Journal: Frontiers in Immunology

Article Title: PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB

doi: 10.3389/fimmu.2020.01559

Figure Lengend Snippet: PDLIM7 promotes p65 degradation in a LIM3 domain-dependent manner. (A) PDLIM7 interacts with p65 but not with p50 subunit of NF- κ B. 293T cells were transfected with a FLAG-tagged p65 or p50 expression plasmid along with or without PDLIM7. Whole cell extracts were immunoprecipitated with anti-c-Myc and immunoblotted with anti-FLAG. Western blots are representative of four independent experiments. (B) Effect of PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding FLAG-tagged p65 and increasing amounts of c-Myc-tagged PDLIM7. Cells were subjected to fractionation and analyzed with the indicated antibody. Western blots are representative of three independent experiments. (C) Western blot analysis for soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65 without or with PDLIM7, then left untreated or treated for 4 h with MG132 (10 μM) and analyzed as in (B) . (D) NIH3T3 cells were transfected with FLAG-p65 along with or without c-Myc-PDLIM7, left untreated or treated for 4 h with MG132 (10 μM) or bafilomycin (100 μM). Cells were subjected to fractionation and analyzed with anti-FLAG antibody. Western blots are representative of three independent experiments. (E) Ubiquitination assay for p65 in 293T cells cotransfected with plasmids encoding His-tagged ubiquitin (His-Ub) and p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in . Western blots are representative of three independent experiments. (F) Effect of ΔLIM3 PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in (B) . Western blots are representative of five independent experiments. (G) Luciferase activity in MEFs transfected with an ELAM-1-luc and pGL4.32-Null renilla construct with or without plasmids encoding p65 in the absence or presence of expression plasmids encoding wild-type or ΔLIM3 PDLIM7. Data are representative of three independent experiments. Shown are the mean values ± SD. ** P < 0.01.

Article Snippet: FLAG-tagged p50 expression plasmid (p50 cFLAG pcDNA3) was purchased from Addgene (#20018).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Fractionation, Ubiquitin Assay, Luciferase, Activity Assay, Construct

Journal: Oncotarget

Article Title: A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

doi: 10.18632/oncotarget.20581

Figure Lengend Snippet: (A) Cells were treated with 5μM JNK-IN-8 (JIN) and/or 3μM lapatinib (Lap) for MDA-MB-231 cells, and 4μM JIN and/or 7μM Lap for MDA-MB-436 cells for various time points. Cells were also transfected with NFκB luciferase reporter plasmids. Line graph points represent normalized luciferase values from 3 technical replicates. (B) MDA-MB-231 cells were treated with either vehicle (DMSO), 5μM JIN and/or 3μM Lap for 48 hours in full media. Whole cell lysates were probed for phospho-p65(ser536), p65, p50, IKKβ, and IKKα. (C) MDA-MB-231 cells were treated with 5μM JIN and/or 3μM Lap for various time points and transfected with a luciferase reporter plasmid under the control of the NQO1 promoter containing Nrf2/ antioxidant response elements (ARE). Line graph points represent normalized luciferase values from 3 technical replicates. (D) Relative levels of glutathione were measured in MDA-MB-231 and MDA-MB-436 cells after 72 hours of treatment in DMSO, JIN, Lap, or JNK-IN-8 and lapatinib (J+L). (E) Levels of NADP + and NADPH were measured in MDA-MB-231 and MDA-MB-436 cells after 72 hours of treatment in DMSO, JIN, Lap, or J+L. (F) MDA-MB-231 cells were non-transfected, transfected with empty vector (EV), or expression plasmids for p65 (p65 OE) and Nrf2 (Nrf2 OE). Twenty four hours after transfection cells were treated with either vehicle (DMSO), 5μM JIN and/or 3μM Lap for 72 hours in full media. Cell viability was assayed using MTS.

Article Snippet: The next day cells were transfected with 200ng (96 well) or 2μg (6 well) of empty vector (pcDNA.3), p65 (RelA cFlag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20012)), or Nrf2 (pCDNA3-Myc3-Nrf2 was a gift from Yue Xiong (Addgene plasmid # 21555)).

Techniques: Transfection, Luciferase, Plasmid Preparation, Control, Expressing

Journal: Oncotarget

Article Title: A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

doi: 10.18632/oncotarget.20581

Figure Lengend Snippet: Lapatinib and JNK-IN-8 jointly inhibit the transcriptional activities of AP-1, NFκB, and Nrf2. This disrupts the cell's natural antioxidant response resulting in accumulation of ROS leading to apoptosis.

Article Snippet: The next day cells were transfected with 200ng (96 well) or 2μg (6 well) of empty vector (pcDNA.3), p65 (RelA cFlag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20012)), or Nrf2 (pCDNA3-Myc3-Nrf2 was a gift from Yue Xiong (Addgene plasmid # 21555)).

Techniques:

Journal: Cell reports

Article Title: High-content image-based pooled screens reveal regulators of synaptogenesis

doi: 10.1016/j.celrep.2025.115889

Figure Lengend Snippet: (A) Workflow of optical pooled CRISPR knockout screen. (B) Example field of view from the screen. Insets show the stains used. Arrows indicate presynaptic specializations induced by NLGN1. Scale bar, 50 μm. (C) Images of in situ sequencing (Laplacian-of-Gaussian filtered) for the same field of view as the insets in (B). Scale bar, 50 μm. (D) Histogram of the number of cells analyzed for each highly expressed gene target ( n = 1,843,247 cells in the primary screen). (E) Example images of Bassoon (red) and HA-NLGN1 (gray) staining from the screen show decreased induction of synaptogenesis by HEK293 cells with CDH2 or NLGN1 knockouts. Scale bar, 25 μm. (F) Boxplots show the number of Bassoon puncta per HEK293 cell in the primary screen (center line, median; box, upper and lower quartiles, Q1–Q3; whiskers, 1.5× interquartile range [IQR]) (non-targeting sgRNAs, n = 26,920 cells; CDH2 knockout, n = 3,899 cells; NLGN1 knockout, n = 1,648 cells). Statistical significance was measured by a one-way ANOVA test, followed by Tukey’s test for multiple comparisons (*** p < 0.001).

Article Snippet: To create knockout cells, wild-type HEK293FT cells or HEK293 cells previously transduced with HA-tagged NLGN1 (pLV-SV40-puro-EF1a-HA-NLGN1-bc, Addgene #200167) were then transiently transfected using Lipofectamine-3000 (Thermo Fisher Scientific) with lentiCas9-Blast (Addgene #52962) and a guide-containing CROPseq-Guide-Zeo vector at a plasmid mass ratio of 3:1, following the manufacturer’s protocol.

Techniques: CRISPR, Knock-Out, In Situ, Sequencing, Staining

Journal: Cell reports

Article Title: High-content image-based pooled screens reveal regulators of synaptogenesis

doi: 10.1016/j.celrep.2025.115889

Figure Lengend Snippet: (A) Scores plot of principal-component analysis (PCA) performed on phenotypic profiles of 644 gene knockouts and 50 non-targeting sgRNAs. (B) Top 15 loadings of PC1 include puncta area, count, and intensity features for Bassoon, VGLUT1, and VGAT stains. Bolded features were summed into a synaptogenesis score for each cell. (C) Two-dimensional PHATE visualization of phenotypic profiles of all gene knockouts. Colors indicate Leiden clusters, with non-targeting sgRNAs in black. (D) PHATE plot shown in (C) with genes colored by the HA-NLGN1 median cell intensity (left) and synaptogenesis score (right). (E) Receiver operating characteristic (ROC) curve of random forest classifier (trained on 70% of the data) predicting cluster labels in (C) using the HA-NLGN1 median cell intensity and component features of the synaptogenesis score. (F) Boxplot of feature importances, calculated from 100 permutations, for the random forest model in (E) (center line, median; box, Q1–Q3; whiskers, 1.5× IQR). (G) Scatterplot comparing HA-NLGN1 median cell intensities and synaptogenesis scores ( Z scored) for all genes, Pearson correlation coefficient r = 0.24. (H) Illustrative images show gene knockouts labeled in (G) with increased expression of HA-NLGN1 and modestly increased induction of presynaptic specializations. Scale bar, 25 μm.

Article Snippet: To create knockout cells, wild-type HEK293FT cells or HEK293 cells previously transduced with HA-tagged NLGN1 (pLV-SV40-puro-EF1a-HA-NLGN1-bc, Addgene #200167) were then transiently transfected using Lipofectamine-3000 (Thermo Fisher Scientific) with lentiCas9-Blast (Addgene #52962) and a guide-containing CROPseq-Guide-Zeo vector at a plasmid mass ratio of 3:1, following the manufacturer’s protocol.

Techniques: Labeling, Expressing

Journal: Cell reports

Article Title: High-content image-based pooled screens reveal regulators of synaptogenesis

doi: 10.1016/j.celrep.2025.115889

Figure Lengend Snippet: (A) Volcano plot of synaptogenesis scores ( Z scored), highlighting genes whose knockouts resulted in decreased (blue; positive controls in navy) or increased (red) presynaptic specializations (false discovery rate [FDR] < 0.1, dashed line). Orange dots represent 50 random samples of 6 non-targeting sgRNAs. (B) Illustrative images of cells with gene knockouts showing decreased induction of presynaptic specializations. Scale bar, 25 μm. (C) Empirical cumulative distribution functions for the normalized Bassoon puncta area of single cells with CRKL , PFN1 , PTEN , CDH2 , and NLGN1 knockouts (shades of blue), compared to cells with non-targeting control sgRNAs (gray). Circles indicate cells shown in (B). (D) Illustrative images of cells with gene knockouts showing increased heterologous synapse formation. Scale bar, 25 μm. (E) Empirical cumulative distribution functions for the normalized Bassoon puncta area of single cells with CADM1 , NSF , and SFPQ knockouts (shades of red) compared to cells with non-targeting sgRNAs (gray). Circles indicate cells shown in (D). (F) Scatterplot comparing VGLUT1 and VGAT puncta scores ( Z scored sum of normalized area and puncta count) for all genes, Pearson’s r = 0.49. (G) Example images of cells with gene knockouts showing a preferential decrease in excitatory (VGLUT1) (left) or inhibitory (VGAT) (right) synapse formation. Scale bar, 25 μm. (H) Heatmap of the synaptogenesis score and its component features for top-scoring genes from the primary screen (FDR < 0.05 and Z score > 1.5 for at least two features). Color indicates the corresponding feature Z score, and circle size indicates the feature FDR. Gene order was obtained from hierarchical clustering.

Article Snippet: To create knockout cells, wild-type HEK293FT cells or HEK293 cells previously transduced with HA-tagged NLGN1 (pLV-SV40-puro-EF1a-HA-NLGN1-bc, Addgene #200167) were then transiently transfected using Lipofectamine-3000 (Thermo Fisher Scientific) with lentiCas9-Blast (Addgene #52962) and a guide-containing CROPseq-Guide-Zeo vector at a plasmid mass ratio of 3:1, following the manufacturer’s protocol.

Techniques: Control

Journal: Cell reports

Article Title: High-content image-based pooled screens reveal regulators of synaptogenesis

doi: 10.1016/j.celrep.2025.115889

Figure Lengend Snippet: (A) Workflow of arrayed co-cultures with clonal knockout HEK293 cells. (B–D) Quantification of presynaptic specializations induced by knockout HEK293 cells transfected with HA-tagged BFP, wild-type NLGN1, or NLGN1 AChE mutant with inactivating K578A/V579A substitutions. Bar plots of the number of (B) Bassoon, (C) VGLUT1, and (D) VGAT puncta per cell show means ± SEM from 4–8 independent experiments (numbers of cells analyzed are listed in ). Statistical significance was calculated by a one-way ANOVA test, followed by Tukey’s test for multiple comparisons (ns = p ≥ 0.05 and *** p < 0.001).

Article Snippet: To create knockout cells, wild-type HEK293FT cells or HEK293 cells previously transduced with HA-tagged NLGN1 (pLV-SV40-puro-EF1a-HA-NLGN1-bc, Addgene #200167) were then transiently transfected using Lipofectamine-3000 (Thermo Fisher Scientific) with lentiCas9-Blast (Addgene #52962) and a guide-containing CROPseq-Guide-Zeo vector at a plasmid mass ratio of 3:1, following the manufacturer’s protocol.

Techniques: Knock-Out, Transfection, Mutagenesis