qb3 macrolab  (Addgene inc)

Journal: eLife

Article Title: Molecular determinants of phase separation for Drosophila DNA replication licensing factors

doi: 10.7554/eLife.70535

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , 2Cc-T , QB3 Macrolab (UC Berkeley) , RRID: Addgene_37237 , Ligation independent cloning (LIC); E. coli expression vector.

Techniques: Recombinant, Ligation, Cloning, Expressing, Plasmid Preparation

Journal: eLife

Article Title: A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons

doi: 10.7554/elife.69795

Figure Lengend Snippet: Figure 4. The AGTR1 Inhibitor olmesartan is neuroprotective in a chemically induced Gaucher disease model. (A) Locomotor tracks of 5 dpf larvae treated 24 hr with 0.2 % DMSO, 500 µM CBE, 10 µM olmesartan, and 500 µM levodopa. The background subtraction method was used to identify and track movement for 5 min duration. (B) Quantification of total distance (in millimeters, mm) travelled during 5 min recordings for each sample group. Drugs were added at the indicated concentrations and incubated for 24 hr before behavioral recording (n = 12–13; *p < 0.05, ***p < 0.001, unpaired t test) (C) Confocal images of TH-immunoreactive DA neurons (red) and 5HT-immunoreactive serotonin neurons (green) in 6dpf larval zebrafish brains after treatments as indicated in (B). (D–E) Quantification of neurons in the demarcated regions as shown in (C). Fluorescent intensity was quantified using ImageJ and normalized against the control (0.2 % DMSO) (n = 8; *p < 0.05, **p < 0.01, unpaired t test).

Article Snippet: 69795 21 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Drosophila melanogaster) UAS- mito- GFP doi: 10.1091/mbc.e05-060526 Gift from the Saxton Lab. Genetic reagent (Morpholino Oligonucleotide) agtr1a Gene Tools LLC agtr1a_MO 0.5 mM (5'- GACG TTGT CCAT TTTG GAGA TTTGT- 3') Genetic reagent (Morpholino Oligonucleotide) agtr1b Gene Tools LLC agtr1b_MO 0.5 mM (5'- TCAT TGCT GATG TTTG GTTC TCCAT- 3') Genetic reagent (PCR Master mix) GoTaq Green Master Mix Promega M7122 Sequence- based reagent Renin Angiotensin Pathway Primers This Paper PCR primers Refer to Supplementary file 1 Sequence- based reagent Nuclear DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- AGAG CGCG ATTG CTGG ATTCAC-3’ Sequence- based reagent Nuclear DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GTCC TTGC AGGT TGGC AAATGG-3’ Sequence- based reagent Mitochondria DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- TTAA AGCC CCGA ATCC AGGTGAGC-3’ Sequence- based reagent Mitochondria DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GAGA TGTT CTCG GGTG TGGGATGG –3’ Sequence- based reagent sgRNA primers for agtr1 This Paper PCR primers Refer to Figure 3—figure supplement 1 Recombinant DNA reagent agtr1a_1b sgRNA plasmid This Paper Agtr1a and agtr1b sgRNA cloned into addgene: 74,009 Recombinant DNA reagent Agtr1a_1b sgRNA scrambled This Paper Agtr1a and agtr1b scrambled sgRNA cloned into addgene: 74,009 Peptide, recombinant protein Cas9- NLS protein UC Berkeley https:// qb3. berkeley. edu/ facility/qb3-macrolab/ Antibody anti- AGTR1 (Rabbit polyclonal) Proteintech 25343–1- AP WB(1:500) IF(1:500) Antibody anti- 5HT (Rabbit polyclonal) Immunostar cat#20,080 IF(1:1000) Antibody anti- TH (Mouse monoclonal) Immunostar cat#22,941 IF(1:500) Antibody Goat anti- Mouse IgG (H + L) CrossAdsorbed Secondary Antibody Thermofisher Alexa Fluor 488 (cat# A- 11001) IF(1:500) Antibody Goat anti- Rabbit IgG (H + L) Highly CrossAdsorbed Secondary Antibody, Thermofisher Alexa Fluor 568 (cat# A- 11036) IF(1:1000) Antibody chicken anti- GFP Abcam ab92456 IF(1:500) Antibody rabbit anti- TH Pel- Freez P41301 IF(1:1000) Antibody Goat Anti- Rabbit IgG H&L Horseradish Peroxidase conjugated antibody Abcam ab6721 WB(1:1000) Continued on next page Continued Kim et al. eLife 2021;0:e69795.

Techniques: Incubation, Control

Journal: eLife

Article Title: A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons

doi: 10.7554/elife.69795

Figure Lengend Snippet: Figure 5. DA neuron-specific RNA-seq uncovers neurotoxic insult-induced alterations of mitochondrial pathway gene expression that is in part restored by the AGTR1 inhibitor olmesartan. (A) A schematic showing the RNA-seq procedure of larval samples from chemical treatment to FACs, library preparation, and differential gene expression analysis. (B) A heatmap of clustering analysis comparing the differential gene expression in DMSO control, olmesartan, CBE, MTZ, MTZ+ olmesartan, and CBE+ olmesartan treatment groups. Gene counts were normalized and analyzed with the R program DESeq2 package. All samples are numbered 1, 2, and 3 to indicate biological replicates. (C–D) Venn diagrams showing the overlapping gene expression alterations between different conditions: MTZ/control and CBE/control (C) and MTZ+ Olmesartan/MTZ and CBE+ Olmesartan/CBE (D) (α = 0.05, FDR

Article Snippet: 69795 21 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Drosophila melanogaster) UAS- mito- GFP doi: 10.1091/mbc.e05-060526 Gift from the Saxton Lab. Genetic reagent (Morpholino Oligonucleotide) agtr1a Gene Tools LLC agtr1a_MO 0.5 mM (5'- GACG TTGT CCAT TTTG GAGA TTTGT- 3') Genetic reagent (Morpholino Oligonucleotide) agtr1b Gene Tools LLC agtr1b_MO 0.5 mM (5'- TCAT TGCT GATG TTTG GTTC TCCAT- 3') Genetic reagent (PCR Master mix) GoTaq Green Master Mix Promega M7122 Sequence- based reagent Renin Angiotensin Pathway Primers This Paper PCR primers Refer to Supplementary file 1 Sequence- based reagent Nuclear DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- AGAG CGCG ATTG CTGG ATTCAC-3’ Sequence- based reagent Nuclear DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GTCC TTGC AGGT TGGC AAATGG-3’ Sequence- based reagent Mitochondria DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- TTAA AGCC CCGA ATCC AGGTGAGC-3’ Sequence- based reagent Mitochondria DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GAGA TGTT CTCG GGTG TGGGATGG –3’ Sequence- based reagent sgRNA primers for agtr1 This Paper PCR primers Refer to Figure 3—figure supplement 1 Recombinant DNA reagent agtr1a_1b sgRNA plasmid This Paper Agtr1a and agtr1b sgRNA cloned into addgene: 74,009 Recombinant DNA reagent Agtr1a_1b sgRNA scrambled This Paper Agtr1a and agtr1b scrambled sgRNA cloned into addgene: 74,009 Peptide, recombinant protein Cas9- NLS protein UC Berkeley https:// qb3. berkeley. edu/ facility/qb3-macrolab/ Antibody anti- AGTR1 (Rabbit polyclonal) Proteintech 25343–1- AP WB(1:500) IF(1:500) Antibody anti- 5HT (Rabbit polyclonal) Immunostar cat#20,080 IF(1:1000) Antibody anti- TH (Mouse monoclonal) Immunostar cat#22,941 IF(1:500) Antibody Goat anti- Mouse IgG (H + L) CrossAdsorbed Secondary Antibody Thermofisher Alexa Fluor 488 (cat# A- 11001) IF(1:500) Antibody Goat anti- Rabbit IgG (H + L) Highly CrossAdsorbed Secondary Antibody, Thermofisher Alexa Fluor 568 (cat# A- 11036) IF(1:1000) Antibody chicken anti- GFP Abcam ab92456 IF(1:500) Antibody rabbit anti- TH Pel- Freez P41301 IF(1:1000) Antibody Goat Anti- Rabbit IgG H&L Horseradish Peroxidase conjugated antibody Abcam ab6721 WB(1:1000) Continued on next page Continued Kim et al. eLife 2021;0:e69795.

Techniques: RNA Sequencing, Gene Expression, Control

Journal: eLife

Article Title: A zebrafish screen reveals Renin-angiotensin system inhibitors as neuroprotective via mitochondrial restoration in dopamine neurons

doi: 10.7554/elife.69795

Figure Lengend Snippet: Figure 6. The AGTR1 inhibitor olmesartan significantly rescues phenotypes in the Drosophila pink1 mutant model. (A–F) images show the abnormal wing posture (B) and thoracic indentation (E) in the mutant compared to wild-type siblings (A,D). Quantification of %mutant individuals with abnormal wing posture (C) and thoracic indentation (F) showed a significant difference between vehicle- and drug (olmesartan)-treated samples. (G–J) Effect of olmesartan on the mitochondrial aggregation and DA neuron loss phenotypes of pink1 mutant, in comparison to DMSO control. Mitochondria are labeled with mito-GFP reporter. Data quantification shown in I, J. (n = 12; **, p < 0.01, *** p < 0.001, unpaired t-test).

Article Snippet: 69795 21 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Drosophila melanogaster) UAS- mito- GFP doi: 10.1091/mbc.e05-060526 Gift from the Saxton Lab. Genetic reagent (Morpholino Oligonucleotide) agtr1a Gene Tools LLC agtr1a_MO 0.5 mM (5'- GACG TTGT CCAT TTTG GAGA TTTGT- 3') Genetic reagent (Morpholino Oligonucleotide) agtr1b Gene Tools LLC agtr1b_MO 0.5 mM (5'- TCAT TGCT GATG TTTG GTTC TCCAT- 3') Genetic reagent (PCR Master mix) GoTaq Green Master Mix Promega M7122 Sequence- based reagent Renin Angiotensin Pathway Primers This Paper PCR primers Refer to Supplementary file 1 Sequence- based reagent Nuclear DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- AGAG CGCG ATTG CTGG ATTCAC-3’ Sequence- based reagent Nuclear DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GTCC TTGC AGGT TGGC AAATGG-3’ Sequence- based reagent Mitochondria DNA_F doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- TTAA AGCC CCGA ATCC AGGTGAGC-3’ Sequence- based reagent Mitochondria DNA_R doi: 10.1016/j. ymeth.2010.01.033 PCR primers 5’- GAGA TGTT CTCG GGTG TGGGATGG –3’ Sequence- based reagent sgRNA primers for agtr1 This Paper PCR primers Refer to Figure 3—figure supplement 1 Recombinant DNA reagent agtr1a_1b sgRNA plasmid This Paper Agtr1a and agtr1b sgRNA cloned into addgene: 74,009 Recombinant DNA reagent Agtr1a_1b sgRNA scrambled This Paper Agtr1a and agtr1b scrambled sgRNA cloned into addgene: 74,009 Peptide, recombinant protein Cas9- NLS protein UC Berkeley https:// qb3. berkeley. edu/ facility/qb3-macrolab/ Antibody anti- AGTR1 (Rabbit polyclonal) Proteintech 25343–1- AP WB(1:500) IF(1:500) Antibody anti- 5HT (Rabbit polyclonal) Immunostar cat#20,080 IF(1:1000) Antibody anti- TH (Mouse monoclonal) Immunostar cat#22,941 IF(1:500) Antibody Goat anti- Mouse IgG (H + L) CrossAdsorbed Secondary Antibody Thermofisher Alexa Fluor 488 (cat# A- 11001) IF(1:500) Antibody Goat anti- Rabbit IgG (H + L) Highly CrossAdsorbed Secondary Antibody, Thermofisher Alexa Fluor 568 (cat# A- 11036) IF(1:1000) Antibody chicken anti- GFP Abcam ab92456 IF(1:500) Antibody rabbit anti- TH Pel- Freez P41301 IF(1:1000) Antibody Goat Anti- Rabbit IgG H&L Horseradish Peroxidase conjugated antibody Abcam ab6721 WB(1:1000) Continued on next page Continued Kim et al. eLife 2021;0:e69795.

Techniques: Mutagenesis, Comparison, Control, Labeling