Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Expression of cell cycle checkpoint genes are altered by heme treatment. RNA-seq was performed on activated B cells (ActB) and plasma cells (PCs) from naïve B cells activated with LPS, IL-2, and IL-5 for 72 h with heme or vehicle added for the last 48 h. (A) GSEA analysis of the indicated gene set using RNA-seq data from Fig. 2 . GSEA analysis of p53 and Rb targets were previously described. (B) Scatterplot showing the correlation in log 2 FC of the 195 shared DEGs between PCs and ActB from Fig. 2C with select pathways and corresponding genes highlighted. (C) Bar plots showing the RPKM normalized values for the indicated genes. Error bars represent mean ± SD and each circle represents data from an individual biological replicate. Asterisks (*) indicate statistically significant differences between the indicated groups. GOBP, Gene Ontology Biological Process; OAS, oligoadenylate synthase; RPKM, reads per kilobase million.

Article Snippet: After blocking, membranes were incubated overnight at 4 °C with rabbit polyclonal anti-p53 (1:5,000; Proteintech, 10442-1-AP) or mouse monoclonal anti-β-actin (1:10,000; clone C4, Santa Cruz Biotechnology, sc-47778) primary antibodies.

Techniques: Expressing, RNA Sequencing, Clinical Proteomics

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme represses the p21-Rb cell cycle regulatory axis. (A) Model depicting how p53, p21, and Rb function to regulate the G1 to S phase transition of the cell cycle under conditions of cell cycle arrest (left) or cell cycle progression (right). Naïve B cells were stimulated with CD40L, IL-4, and IL-5, and heme or vehicle control was added at 24 h. (B) Bar plot representing Trp53 transcript abundance at 54 h and 72 h measured by RT-qPCR. (C) Western blot for p53 protein and β-actin. Images for p53 protein were adjusted for brightness (+40%) and contrast (−40%). (D) Bar plot representing p53 expression relative to β-actin from (C). (E) Boxplot quantitating the ATAC-seq signal at p53 motifs in the indicated condition. (F) Genome plot of the Cdkn1a locus depicting the ATAC-seq signal and significant differential accessible region (box) in heme- vs vehicle-treated activated B cells (ActB) and plasma cells (PCs). The location of a previously identified p53 binding site is indicated in red. (G) Bar plot representing Cdkn1a transcript abundance at 54 h and 72 h measured by RT-qPCR. (H) Representative flow cytometry histogram showing intracellular staining for p21 protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (I) Bar plot quantitating MFI of p21 expression from (H). (J) Representative flow cytometry histogram showing intracellular staining for Rb protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (K) Bar plot quantitating MFI of Rb expression from (J). Data represent the combination of at least 2 independent experiments with the indicated P value calculated with paired Student t- tests. FMO, Fluorescence Minus One; MFI, median fluorescence intensity; RPPM, reads per peak per million.

Article Snippet: After blocking, membranes were incubated overnight at 4 °C with rabbit polyclonal anti-p53 (1:5,000; Proteintech, 10442-1-AP) or mouse monoclonal anti-β-actin (1:10,000; clone C4, Santa Cruz Biotechnology, sc-47778) primary antibodies.

Techniques: Sublimation, Control, Quantitative RT-PCR, Western Blot, Expressing, Clinical Proteomics, Binding Assay, Flow Cytometry, Staining, Fluorescence

Journal: bioRxiv

Article Title: Centrosome architecture and m6A-dependent gating of p53 surveillance after whole-genome doubling

doi: 10.64898/2026.02.25.707964

Figure Lengend Snippet: (A) Schematic representation of the V5-mScarlet knock-in at the endogenous MDM2 locus in Cal51 cells. The tag was inserted downstream of the second translation initiation codon (ATG) and the p53-responsive elements (p53REs). The protospacer adjacent motif (PAM) and protospacer sequences are reported. LHA: left homology arm; NeoR: neomycin resistance; RHA: right homology arm. (B) Immunoblot analysis of the Caspase-2–MDM2–p53 pathway activation in unedited Cal51 and in two independent Cal51 mScarlet-MDM2 clones treated with ZM. (C) Immunofluorescence analysis of Cal51 mScarlet-MDM2 cells treated with ZM, showing mScarlet fluorescence and anti-V5 immunostaining. Scale bar: 20 μm. (D) Quantification of mScarlet fluorescence over time in individual Cal51 mScarlet-MDM2 cells imaged live following ZM or vehicle-only treatment. Traces were aligned by setting time zero to the onset of pseudo-anaphase for each cell. The plot shows the population mean ± s.e.m.; n = 25 cells per condition. (E) Immunoblot analysis of mScarlet-positive and mScarlet-negative Cal51 mScarlet-MDM2 cells isolated by FACS following ZM treatment. (F) Flow cytometry analysis of mScarlet fluorescence in unedited Cal51, wild-type (WT), and CEP83 -/- Cal51 mScarlet-MDM2 cells following ZM treatment.

Article Snippet: The following antibodies were used: rat monoclonal anti-Caspase-2 (a gift from Dr. Andreas Strasser, Walter and Eliza Hall Institute of Medical Research, clone 11B4, 1:1000), mouse monoclonal anti-MDM2 (Thermo Fisher Scientific, MA1-113, clone IF2, 1:1000), rabbit monoclonal anti-V5-tag (CST®, 13202, clone D3H8Q, 1:1000), rabbit polyclonal anti-p53 (CST®, 9282, 1:1000), mouse monoclonal anti-MCL1 (SCBT, sc-12756, clone 22, 1:500), rabbit polyclonal anti-CEP83 (Atlas antibodies, HPA038161, 1:1000), rabbit polyclonal anti-PARP1 (CST®, 9542, 1:1000), rabbit polyclonal anti-Caspase-3 (CST®, 9662, 1:1000), rabbit polyclonal anti-Caspase-7 (CST®, 9492, 1:1000), mouse monoclonal anti-CDC27 (BD Biosciences, 610455, clone 35/CDC27, 1:1000), mouse monoclonal anti-p21 (BD Biosciences 564262, clone 2G12, 1:1000), rabbit monoclonal anti-METTL3 (CST®, 96391, clone D2I6O, 1:1000), rabbit polyclonal anti-METTL14 (ProteinTech®, 26158-1-AP, 1:1000), rabbit polyclonal anti-CRADD/RAIDD (ProteinTech®, 10401-1-AP, 1:1000), rabbit monoclonal anti-VIRMA (CST®, 88358, clone D4N8B, 1:1000), rabbit polyclonal anti-WTAP (CST®, 56501, 1:1000), rabbit polyclonal anti-ZC3H13 (Boster Bio, A11022-1, 1:500), rabbit polyclonal anti-CBLL1 (Bethyl Laboratories®, A302-968A, 1:1000), mouse monoclonal anti-RBM15 (ProteinTech®, 66059-1-Ig, clone 4A1A4, 1:1000), mouse monoclonal anti-HSP90 (SCBT, sc-13119, clone F-8, 1:10000), rabbit monoclonal anti-GAPDH (CST®, 2118, clone 14C10, 1:10000), polyclonal goat anti-rat IgG/HRP (Thermo Fisher Scientific, 31470, 1:5000), polyclonal goat anti-rabbit IgG/HRP (Agilent, P0448, 1:5000), polyclonal rabbit anti-mouse IgG/HRP (Agilent, P0161, 1:5000).

Techniques: Knock-In, Western Blot, Activation Assay, Clone Assay, Immunofluorescence, Fluorescence, Immunostaining, Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: Centrosome architecture and m6A-dependent gating of p53 surveillance after whole-genome doubling

doi: 10.64898/2026.02.25.707964

Figure Lengend Snippet: (A) Schematic overview of the genome-wide CRISPR knockout screen in Cal51 mScarlet-MDM2 cells. Cells were transduced with the Brunello sgRNA library, subjected to puromycin selection, treated with ZM for 40 h, and sorted by FACS based on reporter fluorescence. Genomic DNA from sorted populations was processed for sgRNA amplification and Illumina-based next-generation sequencing. (B) Waterfall plot showing genome-wide ranking of genes based on MAGeCK analysis of sgRNA enrichment in the reporter-negative population. Genes are ordered by rank, and the y-axis represents the -log10-transformed MAGeCK enrichment score (robust rank aggregation score). Selected pathway components are highlighted. (C) sgRNA-level log 2 fold-change (LFC) values for selected genes identified in the CRISPR screen. Each point represents an individual sgRNA from the Brunello library targeting the indicated gene, illustrating the consistency of sgRNA behavior across hits. NTC: non-targeting control. (D) Immunoblot analysis of an isogenic panel of RPE1 cells comprising wild-type p53 cells (WT), TP53 -/- cells, or cells expressing hotspot mutant p53 alleles (R175H or R248Q) following ZM treatment. Two independent clones are shown for each TP53 -/- , R175H, and R248Q genotype. (E) Immunoblot analysis of wild-type (WT) and CRISPR-engineered Cal51 cells harboring point mutations disrupting the p53 response elements (p53REs) in the PIDD1 or MDM2 promoters, as schematized in Fig. S4A, following ZM treatment. Two independently derived clonal cell lines are shown for each genotype. (F) Dot plot of Gene Ontology (GO) Cellular Component terms enriched among the top 200 genes identified in the CRISPR screen. Dot size represents the number of genes associated with each term, and color indicates enrichment significance. (G) Jaccard similarity heatmap showing pairwise overlap between the five top-ranked enriched GO Cellular Component terms shown in (F). Each square reports the Jaccard similarity index, and colour intensity reflects similarity magnitude, highlighting extensive overlap among m6A writer-associated terms and no overlap with the centriole term.

Article Snippet: The following antibodies were used: rat monoclonal anti-Caspase-2 (a gift from Dr. Andreas Strasser, Walter and Eliza Hall Institute of Medical Research, clone 11B4, 1:1000), mouse monoclonal anti-MDM2 (Thermo Fisher Scientific, MA1-113, clone IF2, 1:1000), rabbit monoclonal anti-V5-tag (CST®, 13202, clone D3H8Q, 1:1000), rabbit polyclonal anti-p53 (CST®, 9282, 1:1000), mouse monoclonal anti-MCL1 (SCBT, sc-12756, clone 22, 1:500), rabbit polyclonal anti-CEP83 (Atlas antibodies, HPA038161, 1:1000), rabbit polyclonal anti-PARP1 (CST®, 9542, 1:1000), rabbit polyclonal anti-Caspase-3 (CST®, 9662, 1:1000), rabbit polyclonal anti-Caspase-7 (CST®, 9492, 1:1000), mouse monoclonal anti-CDC27 (BD Biosciences, 610455, clone 35/CDC27, 1:1000), mouse monoclonal anti-p21 (BD Biosciences 564262, clone 2G12, 1:1000), rabbit monoclonal anti-METTL3 (CST®, 96391, clone D2I6O, 1:1000), rabbit polyclonal anti-METTL14 (ProteinTech®, 26158-1-AP, 1:1000), rabbit polyclonal anti-CRADD/RAIDD (ProteinTech®, 10401-1-AP, 1:1000), rabbit monoclonal anti-VIRMA (CST®, 88358, clone D4N8B, 1:1000), rabbit polyclonal anti-WTAP (CST®, 56501, 1:1000), rabbit polyclonal anti-ZC3H13 (Boster Bio, A11022-1, 1:500), rabbit polyclonal anti-CBLL1 (Bethyl Laboratories®, A302-968A, 1:1000), mouse monoclonal anti-RBM15 (ProteinTech®, 66059-1-Ig, clone 4A1A4, 1:1000), mouse monoclonal anti-HSP90 (SCBT, sc-13119, clone F-8, 1:10000), rabbit monoclonal anti-GAPDH (CST®, 2118, clone 14C10, 1:10000), polyclonal goat anti-rat IgG/HRP (Thermo Fisher Scientific, 31470, 1:5000), polyclonal goat anti-rabbit IgG/HRP (Agilent, P0448, 1:5000), polyclonal rabbit anti-mouse IgG/HRP (Agilent, P0161, 1:5000).

Techniques: Genome Wide, CRISPR, Knock-Out, Transduction, Selection, Fluorescence, Amplification, Next-Generation Sequencing, Transformation Assay, Control, Western Blot, Expressing, Mutagenesis, Clone Assay, Derivative Assay

Journal: bioRxiv

Article Title: Centrosome architecture and m6A-dependent gating of p53 surveillance after whole-genome doubling

doi: 10.64898/2026.02.25.707964

Figure Lengend Snippet: (A) Waterfall plot of the genome-wide CRISPR knockout screen showing ranked gene enrichment based on MAGeCK analysis of sgRNA representation in the mScarlet-low population following ZM treatment. The seven core components of the m6A writer complex ( METTL3, METTL14, WTAP, VIRMA, ZC3H13, RBM15 , and CBLL1 ) are highlighted. (B) sgRNA-level log 2 fold-change (LFC) values for each m6A writer complex gene identified in the CRISPR screen. Each point represents an individual sgRNA from the Brunello library targeting the indicated gene. NTC: non-targeting control. (C) Immunoblot analysis of Cal51 cells following individual knockout of each m6A writer complex component and ZM treatment. CRADD (RAIDD) knockout is shown as a positive control for suppression of pathway output. (D) Flow cytometry analysis of mScarlet fluorescence in Cal51 mScarlet-MDM2 cells following knockout of the indicated m6A writer components and ZM treatment. Bars represent mean ± standard deviation (N = 3 independent replicates). (E) Immunoblot analysis of METTL3-deficient cells reconstituted with wild-type (WT) or catalytic-dead METTL3 (DPPW→APPA) following ZM treatment. (F) Dose-response curves for four chemically distinct METTL3 inhibitors based on nuclear mScarlet fluorescence measured in Cal51 mScarlet-MDM2 cells following ZM treatment. IC 50 values were estimated for each compound. (G) Immunoblot analysis of Cal51 cells treated with ZM and the indicated METTL3 inhibitors (each at 2.5 µM). (H) Global m6A levels measured by ELISA on poly(A)-enriched mRNA isolated from Cal51 cells following treatment with METTL3 inhibitors. Box plots display medians (horizontal lines), the interquartile range (grey boxes), maximum-to-minimum range (whiskers) and individual data points (N = 5 independent replicates); m6A content is normalized to vehicle-treated cells. (I-J) RT–qPCR analysis of p53 (I) and MDM2 (J) transcript levels in Cal51 cells under basal conditions and following ZM treatment, in the presence or absence of METTL3 inhibition. Transcript levels are shown relative to the untreated condition. One-way ANOVA test. (K) Immunoblot analysis corresponding to the samples shown in (I–J).

Article Snippet: The following antibodies were used: rat monoclonal anti-Caspase-2 (a gift from Dr. Andreas Strasser, Walter and Eliza Hall Institute of Medical Research, clone 11B4, 1:1000), mouse monoclonal anti-MDM2 (Thermo Fisher Scientific, MA1-113, clone IF2, 1:1000), rabbit monoclonal anti-V5-tag (CST®, 13202, clone D3H8Q, 1:1000), rabbit polyclonal anti-p53 (CST®, 9282, 1:1000), mouse monoclonal anti-MCL1 (SCBT, sc-12756, clone 22, 1:500), rabbit polyclonal anti-CEP83 (Atlas antibodies, HPA038161, 1:1000), rabbit polyclonal anti-PARP1 (CST®, 9542, 1:1000), rabbit polyclonal anti-Caspase-3 (CST®, 9662, 1:1000), rabbit polyclonal anti-Caspase-7 (CST®, 9492, 1:1000), mouse monoclonal anti-CDC27 (BD Biosciences, 610455, clone 35/CDC27, 1:1000), mouse monoclonal anti-p21 (BD Biosciences 564262, clone 2G12, 1:1000), rabbit monoclonal anti-METTL3 (CST®, 96391, clone D2I6O, 1:1000), rabbit polyclonal anti-METTL14 (ProteinTech®, 26158-1-AP, 1:1000), rabbit polyclonal anti-CRADD/RAIDD (ProteinTech®, 10401-1-AP, 1:1000), rabbit monoclonal anti-VIRMA (CST®, 88358, clone D4N8B, 1:1000), rabbit polyclonal anti-WTAP (CST®, 56501, 1:1000), rabbit polyclonal anti-ZC3H13 (Boster Bio, A11022-1, 1:500), rabbit polyclonal anti-CBLL1 (Bethyl Laboratories®, A302-968A, 1:1000), mouse monoclonal anti-RBM15 (ProteinTech®, 66059-1-Ig, clone 4A1A4, 1:1000), mouse monoclonal anti-HSP90 (SCBT, sc-13119, clone F-8, 1:10000), rabbit monoclonal anti-GAPDH (CST®, 2118, clone 14C10, 1:10000), polyclonal goat anti-rat IgG/HRP (Thermo Fisher Scientific, 31470, 1:5000), polyclonal goat anti-rabbit IgG/HRP (Agilent, P0448, 1:5000), polyclonal rabbit anti-mouse IgG/HRP (Agilent, P0161, 1:5000).

Techniques: Genome Wide, CRISPR, Knock-Out, Control, Western Blot, Positive Control, Flow Cytometry, Fluorescence, Standard Deviation, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR, Inhibition

Journal: bioRxiv

Article Title: Centrosome architecture and m6A-dependent gating of p53 surveillance after whole-genome doubling

doi: 10.64898/2026.02.25.707964

Figure Lengend Snippet: (A) Immunoblot analysis of RPE1 cells of the indicated genotypes following ZM treatment. (B) Immunofluorescence analysis of PIDD1 localization in RPE1 cells of the indicated genotypes following ZM treatment. Magnified insets of boxed regions (without the DNA channel) are shown. Scale bar: 5 μm. (C) Ultrastructure expansion microscopy (U-ExM) coupled with Structured Illumination Microscopy (SIM) was used to analyze distal (CEP83-positive, top panels) and subdistal (CEP128-positive, bottom panels) appendage organization in RPE1 cells of the indicated genotypes. Representative images are shown. Scale bar: 500 nm. (D) U-ExM/SIM analysis of centrosome spatial organization in ZM-treated RPE1 cells. Mature mother centrioles were visualized by CEP83 staining. Representative images illustrate tightly clustered or spatially separated CEP83-positive centrioles within centrosome assemblies. Scale bar: 500 nm. (E) Quantification of centrosome clustering in ZM-treated RPE1 cells of the indicated genotypes. The three-dimensional distance between the two closest CEP83-positive distal appendage signals was measured. Each small dot represents a single cell measurement; same-symbol colors distinguish biological replicates. The mean of each biological replicate is reported (larger dots) ± standard deviation. N = 4 biological replicates, n = 30 cells per condition; 120 cells total. Mann-Whitney test. (F) Proposed model for multi-layered regulation of p53 surveillance following whole-genome doubling (WGD). WGD is associated with the presence of supernumerary centrosomes that engage a centrosome-based surveillance pathway. At an upstream structural layer, subdistal appendage-dependent centrosome architecture is associated with a spatial organization of centrosomes permissive for Caspase-2 activation. Caspase-2-mediated cleavage of MDM2 alters the p53-MDM2 circuit, converting negative feedback into positive feedback and amplifying p53 signaling. At a downstream competence layer, the m6A writer complex modulates the ability of this circuit to sustain pathway output independently of Caspase-2 activation.

Article Snippet: The following antibodies were used: rat monoclonal anti-Caspase-2 (a gift from Dr. Andreas Strasser, Walter and Eliza Hall Institute of Medical Research, clone 11B4, 1:1000), mouse monoclonal anti-MDM2 (Thermo Fisher Scientific, MA1-113, clone IF2, 1:1000), rabbit monoclonal anti-V5-tag (CST®, 13202, clone D3H8Q, 1:1000), rabbit polyclonal anti-p53 (CST®, 9282, 1:1000), mouse monoclonal anti-MCL1 (SCBT, sc-12756, clone 22, 1:500), rabbit polyclonal anti-CEP83 (Atlas antibodies, HPA038161, 1:1000), rabbit polyclonal anti-PARP1 (CST®, 9542, 1:1000), rabbit polyclonal anti-Caspase-3 (CST®, 9662, 1:1000), rabbit polyclonal anti-Caspase-7 (CST®, 9492, 1:1000), mouse monoclonal anti-CDC27 (BD Biosciences, 610455, clone 35/CDC27, 1:1000), mouse monoclonal anti-p21 (BD Biosciences 564262, clone 2G12, 1:1000), rabbit monoclonal anti-METTL3 (CST®, 96391, clone D2I6O, 1:1000), rabbit polyclonal anti-METTL14 (ProteinTech®, 26158-1-AP, 1:1000), rabbit polyclonal anti-CRADD/RAIDD (ProteinTech®, 10401-1-AP, 1:1000), rabbit monoclonal anti-VIRMA (CST®, 88358, clone D4N8B, 1:1000), rabbit polyclonal anti-WTAP (CST®, 56501, 1:1000), rabbit polyclonal anti-ZC3H13 (Boster Bio, A11022-1, 1:500), rabbit polyclonal anti-CBLL1 (Bethyl Laboratories®, A302-968A, 1:1000), mouse monoclonal anti-RBM15 (ProteinTech®, 66059-1-Ig, clone 4A1A4, 1:1000), mouse monoclonal anti-HSP90 (SCBT, sc-13119, clone F-8, 1:10000), rabbit monoclonal anti-GAPDH (CST®, 2118, clone 14C10, 1:10000), polyclonal goat anti-rat IgG/HRP (Thermo Fisher Scientific, 31470, 1:5000), polyclonal goat anti-rabbit IgG/HRP (Agilent, P0448, 1:5000), polyclonal rabbit anti-mouse IgG/HRP (Agilent, P0161, 1:5000).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Single Cell, Standard Deviation, MANN-WHITNEY, Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Caspase-2 inhibits mitochondrial respiration in colorectal adenocarcinoma cells

doi: 10.1186/s12964-026-02671-z

Figure Lengend Snippet: Identification of caspase-2-downstream proteins regulating mitochondria-related gene transcription. A Protein–protein interaction network of the predicted transcription factors and caspase-2, based on the STRING database. B Representative immunoblots and relative protein levels of CASP2 and p53 in SW620 WT and SW620 Casp2 −/− cells. C Relative gene expression of Tp53, Sdhb, Timm21, Timm23, Tomm20 , and Tomm40 in SW620 Casp2 . −/− cells transfected with siRNA targeting Tp53 or negative control (NC). D Representative immunoblots and ( E ) relative protein levels of CASP2, p53, SDHA, SDHB, TIMM23, TOMM20, and TOMM40 in SW620 Casp2-KO and SW620 Casp2-KO + shp53 cells. Results are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: anti-caspase-2 mouse monoclonal antibody (Cat. No. 611023; BD Biosciences, San Diego, CA, USA); anti-SDHA (D6J9M) XP® rabbit monoclonal antibody (Cat. No. 11998); anti-SDHB (E3H9Z) XP® rabbit monoclonal antibody (Cat. No. 92649), anti-TIMM23 (E1Q7L) rabbit monoclonal antibody (Cat. No. 34822), anti-GAPDH (14C10) rabbit monoclonal antibody (Cat. No. 2118), and anti-c-Jun rabbit monoclonal antibody (Cat. No. 9165) (all from Cell Signaling Technology, Danvers, MA, USA); anti-TOMM20 rabbit monoclonal antibody (Cat. No. ab186735) and anti-TOMM40 rabbit monoclonal antibody (Cat. No. ab185543) (both from Abcam, Cambridge, UK); anti-TIMM21 mouse polyclonal antibody (Cat. No. PA5-100,224; Invitrogen, Thermo Fisher Scientific); anti-p53 (FL-393) rabbit polyclonal antibody (Cat. No. SC-6243; Santa Cruz Biotechnology, Dallas, TX, USA); anti-JNK/SAPK-1/2 mouse monoclonal antibody (Cat. No. AT-7041; MBL International, Schaumburg, IL, USA).

Techniques: Western Blot, Gene Expression, Transfection, Negative Control