selected for GP index quantification. Scale bars represent 10 µm. GP index derived from the average intensity spectra from Laurdan- (C) or Di-4-ANEPPDHQ-stained (F) cells for each cell type and indicated treatment. Data from three separate experiments were pooled, plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (**** p < 0.0001). Each data point represents the average value measured for a ROI at the PM in one cell (Tables S3 and S4).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Infection, Fluorescence, Staining, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: Quantitative analysis of protein diffusion via fluorescence correlation spectroscopy (sFCS) in non-infected (MOCK) and FPV-/WSN-infected HEK293T and DF1 cells expressing three model proteins labelled with green fluorescent proteins (mEGFPs) and associated to the plasma membrane (PM). Specifically, we investigated: i) a construct anchored to the inner-leaflet of the PM via a myristoylated and palmitoylated (mp) peptide (mp-mEGFP), ii) a construct anchored to the outer-leaflet of the PM via a glycosylphosphatidylinositol (GPI) anchor (GPI-mEGFP) and iii) one representative transmembrane protein, i.e. the influenza envelope protein hemagglutinin (HA-mEGFP). Measurements were performed 16 hpi. The box plots show the diffusion coefficients calculated from sFCS diffusion times. Data from three separate experiments were plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (* p < 0.05, *** p < 0.001, **** p < 0.0001). Each data point represents the value measured at the PM in one cell (Table S5).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Diffusion-based Assay, Fluorescence, Spectroscopy, Infection, Expressing, Clinical Proteomics, Membrane, Construct, Comparison

Journal: Cell

Article Title: METTL13 methylation of eEF1A increases translational output to promote tumorigenesis

doi: 10.1016/j.cell.2018.11.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pWZL Blast GFP , ( Orimo et al., 2005 ) , Addgene #12269.

Techniques: Virus, Plasmid Preparation, Recombinant, Protease Inhibitor, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Silver Staining, Mutagenesis, Control, Software, Membrane

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: MCF10DCIS.com cells express a mutated allele of PCSK5 that is predicted to be damaging. A, Computational predictions for the PCSK5 M452I mutation of MCF10DCIS.com alongside two single-nucleotide polymorphisms (SNPs; R486H and A565T) and a somatic mutation (T288P) confirmed experimentally to be inactive ( 28 ). Outputs for each algorithm were grouped as Neutral (N), Likely Neutral (LN), Unknown (U), Likely Damaging (LD), or Damaging (D) as described in Supplementary Table S1 . B and C, Confirmation of the M452I mutation in genomic DNA (gDNA) ( B ) and mRNA ( C ) from MCF10DCIS.com cells. MCF10A-5E cells ( 32 ) provide a wildtype reference.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Mutagenesis

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: 293T cells co-expressing PCSK5 M452I secrete mature ectopic GDF11 with intermediate efficiency. A, Catalytically active PCSK5 promotes ectopic GDF11 secretion. Cells were lipofected with 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) plus 100 ng of GDF11, and conditioned medium was collected after 24 hours to measure GDF11 release by ELISA. PCSK5 T288P was included as a catalytically dead control ( 28 ). B, GDF11 release by PCSK5 M452I plus wildtype PCSK5 (PCSK5 WT ) is additive. Cells were treated as in ( A ) and compared with 1.5 ng PCSK5 WT plus 1.5 ng PCSK5 M452I . C, Cotransfection with oncogenic HRAS G12V approximates the level of prenylated HRAS (HRAS prenyl ) in MCF10DCIS.com. 293T cells were lipofected with 2 ng of HRAS G12V and 3 ng of the indicated PCSK5 allele (or EGFP overexpression control) and immunoblotted for HRAS with vinculin and p38 used as loading controls. MCF10A-5E cells are a negative control for HRAS overexpression. D, HRAS G12V cotransfection does not alter the relative GDF11 secretion efficiencies of wildtype PCSK5, PCSK5 M452I , and PCSK5 T288P . Cells were treated as in ( C ) and measured for GDF11 release by ELISA. For ( A ), ( B ), and ( D ), GDF11 ELISA results are normalized to the GDF11-only condition [gray dashed in ( A )] and shown as the mean ± SEM of N = 4 biological replicates. Differences among +GDF11 groups were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Expressing, Over Expression, Control, Enzyme-linked Immunosorbent Assay, Cotransfection, Negative Control

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: Inducible reconstitution of PCSK5 alleles in PCSK5 −/− MCF10DCIS.com cells. A, Approach to MCF10DCIS.com engineering. Cells were transduced with a destabilizing domain (DD)-containing Cas9-P2A-Venus ( 61 ) and a single-guide RNA targeting Exon 4 of PCSK5 (sgPCSK5). Transduced cells were treated with 200 nM Shield-1 ( 61 ) to stabilize Cas9-P2A-Venus and 2% matrigel to promote PCSK5 accessibility before sorting single Venus-positive cells into 10 ng/ml GDF11 (to aid recovery upon PCSK5 loss) and screening genomic DNA (gDNA) of expanded clones for knockout. One confirmed PCSK5 −/− clone was then transduced with sgPCSK5-resistant, tetracycline (tet)-regulated, V5-tagged alleles of PCSK5 and selected polyclonally for hygromycin resistance. B, Sequence-confirmed knockout alleles of MCF10DCIS.com clone 3D8. The PCSK5 coding sequence (CDS) is shown with annotations for the signal peptide (SP, purple), proprotein sequence (Pro, green), and peptidase domain (blue) including its catalytic triad (yellow stars). The protospacer adjacent motif (PAM) of sgPCSK5 is just upstream of the first triad amino acid, and deletions (white, Allele 1) or insertions (pink, Allele 2) induce frameshift mutations (gray) removing the first amino acid in the catalytic triad (black outlined stars) and producing premature stop codons (red). C, Quantification of reconstituted PCSK5 alleles by calibrating against recombinant V5-containing Multitag protein at the indicated copies per cell ( 63 , 72 ). Cells were treated with 1 μg/ml doxycycline for 24 hours, and total protein from counted cells was immunoblotted by two-color fluorescence detection for V5 (800 channel) with tubulin and p38 (700 channel) used as loading controls for cells. Copy number estimates are: PCSK5 WT , 136,000 ± 11,000 copies per cell; PCSK5 M452I , 164,000 ± 6,000 copies per cell; PCSK5 T288P , 176,000 ± 15,000 copies per cell ( N = 4 biological replicates).

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Transduction, Clone Assay, Knock-Out, Sequencing, Recombinant, Fluorescence

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: Impaired anterograde transport of catalytically deficient PCSK5 alleles in PCSK5 −/− MCF10DCIS.com cells. A, Immuno-colocalization of V5 epitope tag (magenta) with the endoplasmic reticulum (ER) marker PDI (green) for the indicated PCSK5 addback allele. B, Whole-cell V5–PDI colocalization quantified by Manders colocalization coefficient as a fraction of total per-cell V5 immunoreactivity in N = 348 (wildtype PCSK5), 322 (PCSK5 M452I ), and 324 (PCSK5 T288P ) cells. C, Immuno-colocalization of V5 epitope tag (magenta) with the cis Golgi marker GM130 (green) for the indicated PCSK5 addback allele. D, Whole-cell V5–GM130 colocalization quantified by Manders colocalization coefficient as a fraction of total per-cell V5 immunoreactivity in N = 398 (wildtype PCSK5), 373 (PCSK5 M452I ), and 431 (PCSK5 T288P ) cells. E, Immuno-colocalization of V5 epitope tag (magenta) with the trans Golgi marker TGN38 (green) for the indicated PCSK5 addback allele. F, Whole-cell V5–TGN38 colocalization quantified by Manders colocalization coefficient as a fraction of total per-cell V5 immunoreactivity in N = 361 (wildtype PCSK5), 358 (PCSK5 M452I ), and 324 (PCSK5 T288P ) cells. For ( A ), ( C ), and ( E ), cells were immunostained for the indicated targets along with tubulin for cell segmentation, counterstained with DAPI (blue), and imaged on a laser-scanning confocal microscope followed by adaptive image deconvolution. Scale bars are 5 μm (upper) and 1 μm (lower). For ( B ), ( D ), and ( F ), arcsine-transformed coefficients were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Marker, Microscopy, Transformation Assay

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: PCSK5 activity promotes rounded multi-cell organization in 3D matrigel cultures of MCF10DCIS.com. A, Spheroid growth rates for the indicated PCSK5 addback lines estimated by nonlinear least-squares regression of cross-sectional area ( 50 ) at 4, 8, and 12 days from N = 8 biological replicates (gray dashed). B and C , Reduced multi-cell circularity of MCF10DCIS.com upon loss of PCSK5. For ( B ), the scale bar is 100 μm. For ( C ), circularities were calculated from N = 1819 ( DCIS.com ) and 2028 (PCSK5 −/− ) segmented spheroids collected from 4 biological replicates at 16 days. Arcsine-transformed circularities were analyzed by two-sample homoscedastic t test. D and E, Multi-cell circularity of PCSK5 −/− cells is restored by wildtype PCSK5 or addition of 250 ng/ml recombinant GDF11, but not PCSK5 M452I or PCSK5 T288P . For ( D ), the scale bar is 100 μm. For ( E ), circularities were calculated from N = 5503 (wildtype PCSK5), 5016 (PCSK5 M452I ), 3495 (PCSK5 T288P ), and 3815 (PCSK5 T288P +GDF11) segmented spheroids collected from 8 biological replicates at 8 days, and arcsine-transformed circularities were analyzed by multiway ANOVA with PCSK5 genotype as a fixed effect. Significant factors were followed up pairwise by Tukey-Kramer post hoc analysis. F and G , PCSK5 alleles do not alter the differentiation phenotypes of MCF10DCIS.com cells in 3D matrigel culture. Cultures in ( A ) plus PCSK5 T288P +GDF11 cultures were lysed and immunoblotted for CDH1, TP63, and VIM with vinculin, tubulin, ERK1/2, and p38 used as loading controls. For ( G ), data from N = 4 biological replicates were normalized to the mean of wildtype PCSK5 cultures, and the three unstimulated genotypes were Box-Cox-transformed and compared by multiway ANOVA with PCSK5 genotype as a fixed effect.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Activity Assay, Transformation Assay, Recombinant

Journal: Molecular cancer research : MCR

Article Title: PCSK5 M452I is a recessive hypomorph exclusive to MCF10DCIS.com cells

doi: 10.1158/1541-7786.MCR-25-0211

Figure Lengend Snippet: PCSK5 activity promotes comedo and stromal phenotypes in MCF10DCIS.com intraductal xenografts. A, Representative hematoxylin–eosin images of wildtype PCSK5 lesions exhibiting small (less than 300 μm; left), punctate (yellow; middle), or large (greater than 300 μm; right) comedo necrosis at 54 days post-injection. B, Prevalence of comedo necrosis phenotypes among lesions from N = 6–8 animals per PCSK5 genotype. C, Representative hematoxylin–eosin images of wildtype PCSK5 lesions exhibiting hypercellular stroma (left), irregularity at the peripheral DCIS-stromal interface (middle), or intraductal stromal expansion (right) at 54 days post-injection. D, Prevalence of stromal phenotypes among lesions from N = 6–8 animals per PCSK5 genotype. For ( A ) and ( C ), the scale bar is 100 μm. For ( B ) and ( D ), arcsine-transformed fractions were analyzed by multiway ANOVA with PCSK5 genotype and sub-phenotype as fixed effects. Significant differences by genotype were followed up by Tukey-Kramer post hoc analysis.

Article Snippet: GDF11 secretion assay—pLX302 GDF11-V5 puro (RRID:Addgene_83097), pLX304 (wildtype) PCSK5-V5 blast (RRID:Addgene_83100), and pLX304 PCSK5 (T288P)-V5 blast (RRID:Addgene_83101) were described previously ( 28 ). pBabe GFP (RRID:Addgene_10668), pBabe puro HA PIK3CA H1047R (RRID:Addgene_12524), pHAGE GFP (RRID:Addgene_106281), and pHAGE PIK3CA H1047R (RRID:Addgene_116500) were commercially obtained. pDONR223 PCSK5 (M452I) (RRID:Addgene_232445) was prepared by QuikChange II XL site-directed mutagenesis (Agilent, 200521) of pDONR223 (wildtype) PCSK5 from the human ORFeome v5.1 and recombined into pLX304 (RRID:Addgene_25890) with LR clonase II (Invitrogen, 11791020) to yield pLX304 PCSK5 (M452I)-V5 blast (RRID:Addgene_232446). pDONR223 BMP2 and pDONR223 BMP4 from the human ORFeome v5.1 were similarly recombined into pLX302 (RRID:Addgene_25896) with LR clonase II (Invitrogen, 11791020) to yield pLX302 BMP2-V5 puro (RRID:Addgene_246525) and pLX302 BMP4-V5 puro (RRID:Addgene_246526). pcDNA3 was used as a carrier plasmid for lipofections, and pLX302 EGFP-V5 puro (RRID:Addgene_141348) or pLX304 EGFP-V5 blast (RRID:Addgene_232447) was used when diluting GDF11 or PCSK5 plasmid dosage and for negative controls. pcDNA3.1 HRAS (G12V) was kindly provided by David Kashatus.

Techniques: Activity Assay, Injection, Transformation Assay