plko 1 control shrna vector targeting gfp  (Addgene inc)

Journal: Disease Models & Mechanisms

Article Title: A common cellular response to broad splicing perturbations is characterized by metabolic transcript downregulation driven by the Mdm2–p53 axis

doi: 10.1242/dmm.050356

Figure Lengend Snippet: Dosage-dependent alternative splicing of Mdm2 following Eftud2 KD. (A) Plotted are levels of knocked down Eftud2 ( Eftud2 KD) as assayed by qPCR for two independent primer pairs for Eftud2, after using two independent Eftud2- targeting shRNAs (shRNA1 or shRNA2) or nt-shRNA as control. Bar graphs show 2 −ΔΔCt , representing Eftud2 KD compared to Eftud2 levels after control treatment. Each biological replicate of shRNA KD was compaired pairwise to the corresponding nt-shRNA biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. Error bars represent ±standard deviation of biological replicates. Significance values were determined using Student's t -test on paired ΔCt values. ΔCt values were calculated by normalizing raw values to mitochondrial rRNA levels. ** P <0.01. (B) Western blots of mESC lysates, showing levels of EFTUD2 and p53 after treatment with shRNA1 or shRNA2 to knock down Eftud2 or with control nt-shRNA; levels of β-tubulin were used as loading control. Relative intensity values, standardized to those of β-tubulin and normalized to nt-shRNA, are listed below each protein band for both EFTUD2 and p53. (C) Summary of significant [false discovery rate (FDR)<0.01] alternative splicing events observed in response to shRNAs targeting Eftud2, calculated by using the rMATS tool. (D,E) Plotted are percent-spliced-in (PSI) of skipped-exon events detected by rMATs in mESCs treated with either shRNA1 (D) or shRNA1 (E) ( x -axis) to knock down Eftud2 , or with control nt-shRNA ( y -axis). Plotted points represent skipped-exon events present in response to either KD, with a higher number of skipped-exon events in shRNA2 than in shRNA1. Darker colors indicate more-significant P -values, NS, not significant. Both Mdm2 skipped-exon events were skipping Exon 3, defined as separate events by rMATS due to slight differences in the upstream exon. (F) Exon-skipping frequency ((defined as 1 minus the percent-spliced-in value calculated by rMATS) of Mdm2 Exon 3 skipping event in mESCs treated with shRNA1 or shRNA2 to knock down Eftud2 , or with nt-shRNA (control). Significance values were determined by rMATS with a likelihood-ratio test. **** P <0.0001. (G) Schematic of alternative Mdm2 splicing events of interest (top) and their resulting protein products MDM2-p90 and MDM2-p76 (middle and bottom, respectively). Numbered boxes indicate exons. Blue diagonal lines connecting exons 2 and 4 indicate the exon-skipping event. Green bent arrows indicate start codons. Skipping of Exon 3 gives rise to an MDM2 isoform (i.e. MDM2-p76) that lacks the complete p53-binding domain. p53-BD, p53-binding domain; Acidic, acidic domain; Zn, Zinc finger domain; RING, really interesting new gene (RING) finger domain. Drawings are not to scale.

Article Snippet: shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027).

Techniques: Alternative Splicing, shRNA, Control, Standard Deviation, Western Blot, Knockdown, Binding Assay

Journal: Disease Models & Mechanisms

Article Title: A common cellular response to broad splicing perturbations is characterized by metabolic transcript downregulation driven by the Mdm2–p53 axis

doi: 10.1242/dmm.050356

Figure Lengend Snippet: Mdm2 alternative splicing occurs in response to various splicing perturbations. (A) Top: Schematic of the region surrounding the Mdm2 alternative splicing event. Black arrows represent primer binding sites. Green bent arrows represent start codons. Blue diagonal lines connecting exons 2 and 4 indicate exon-skipping events. Bottom: Agarose gel of semi-quantitative reverse transcription PCR (sqRT-PCR) to analyze alternative splicing of Mdm2 in response to individual shRNA knockdown (KD) of splicing factors Sf3b4, Txnl4a or Prpf8. Approximate size of sqRT-PCR products is as indicated (∼200 bp, ∼100 bp). ΔE3 indicates the product resulting from Mdm2 exon 3 skipping. (B) Western blotting of mESC lysates. mESCs were infected with different shRNAs specifically targeting splicing factors Eftud2 , Sf3b4 , Txnl4a or Prpf8 , or with nt-shRNA (control) as indicated. Bands show levels of p53 (top); β-tubulin (bottom) was used as loading control. Relative intensity ( rel. intensity ) values standardized to β-tubulin and normalized to nt-shRNA are provided under each band. (C) Representative sqRT-PCR (top) of Mdm2 alternative splicing in response to 4 h of TG003 treatment, and corresponding quantification of sqRT-PCR band intensities (bottom). Error bars represent ±standard deviation of two biological replicates. (D) Western blot of p53 in response to TG003 treatment; β-tubulin was used as loading control. (E) sqRT-PCR of Mdm2 alternatively spliced in response to 4 h-treatment with either Doxorubicin (left panels) or Nutlin-3a (right panels). The corresponding quantification of Mdm2 sqRT-PCR band intensities is plotted below.

Article Snippet: shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027).

Techniques: Alternative Splicing, Binding Assay, Agarose Gel Electrophoresis, Reverse Transcription, shRNA, Knockdown, Western Blot, Infection, Control, Standard Deviation

Journal: Disease Models & Mechanisms

Article Title: A common cellular response to broad splicing perturbations is characterized by metabolic transcript downregulation driven by the Mdm2–p53 axis

doi: 10.1242/dmm.050356

Figure Lengend Snippet: RNA-seq reveals metabolic transcript downregulation in Eftud2 KD mESCs. (A,D) Over-representation analysis of 1000 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway genes, comparing those most significantly upregulated (A) or downregulated (D) under shRNA2 knockdown ( KD) and under nt-shRNA. (B,C) Differential expression of all genes analyzed in A and D, comparing the effect of shRNA2 (B) or of shRNA1 (C) with that of nt-shRNA as assayed by DESeq2. Blue, genes involved in hexose sugar metabolism (Glycolysis); red, genes involved in sterol biosynthesis (Sterol); yellow, p53 transcriptional targets (p53 responsive). Components of gene sets and their compilation are described under Compilation of gene set lists in Materials and Methods. (E) Plotted is the log2-fold change of all glycolytic genes significantly differentially expressed in under both shRNA1 and shRNA2. (F) Quantification of qPCR analysis. Plotted is the downregulation of select glycolytic gene transcripts as indicated, following Eftud2 KD in p53-expressing and p53-null cell lines. Bar graphs show 2 −ΔΔCt , representing KD relative to control nt-shRNA in the given p53 status (denoted by the dotted line). Each biological replicate of shRNA KD was compaired pairwise to the corresponding nt-shRNA biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. Error bars represent ±standard deviation of biological replicates. Significance values determined by Student's t -test on ΔΔCt values. ΔCt values calculated by normalizing raw values to mitochondrial rRNA levels. * P <0.05, ** P <0.01.

Article Snippet: shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027).

Techniques: RNA Sequencing, Knockdown, shRNA, Quantitative Proteomics, Expressing, Control, Standard Deviation

Journal: Disease Models & Mechanisms

Article Title: A common cellular response to broad splicing perturbations is characterized by metabolic transcript downregulation driven by the Mdm2–p53 axis

doi: 10.1242/dmm.050356

Figure Lengend Snippet: Select glycolytic transcripts are downregulated under splicing perturbations. (A) Quantification of qPCR of select glycolytic genes ( Hk1 , Pfkfb3 , Pfkm ) in mESCs infected with the stated shRNA. Bar graphs show 2 −ΔΔCt , representing knockdown (KD) relative to control nt-shRNA treatment. Each biological replicate of shRNA KD was compaired pairwise to the corresponding nt-shRNA biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. (B) Quantification of qPCR of select glycolytic genes in mESCs infected with Sf3b4 shRNA, in p53-expressing (p53 Proficient) and p53-null mESC lines. Bar graphs show 2 −ΔΔCt , representing KD relative to control nt-shRNA treatment in the given p53 status (denoted by dotted line). Each biological replicate of shRNA KD was compaired pairwise to the corresponding nt-shRNA biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. (C) Quantification of qPCR of select glycolytic genes in mESCs treated with 100 µM TG003 for 3 days. Each biological replicate of TG003 treatment was compaired pairwise to the corresponding DMSO treatment biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. (D) Quantification of qPCR of select glycolytic genes in mESCs treated with 100 µM TG003 for 3 days, in p53-expressing (p53 Proficient) and p53-null cell lines. Bar graphs show 2 −ΔΔCt , representing KD relative to control DMSO treatment in the given p53 status (denoted by dotted line). Each biological replicate of TG003 treatment was compaired pairwise to the corresponding DMSO treatment biological replicate to calculate KD levels, and each biological replicate is the average of three technical replicates. Error bars represent ± standard deviation of biological replicates. Significance values determined by Student's t -test on paired ΔCt (A, C) or ΔΔCt (B, D) values. ΔCt values calculated by normalizing raw values to mitochondrial rRNA levels. * P <0.05, ** P <0.01, *** P <0.001; ∼, not significant.

Article Snippet: shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027).

Techniques: Infection, shRNA, Knockdown, Control, Expressing, Standard Deviation

Journal: Nature communications

Article Title: Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

doi: 10.1038/s41467-021-21938-2

Figure Lengend Snippet: Fig. 3 FTH1 as a microtubule-stabilizing factor controlled by iron and isocitrate. a Levels of major microtubule-associated proteins (MAPs) in erythroid cells cultured from human CD34+ progenitors, from proteomic data of Gautier18. Graph: mean protein copy number per cell for five most abundant MAPs at indicated stages of differentiation. Red: Stathmin 1 (STMN1); green: ferritin heavy chain (FTH1); blue: microtubule-associated protein RP/EB family member 1 (MAPRE1); grey: microtubule-associated protein RP/EB family member 2 (MAPRE2); black: regulator of microtubule dynamics protein 1 (RMDN1); purple: microtubule-associated protein 4 (MAP4). Stages: progenitor 1 (Prog1), progenitor 2 (Prog2), proerythroblast (ProE), basophilic erythroblast 1 (Baso1), basophilic erythroblast 2 (Baso2), polychromatophilic erythroblast (Poly), and orthchromatic erythroblast (Ortho). Error bars, SEM; n = 3 biologically independent replicates for Prog1 and Prog2, and four biologically independent replicates for all other stages. b FTH1 levels by immunoblot on whole cell lysates from human CD34+ progenitors cultured 1–3 days in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium ± isocitrate (IC). Red arrow: lysosomal proteolytic fragment. Graph: mean normalized FTH1 signal (day 2), relative to value with 100% TSAT. Error bars, SEM; n = 3 independent experiments; ***, ****P = 0.0008, 0.0003, one-way ANOVA with Tukey post hoc. c FTH1 levels by immunoblot on human CD34+ progenitors cultured 1–3 days in iron-replete or deficient erythroid medium ± protease inhibitor (5 µM CA074-me). Red arrow: proteolytic fragment. Graph: mean normalized FTH1 signal (day 2), relative to value with 100% TSAT. Error bars, SEM; n = 3 independent experiments; *, **, ***P = 0.028, 0.013, 0.0006, one-way ANOVA with Tukey post hoc. d FTH1 knockdown with lentiviral shRNA. Immunoblot of transduced human CD34+ progenitors cultured 3 days in iron-replete erythroid medium. e Microtubule alterations demonstrated by immunofluorescence on cells as in d. Red: β-tubulin; blue: DAPI. Graphs: microtubule (MT) density, reflected as averaged microtubule signal per cell in arbitrary units. Numbers indicate mean ± SEM; n = 4 independent experiments for assessment of FTH1#1 and three independent experiments for assessment of FTH1#2; *, ***P = 0.029, 0.0014, unpaired two-sided Student’s t test. Note: most experiments separately assessed FTH1#1 and FTH1#2. See also Supplementary Figs. 5–7 and Supplementary Data Table 1. Source data are provided as a Source data file.

Article Snippet: GFP-targeting control shRNA plasmid, pLKO.1 GFP shRNA, was purchased from Addgene (#30323).

Techniques: Cell Culture, Western Blot, Protease Inhibitor, Knockdown, shRNA

Journal: Nature communications

Article Title: Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

doi: 10.1038/s41467-021-21938-2

Figure Lengend Snippet: Fig. 4 Implication of FTH1 in the erythroid iron restriction response. a, b Influence of FTH1 levels on viability and proliferation. Graphs: mean % viable cells and fold increases in cell number for human CD34+ progenitors transduced with lentiviral shRNA vectors targeting either green fluorescent protein (GFP) or ferritin heavy chain (FTH1), and cultured 4 days in iron-replete erythroid medium. Error bars, SEM; n = 3 biologically independent experiments; *, **P = 0.011, 0.009, one-way ANOVA with Tukey post hoc. NS not significant. GPA glycophorin A. c Influence of FTH1 levels on erythroid differentiation as determined by flow cytometry on cells treated as in a. d Mean fold change in GPA+ cells associated with lentiviral transductions and 4 days culture in iron- replete erythroid medium. Error bars, SEM; n = 3 independent experiments; *, **P = 0.019, 0.013, unpaired two-sided Student’s t test. Note: most experiments separately assessed FTH1#1 and FTH1#2. e Distinct consequences of ferritin enforcement in iron-replete versus iron-restricted progenitors. Flow cytometry analysis of CD34+ progenitors transduced with lentiviral (LV) expression vectors for ferritin heavy (FTH1) and light (FTL) chains followed by 4 days culture in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium. f Mean fold change in GPA+ cells associated with ferritin enforcement in cells cultured as in e. Error bars, SEM; n = 3 independent experiments; *P = 0.02, unpaired two-sided Student’s t test. g Ferritin levels in cells transduced and cultured as in e and f, as assessed by immunoblot. LV-OE lentiviral overexpression; Vec vector. Representative results from three independent experiments. See also Supplementary Fig. 8. Source data are provided as a Source data file.

Article Snippet: GFP-targeting control shRNA plasmid, pLKO.1 GFP shRNA, was purchased from Addgene (#30323).

Techniques: Transduction, shRNA, Cell Culture, Cytometry, Flow Cytometry, Expressing, Western Blot, Over Expression, Plasmid Preparation