gfp-targeting control sirna  (Merck KGaA)

Journal: Molecular Vision

Article Title: Expression of integrin-linked kinase in the murine lens is consistent with its role in epithelial-mesenchymal transition of lens epithelial cells in vitro

doi:

Figure Lengend Snippet: ILK is induced during LEC differentiation and inhibition of ILK retards expression of EMT markers α-SMA and fibronectin. A : Immunoblot of lysates (20 μg/lane) from immortalized and primary mouse LEC that grew out from dissected lens capsules (0-14 days). Lane 1, rhILK (500 ng); lane 2, immortalized LEC; lane 3, primary LEC, 0 days; lane 4, primary LEC, 7 days; lane 5, primary LEC, 14 days. Lysates were probed for ILK and α-SMA as an indicator of EMT. rhILK (lane 1) was used for protein size comparison, and a probe for GAPDH was used to normalize protein loading. B : Densitometry of protein bands from the blot shown in A . Relative expression values are normalized to those of GAPDH. C : Immunoblot of lysates (2 capsules/lane) from primary mouse LEC that grew out from dissected lens capsules (0-14 days). Lane 1, primary LEC + ILK-targeting siRNA, 7 days; lane 2, primary LEC + ILK-targeting siRNA, 14 days; lane 3, primary LEC + GFP-targeting siRNA (control), 7 days; lane 4, primary LEC + GFP-targeting siRNA. Lysates were probed for ILK to verify ILK-targeting siRNA efficiency. Fibronectin and α-SMA were monitored as indicators of EMT. An additional probe for GAPDH was used to normalize protein loading. D : Densitometry of protein bands from the blot shown in C . ILK- and GFP-siRNA-treated explants show a significant difference in protein expression after 14 days of culture (the asterisk indicates a p<0.02). Relative values are normalized to that of GAPDH.

Article Snippet: Six μl of HiPerFect (Qiagen) was incubated with either GFP-targeting siRNA (control) or an equimolar mixture of 3 ILK-targeting siRNA constructs (#SI01076047, #SI01076054, and #SI01076061; Qiagen) in serum-free DMEM, according to the manufacturer's protocol.

Techniques: Inhibition, Expressing, Western Blot

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