polyattract® mrna isolation system iv z5310  (Promega)

Journal: RSC Medicinal Chemistry

Article Title: Structure-guided design of a methyltransferase-like 3 (METTL3) proteolysis targeting chimera (PROTAC) incorporating an indole–nicotinamide chemotype †

doi: 10.1039/d5md00359h

Figure Lengend Snippet: Evaluation of AF151 in MOLM-13 cells. (A) Relative METTL3 and METTL14 mRNA levels (ΔΔCt) after treatment with AF151 (2 μM) or STM2457 (10 μM) for 24 h resp. 48 h. (B) Cell viability inhibition after treatment with varying concentrations of AF151 , WD6305, and STM2457. (C) m 6 A quantification of total mRNA isolated from MOLM-13 cells after treatment with AF151 (2 μM) or STM2457 (10 μM) for the indicated durations. (D) Real-time apoptosis assay (homogeneous luminogenic annexin V binding assay). MOLM-13 cells were treated with AF151 at the indicated concentrations, and apoptosis was monitored via luminescence over 48 h. (E) Time course treatment experiment of Bcl-like proteins performed analogously to . Western blot image after treatment with AF151 at 2 μM at the indicated time points. The corresponding time-response plot can be found in . (F) Zero interaction potency (ZIP) analysis of co-treatment (72 h) with AF151 and venetoclax in MOLM-13 cells. Quantification and illustration were generated with the SynergyFinder+ webserver.

Article Snippet: PolyA mRNA was then isolated using the PolyATtract® mRNA Isolation System (Promega). m 6 A levels were quantified using a colorimetric m 6 A RNA methylation Assay Kit (Abcam) as specified in the manufacturer's protocol.

Techniques: Inhibition, Isolation, Apoptosis Assay, Binding Assay, Western Blot, Generated

Journal: Journal of Experimental Botany

Article Title: Impact of m 6 A modification and transcript quantity on mRNA composition in plant stress granules under hypoxia

doi: 10.1093/jxb/eraf046

Figure Lengend Snippet: Localization of poly(A) RNA and stress granules in meristematic cells of lupin roots in response to hypoxia. Poly(A) RNA was detected by fluorescence in situ hybridization (FISH; red) and stress granules (SGs) were detected by immunofluorescence (IF; green) of Poly(A) Binding Protein 2 (PAB2). (A–C) Cells from roots under normoxia, (D–O) after 1–15 h of hypoxia, and (P–R) after 15 h of hypoxia followed by 6 h of reoxygenation. Merged images of the FISH and IF signals together with DAPI staining are shown. The scale bar is 10 µm. Arrowheads indicate the accumulation of poly(A) RNA during hypoxia; note their absence after the removal of stress. (S–U) Quantification of (S) the number of SGs per cell, (T) the size of SGs, and (U) the percentage of the cytoplasmatic pool of poly(A) RNA that was present in the SGs during the hypoxia stress. Significant differences between pairs of means were determined using Student’s t -test: * P <0.05, ** P <0.01.

Article Snippet: For immunoprecipitation of m 6 A-RNA, total RNA was extracted from the meristematic parts of the lupin roots as described above, followed by poly(A) RNA enrichment using PolyATtract ® mRNA Isolation Systems (Promega).

Techniques: Fluorescence, In Situ Hybridization, Immunofluorescence, Binding Assay, Staining

Journal: Journal of Experimental Botany

Article Title: Impact of m 6 A modification and transcript quantity on mRNA composition in plant stress granules under hypoxia

doi: 10.1093/jxb/eraf046

Figure Lengend Snippet: Localization of poly(A) RNA and stress granules in resin sections of meristematic cells of lupin roots in response to hypoxia. Poly(A) RNA was detected by fluorescence in situ hybridization (FISH; red) and stress granules (SGs) were detected by immunofluorescence (IF; green) of Poly(A) Binding Protein 2 (PAB2). Cells after (A–C) 6 h and (D–F) 15 h of hypoxia. Merged images of the FISH and IF signals together with DAPI staining are shown. Arrowheads indicate SGs. The scale bar is 5 µm. (G) Ultrastructure of the bi-zonal form of the SGs; r , ring of coiled, dense fibers; c , central brighter area; er , endoplasmatic reticulum. Scale bar is 3 µm. (H) Stochastic optical reconstruction microscopy (STORM) analysis. The dots represent individual poly(A) RNA molecules that were detected during the acquisition and are color-coded by depth. The dot size in the image is set to 50 nm. The SG structure in the image is the isosurface of the cluster calculated by performing a Cluster analysis on the data set.

Article Snippet: For immunoprecipitation of m 6 A-RNA, total RNA was extracted from the meristematic parts of the lupin roots as described above, followed by poly(A) RNA enrichment using PolyATtract ® mRNA Isolation Systems (Promega).

Techniques: Fluorescence, In Situ Hybridization, Immunofluorescence, Binding Assay, Staining, Microscopy

Journal: Journal of Experimental Botany

Article Title: Impact of m 6 A modification and transcript quantity on mRNA composition in plant stress granules under hypoxia

doi: 10.1093/jxb/eraf046

Figure Lengend Snippet: Localization of ADH1 and RPB1 mRNA and stress granules in meristematic cells of lupin roots in response to hypoxia and subsequent reoxygenation. Seedlings were submerged for 15 h (hypoxia; ‘15h’) after which the stress was removed for 6 h (reoxygenation; ‘15+6h’); no stress was applied to control seedlings (normoxia). Poly(A) RNA was detected by fluorescence in situ hybridization (FISH; red) and stress granules (SGs) were detected by immunofluorescence (IF; green) of Poly(A) Binding Protein 2 (PAB2). Merged images of the signals with DAPI staining are shown. (A–I) Alcohol Dehydrogenase 1 ( ADH1 ), (J–R) the DNA-directed RNA polymerase II subunit ( RPB1 ), and (S–U) the 5´-UTR of RPB1 . Arrowheads indicate SGs. Scale bars are 10 µm. The panels to the right show magnified images of the SG that is marked with a square. Homogenous distribution of RPB1 transcripts in all areas of the SGs was detected by FISH using a probe targeting the middle sequence of mRNA (arrowheads in O), whereas when a probe targeting the 5´-UTR of RPB1 was used transcripts were only visible in the ring of the SGs (arrowheads in U). ADH1 transcripts were present only in the central zone of the SGs (arrowheads in F). (V, W) The relative fluorescence intensities of mRNA of (V) ADH1 and (W) RPB1 in the cytoplasm and SGs during normoxia and hypoxia. Data are means (±SE) of three independent biological replicates. Significant differences between means were determined using Student’s t -test: * P <0.05, ** P <0.01.

Article Snippet: For immunoprecipitation of m 6 A-RNA, total RNA was extracted from the meristematic parts of the lupin roots as described above, followed by poly(A) RNA enrichment using PolyATtract ® mRNA Isolation Systems (Promega).

Techniques: Control, Fluorescence, In Situ Hybridization, Immunofluorescence, Binding Assay, Staining, Sequencing

Journal: Journal of Experimental Botany

Article Title: Impact of m 6 A modification and transcript quantity on mRNA composition in plant stress granules under hypoxia

doi: 10.1093/jxb/eraf046

Figure Lengend Snippet: Localization of poly(A) RNA and m 6 A in meristematic cells of lupin roots in response to hypoxia. Poly(A) RNA was detected by fluorescence in situ hybridization (FISH; red) and m 6 A was detected by immunofluorescence (IF; green). Merged images of the signals with DAPI staining are shown. (A–C) Normoxia conditions (Control) and (D–O) hypoxia conditions for 3–15 h. The panels to the right show magnified images of the stress granule (SG) that is marked with a square. The scale bar is 10 µm. Accumulation of m 6 A in SGs was observed during 3–6 h of hypoxia (arrowheads in F, I) followed by a reduction during 9–15 h (arrowheads in L, O). (P) Ratio of fluorescence signal in SGs compared with the cytoplasm during 3–15 h of hypoxia. (Q) Quantitative measurements of methylation (m 6 A modification) in transcripts of HUP1 , ADH1 , RPB1 , L37 , L44 , WIN1 , and PCO1 in lupin roots subjected to normoxia (control), hypoxia for 15 h, and hypoxia followed by reoxygenation for 6 h. Gene names are listed in full in . Data are means (±SE) of three independent biological replicates. Significant differences between means were determined using Student’s t -test: * P <0.05, ** P <0.01.

Article Snippet: For immunoprecipitation of m 6 A-RNA, total RNA was extracted from the meristematic parts of the lupin roots as described above, followed by poly(A) RNA enrichment using PolyATtract ® mRNA Isolation Systems (Promega).

Techniques: Fluorescence, In Situ Hybridization, Immunofluorescence, Staining, Control, Methylation, Modification

Journal: Journal of Experimental Botany

Article Title: Impact of m 6 A modification and transcript quantity on mRNA composition in plant stress granules under hypoxia

doi: 10.1093/jxb/eraf046

Figure Lengend Snippet: Localization of poly(A) RNA, the m 6 A-binding protein ECT2, and the DNA-directed RNA polymerase protein RBP47b in Arabidopsis roots cells in response to long-term hypoxia. Seedlings were subjected to normoxia (Control) conditions or 3 d of hypoxia (3D). Poly(A) RNA was detected by fluorescence in situ hybridization (FISH; red), and ECT2 and RBP47b were detected by fluorescence of GFP (green). Merged images of the signals with DAPI staining are shown. (A–F) The ECT2::GFP line and (G–L) the mta mutant transformed with the pABI3::MTA construct ( pABI3::MTA-mta ) crossed with the ECT2::GFP line. (M–P) The RBP47b::GFP line and (Q–T) the pABI3::MTA-mta line crossed with the RBP47b::GFP line. Scale bars are 10 µm. (U) The relative level of poly(A) RNA in the stress granules (SGs) of the ECT2::GFP line and the pABI3::MTA-mta × ECT2::GFP cross under hypoxia. (V) The number of SGs per cell in the RBP47b::GFP line and the pABI3::MTA-mta × RBP47b::GFP cross under hypoxia. Data are means (±SE) of three independent biological replicates. Significant differences were determined using Student’s t -test: ** P < 0.01).

Article Snippet: For immunoprecipitation of m 6 A-RNA, total RNA was extracted from the meristematic parts of the lupin roots as described above, followed by poly(A) RNA enrichment using PolyATtract ® mRNA Isolation Systems (Promega).

Techniques: Binding Assay, Control, Fluorescence, In Situ Hybridization, Staining, Mutagenesis, Transformation Assay, Construct