lentiviral plko 1 shrna construct shgfp  (Addgene inc)

Journal: PLOS Pathogens

Article Title: SARS-CoV-2 defective viral genomes from distinct genomic regions drive divergent interferon responses

doi: 10.1371/journal.ppat.1014099

Figure Lengend Snippet: (A) Genome schematics of DVG-B (left) and DVG-ΔN (right). (B) A549-ACE2 cells were infected with WT, DVG-B or DVG-ΔN at MOI 1 and cells were collected 24-hours post-infection for RT-qPCR. Expression of host genes and viral genes/eGFP were calculated relative to GAPDH. **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. N = 3, mean±SD. (C) A549-ACE2 cells were infected with WT (MOI 0.1), DVG-B or DVG-ΔN (MOI 1) and cells were collected 24-hours post-inoculation for confocal microscopy. Representative maximum intensity projections of merged dsRNA (red) staining. Nuclei were Hoechst stained (blue). GFP (green) indicates DVG-B and DVG-ΔN, respectively. Scale bar indicates 20μm. N = 4 fields for WT, DVG-B and DVG-ΔN, respectively. (D) A549-ACE2 cells were transduced with lentiviral vectors encoding SARS-CoV-2 orf9b (ORF9b) or an empty lentiviral vector control (EV). 48 hours post-transduction, cells were infected with DVG-ΔN (MOI 1). 24 hpi cells were collected for RT-qPCR. Expression of host and viral genes were calculated relative to GAPDH. *p < 0.05, by two-way ANOVA with Bonferroni’s multiple comparisons test. N = 3, mean±SD.

Article Snippet: Lentiviral constructs were obtained from addgene ( https://doi.org/10.1038/s41586-020-2286-9 ).

Techniques: Infection, Quantitative RT-PCR, Expressing, Confocal, Microscopy, Staining, Transduction, Plasmid Preparation, Control

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Significantly differentially expressed ECM molecules in early, mid and late-stage mammary tumor samples from the PyMT mouse model of breast cancer in comparison to healthy fat pad tissue (A) PCA of individual samples ( n = 4 or 5 mice per group). Principal components 1 and 2 account for 81.6% of the variability in the dataset. (B) Heatmap of significantly differentially deposited matrisomal elements between groups within the dataset (left). Missing values are shaded in gray. Four major clusters were identified based on Euclidian hierarchical clustering of proteins (middle) and the matrisome categorical annotation for these clustered matrisomal elements based on the matrisome project is shown (right). Expression of PXDN in each sample (cluster 3) is highlighted in red.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Comparison, Expressing

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Association of PXDN expression with patient overall survival Significance between Kaplan-Meier survival curves were calculated with the log-rank test. (A) Overall survival of 1,082 patients from the TCGA breast cancer cohort stratified by median mRNA expression of PXDN. (B) Overall survival of 1,082 patients from the TCGA breast cancer cohort separated by disease stage, and stratified by median mRNA expression of PXDN (dotted line = low PXDN expression, solid line = high PXDN expression) and stage of disease (stage I, red; II, gold; III, green; IV, blue; or unknown, purple). (C) Extraction of data from (B) showing patients with stage II breast cancer at the time of diagnosis stratified by median mRNA expression of PXDN (dotted line = low PXDN expression, solid line = high PXDN expression) ( n = 615). (D) Examples of PXDN IHC staining intensity and corresponding intensity scores for the stromal and epithelial compartments of tumors ( n = 334). Scale bars, 50 μm. (E) Overall survival of 311 invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by high vs. low Allred-scores for PXDN IHC staining of epithelial compartments of tumors. (F) Overall survival of 309 invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by high vs. low Allred-scores for PXDN IHC staining of stromal compartments of tumors. (G) Overall survival of invasive ductal carcinoma patients from the CREA tumor microarray cohort stratified by combined stromal and epithelial Allred-scores for PXDN IHC staining of cores. Log-rank p values between each curve of (G) are listed in .

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Expressing, Extraction, Biomarker Discovery, Immunohistochemistry, Microarray

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Single cell analysis of breast tumor cells expressing PXDN (A–C) Murine PyMT tumor single cell data (11,490 cells) obtained from Valdés-Mora et al. (A) UMAP of the major cell types identified in the murine single cell data. (B) UMAP of PXDN expression across cells. (C) Violin plots of fold change in PXDN expression according to cell type in the murine dataset. (D and E) Human breast cancer single cell and spatial data containing 130,246 cells from 26 invasive breast cancer patients. (D) UMAP of the major cell types identified in the human single cell data. (E) UMAP of PXDN expression across human cells. (F) Boxplots of fold change in PXDN expression according to cell type in the human dataset.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Single-cell Analysis, Expressing, Single Cell

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: PXDN affects cell proliferation and spheroid formation dynamics in CAFs Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are shown on each graph. p values <0.05 were considered significant. ns = not significant. (A and C) RNA expression of PXDN in shScr and sh#1 (A), or WT and OE (C) CAFs. Error bars show the standard deviation of six independent experiments. (B and D) Representative western blots showing PXDN levels in conditioned media (top) from shScr and sh#1 CAFs (B) or WT and OE CAFs (D), with densitometry quantification of three biological replicate western blots (bottom). Densitometry measurements were normalized to total protein loading (measured by Ponceau S stain) and calculated relative to control (shScr or WT) protein expression. (E and F) 2D proliferation of shScr and sh#1 CAFs (E) or WT and OE CAFs (F) as measured by Alamar blue assay five days of culture (left). Differences between CAF lines were directly compared on day 5 (right). Error bars indicate the standard error of the mean for three biological replicates. (G–L) 3D growth of shScr (blue), sh#1 (red), WT (purple), and OE (gold) CAFs as spheroids over the course of 16 days. (G and I) Spheroid area and (K and L) representative images over the course of 16 days. Scale bars, 400 μm. Dotted lines indicate media changes on days 4, 8, and 12. Error bars indicate the standard error of the mean for three biological replicates. (H and J) The day of spheroid growth at which spheroids reached their smallest size before expanding. Error bars indicate the standard error of 24 spheroids across three biological replicates.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: RNA Expression, Standard Deviation, Western Blot, Staining, Control, Expressing, Alamar Blue Assay

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: PXDN inhibits the ability of CAFs to remodel collagen matrices (A and C) Representative images of collagen matrices contraction over the course of 12 days when seeded with shScr or sh#1 CAFs (A) or WT or OE CAFs (C). Scale bars, 1 cm. (B and D) Area of matrices during contraction over the course of 12 days (left), with differences in final area of matrices compared at day 12 (right). Error bars indicate the standard error of the mean for three biological replicates. p values were calculated using Student’s t tests. p values <0.05 were considered significant.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques:

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Impact of CAF-produced PXDN on cancer cell proliferation and motility Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are indicated on each graph. p values <0.05 were considered significant. ns = not significant. (A) Schematic of generation of CAF-generated CDMs, onto which cancer cells were seeded. (B and C) Proliferation rates of cancer cells seeded on CDMs produced by sh#1 or shScr CAFs (B), or by WT and OE CAFs (C) as measured by Alamar blue at day 7. Error bars represent the standard deviation of eight replicates. (D and F) Mean velocity of cancer cells when seeded onto CDMs produced by shScr or sh#1 CAFs (D) or by WT or OE CAFs (F). (E and G) Distance traveled by cancer cells when seeded onto CDMs produced by shScr or sh#1 CAFs (E) or by WT or OE CAFs (G). A minimum of ten cells were tracked per biological replicate, across three biological replicates. Error bars indicate the standard deviation.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Produced, Generated, Standard Deviation

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Effect of CAF PXDN expression on breast tumor progression in vivo Differences between shScr CAFs and sh#1 CAFs, or between WT CAFs and OE CAFs, were calculated using Student’s t tests and p values are indicated on each graph. p values <0.05 were considered significant; ns = not significant. (A and B) Latency of tumor formation from the time cells were implanted into mice along with shScr or sh#1 CAFs (A) or with WT or OE CAFs (B) until tumors reached 50 mm 3 in size. (C and D) Final weights of tumors upon collection from mice implanted with cancer cells and shScr or sh#1 CAFs (C) or with WT or OE CAFs (D). (E) Representative images of α-SMA staining in tumors from shScr or sh#1 CAF tumors. Scale bars, 100 μm. (F) Quantification of α-SMA staining in shScr and sh#1 CAF tumors. Error bars indicate the standard deviation. (G) Representative images of α-SMA staining in tumors from WT or OE CAF tumors. Scale bars, 100 μm. (H) Quantification of α-SMA staining in WT and OE CAF tumors. Error bars indicate the standard deviation.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Expressing, In Vivo, Staining, Standard Deviation

Journal: iScience

Article Title: Stromal peroxidasin drives early tumor growth in breast cancer

doi: 10.1016/j.isci.2026.116078

Figure Lengend Snippet: Effect of PXDN inhibition on breast tumor progression using AZD5904 (A) Inhibition of PXDN by a range of concentrations of AZD5904 measured on live cells using a modified Amplex red activity assay. Resorufin fluorescence was monitored continuously for 1 h. Error bars indicate standard error of the mean across three biological replicates, each consisting of eight technical replicates. (B) Final resorufin fluorescence readings at 60 min were used to determine to IC50 inhibitory concentration of AZD5904. (C) Kaplan-Meier curve of mouse survival measured from the start of treatment with AZD5904 or vehicle (when tumors reached a detectable size of 50 mm 3 ) until tumors reached a maximum ethical size of 1 cm by 1 cm and mice were sacrificed. Survival curves were significantly different, with a log-rank p value of 0.0088. (D) Final weights of tumors upon collection from mice in the AZD5904 in vivo study. Error bars indicate the standard deviation. Student’s t test showed no statistical significance between vehicle and AZD5904 treatment groups. (E) Time taken for tumors to reach the detectable size of 50 mm 3 at which point treatment started. Error bars indicate the standard deviation. Student’s t test showed no statistical significance between vehicle and AZD5904 treatment groups.

Article Snippet: Lentiviral PXDN shRNA knockdown constructs and a Scrambled control construct containing GFP sequences were obtained from OriGene (TL513178V).

Techniques: Inhibition, Modification, Activity Assay, Fluorescence, Concentration Assay, In Vivo, Standard Deviation