Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: C -terminal domain truncations affect TDP-43 aggregate formation in the cytoplasm of SH-SY5Y cells: ( A ) A schematic representation of mKO2-tagged constructs and their abbreviations. Only TDPwt has nuclear localization signal (NLS), whereas others lack NLS and except dNLS that holds intact C-terminus, they carry deletions of LCD: IDR2 is deleted in dNLSd343 (ends at 343 aa residue), IDR2 and CR are deleted in dNLSd299 (ends at 299 aa residue); whole C -terminal domain is deleted in dNLSd267 (ends in 267 aa residues). A control construct KO2only holds sequence for mKO2 protein alone. ( B ) Western blot analysis of mKO2-tagged constructs. ( C ) Quantification of transfected cells harboring aggregates. ( D ) Average number of aggregates in an individual cell. ( E ) Average aggregate size within individual cells. ( F ) SH-SY5Y cells, expressing mKO2-tagged constructs. Nuclei are counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Construct, Residue, Control, Sequencing, Western Blot, Transfection, Expressing

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut hnRNPA1. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut hnRNPA1. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut MATR3. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut MATR3. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut VCP. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut VCP. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut UBQLN2. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut UBQLN2. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: Example of practical brightness quantification using the T2A peptide linker. Cells expressing either yoeCFP-T2A-mCherry or mTq2-T2A-mCherry were grown to midlog and visualized using a widefield microscope. yoeCFP shows a low brightness compared to mCherry. In contrast, mTq2 shows a higher brightness than mCherry. Calibration bar indicates the ratio value when dividing the CFP by the RFP channel (i.e. the relative brightness to mCherry).

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Expressing, Microscopy

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: In vivo brightness and photostability of FPs. A) In vivo brightness of FPs measured by normalizing the fluorescence intensity of single-cells expressing FP-T2A-FP and dividing the fluorescence of the FP of interest to the control FP (either mTq2 or mCherry). B) Photostability of FPs. Per FP, a time-lapse movie was recorded and the photostability was measured as the fluorescent fraction of the last time frame compared to the first frame. Dots represent relative brightness or photostability of an individual cell, boxes indicate median with quartiles, whiskers indicate the 0.05–0.95 fraction of the datapoints. C) Overview of the brightness and photostability of all characterised FPs. D) Coefficient of variation (CV) of the mean brightness of 3 days as an indication of day-to-day variation.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: In Vivo, Fluorescence, Expressing

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: pH sensitivity of FPs. A) Yeast cells were incubated for 2 hours in citric-acid/Na 2 HPO 4 buffers set at pH 3-8 with 2 mM 2,4-DNP and fluorescence was measured using a fluorescent plate reader. Per FP, at least 3 technical replicates were measured. Afterwards, fluorescence was normalized to the pH giving the highest fluorescence and a Hillfit was performed to determine the hill coefficient and pKa value, plotted at the y- and x-axis, respectively. B) mTq2 is an example of an FP that shows different pH sensitivity. pH calibration in vitro was performed using purified proteins in a Citric Acid – Sodium Citrate buffer (pH 3 – 5.4) and a NaH 2 PO 4 /Na 2 HPO 4 0.1 M buffer (pH 5.9-8). Dots represent mean of at least 3 replicates, error bars indicate SD. C) pH curve of sYFP2 which shows an offset (fluorescence plateau) at low pH. This offset gives different values of the pKa (red point, which is the pH that gives a 50% decrease between 1 and the offset) and the pH 50% which gives an absolute 50% decrease in fluorescence (blue point). Dots represent mean of at least 3 replicates, error bars indicate SD.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Incubation, Fluorescence, In Vitro, Purification