double up  (Addgene inc)

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: Overexpression of CIP4 or FBP17 decreases migration at E14.5+4 days. (a) Representation of the Double UP overexpression plasmid construct used in these experiments. Neurons that are transfected with only the Double UP plasmid express mNeon- green (Cre(-) cell). When limited amounts of Cre are co-transfected into about half the neurons, these neurons express mScarlet (mNeon-green is excised) and unlabeled CIP4 or FBP17 (Cre(+) cell). (b) Schematic of experimental design and quantitation procedure. (c, e, g) Representative images of empty Double UP (c), Double UP + CIP4 (e), and Double UP + FBP17 (g) four days after electroporation at E14.5 (E14.5◊E18.5). CP= cortical plate, SP/IZ= subplate/intermediate zone, SVZ= subventricular zone, ST= striatum, V= lateral ventricle. Scale bar 100µm. (d, f, h) Dot plots of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all labeled neurons in a single coronal brain section. Connected dots indicate measurements were made in the same coronal section. Black diamonds and bars represent cumulative mean ± SEM. Paired t-test (two-tailed). (d) 7 brains, mNeon: mean 295±45.0µm, 1316 cells, mScarlet: 310±44.4µm, 1055 cells. (f) 6 brains, Control: 294±23.0µm, 745 cells, CIP4: 404±9.8µm, 731 cells. (h) 10 brains, Control: 224±21.0µm, 2144 cells, FBP17: 305±20.5µm, 1828 cells. (b) Created in BioRender. English, L. (2024) BioRender.com/u35d157.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Over Expression, Migration, Plasmid Preparation, Construct, Transfection, Quantitation Assay, Electroporation, Labeling, Two Tailed Test, Control

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: CIP4, but not FBP17, overexpression inhibits migration by decreasing number and length of processes. (a, h, o) Representative images of migration after electroporation with Double UP (a) Double UP CIP4 (h) and Double UP FBP17 (o) two days after electroporation at E14.5 (E14.5◊E16.5). CP= cortical plate, SP/IZ= subplate/ intermediate zone, SVZ= subventricular zone, ST= striatum, V= lateral ventricle. Scale bar 100µm. (b, i, p) Dot plots of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all cortical neurons in a coronal brain section. Connected dots indicate measurements were made in the same coronal section. (c, c’, j, j’, q, q’) Maximum projection of green cells (c, j, q) and magenta cells (c’, j’, q’) from Double UP, Double UP CIP4, and Double UP FBP17 displayed with inverted contrast to emphasize morphology. Scale bar 50 µm. (d, k, r) Representative traces of electroporated neurons from each condition to emphasize morphology. (e, l, s) SuperPlots showing comparison of the number of processes from individual green and magenta cells. Each dot represents an individual cell. Triangles represent the mean number of cell processes per brain connected with a line to indicate means are from the same brain. (f, m, t) SuperPlots showing comparison of the length of longest processes from individual green and magenta cells. Each dot represents an individual cell. Triangles represent the mean number of cell processes for per brain connected with a line to indicate means are from the same brain. (g, h, u) Stacked bar graphs of cell polarity percentages, nonpolar= 0 processes, unipolar= 1 process, bipolar= 2 processes, multipolar= 3+ processes. Error bars show SEM. Paired t-test (two-tailed). (b) 6 brains, mNeon: 424±15.3µm, 693 cells, mScarlet: 427±16.2µm, 554 cells. (e) 5 brains, mNeon: 2.9±0.1 processes, 75 cells, mScarlet: 3.4±0.2 processes, 75 cells. (f) 5 brains, mNeon: 29 ±1.4µm, 72 cells, mScarlet: 27±1.4µm, 73 cells. (g) 5 brains, mNeon: nonpolar 0% of cells mean 0.0±0.0 cells, unipolar 6.7% of cells mean 1.0±0.3 cells, bipolar 39% of cells mean 5.8±1.0 cells, multipolar 55% of cells mean 8.2±0.8 cells, n=75 cells. mScarlet: nonpolar 1.3% of cells mean 0.2±0.2 cells, unipolar 4% of cells mean 0.6±0.4 cells, bipolar 28% of cells mean 4.2±1.5 cells, multipolar 67% of cells mean 10±1.8 cells, 75 cells. (i) 7 brains, Control: 386±27.4µm, 860 cells, CIP4: 407±24.4µm, 873 cells. (l) 7 brains, Control: 3.3±0.3 process, 105 cells, CIP4: 1.0±0.2 process, 105 cells. (m) 7 brains, Control: 25±1.2µm, 104 cells, CIP4: 17±1.6µm, 56 cells. (n) 7 brains, Control: nonpolar 0% of cells mean 0.0±0.0 cells, unipolar 11% of cells mean 1.7±0.6 cells, bipolar 18% of cells mean 2.7±0.9 cells, multipolar 70% of cells mean 11±1.3 cells, 105 cells. CIP4: nonpolar 41% of cells mean 6.2±1.6 cells, unipolar 21% of cells mean 3.1±0.6 cells, bipolar 22% of cells mean 3.3±0.9 cells, multipolar 15% of cells mean 2.3±1.2 of cells, 105 cells. (p) 9 brains, Control: 412±17.5µm, 1308 cells, FBP17: 415 ±18.6µm, 832 cells. (s) 7 brains, Control: 2.8±0.1 processes, 105 cells, FBP17: 2.9±0. processes, 105 cells. (t) 7 brains, Control: 21±1µm, 101 cells, FBP17: 19±1.2µm, 85 cells. (u) brain, Control: nonpolar 3.8% of cells mean 0.6±0.4 cells, unipolar 18% of cells mean 2.7±1.5 cells, bipolar 20% of cells mean 3.0±0.7 cells, multipolar 58% of cells mean 8.7±1.9 cells, 105 cells. FBP17: nonpolar 16% of cells mean 2.4±0.9 cells, unipolar 18% of cells mean 2.7±1.6 cells, bipolar 13% of cells mean 2.0±0.7 cells, multipolar 52% of cells mean 7.8±2.2 cells, 105 cells. All values mean±SEM.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Over Expression, Migration, Electroporation, Comparison, Two Tailed Test, Control

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: Knockdown of CIP4 or FBP17 decreases migration at E14.5+4 days. (a) Representation of the Double UP and pSico plasmid constructs used in these experiments. Neurons that are transfected with both the Double UP plasmid and a floxed, inverted PGK- puromycin cassette express only mNeon-green and no shRNA (Control cell). When limited amounts of Cre are co-transfected into about half the population of neurons, these neurons express mScarlet (mNeon is excised) and shRNA (PGK-puro cassette excised) targeting either CIP4 or FBP17 (Knockdown cell). (b) Representative image of Double UP + pSico scramble shRNA four days after electroporation at E14.5 (E14.5◊E18.5). Scale bar 100µm. (c) Dot plot of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all cortical neurons in a coronal brain section. Connected dots indicate measurements were made in the same coronal section. (d) Western blot of HEK293 cells transfected with mouse CIP4 and post-Cre pSico plasmids expressing either scramble or one of two CIP4-targeted shRNAs. (e) Quantification of western blots in (d). (f, h) Representative images of Double UP + pSico CIP4 shRNA1 (f) and shRNA2 (h) four days after electroporation at E14.5 (E14.5◊E18.5). (g, i) Dot plots of migration of green and magenta cells for CIP4 shRNA1 (g) and shRNA2 (i). (j) Western blot of HEK293 cells transfected with mouse FBP17 and post-Cre pSico plasmids expressing either scramble or one of two FBP17-targeted shRNAs. (k) Quantification of western blots (j). (l, n) Representative image of Double UP + pSico FBP17 shRNA1 (l) and shRNA2 (n) four days after electroporation at E14.5 (E14.5◊E18.5). (m, o) Dot plots of migration of green and magenta cells for FBP17 shRNA1 (m) and shRNA2 (o). Black diamonds and bars represent cumulative mean ± SEM. Paired t-test (two-tailed). (c) 10 brains, Control: 214±25.0µm, 2453 cells, scramble: 227±28.0µm, 2142 cells. (g) 6 brains, Control: 324±27.0µm, 1669 cells, CIP4 shRNA1: 386±27.4µm, 1631 cells. (i) 5 brains, Control: 204±20.5µm, 921 cells, CIP4 shRNA2: 243±24.7µm, 798 cells. (m) 7 brains, Control: 254±22.8µm, 959 cells, FBP17 shRNA1: 352±29.1µm, 1095 cells. (o) 5 brains, Control: 182±35.8µm, 1269 cells, FBP17 shRNA2: 207±34.5µm, 1082 cells.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Knockdown, Migration, Plasmid Preparation, Construct, Transfection, shRNA, Control, Electroporation, Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: Overexpression of CIP4 or FBP17 decreases migration at E14.5+4 days. (a) Representation of the Double UP overexpression plasmid construct used in these experiments. Neurons that are transfected with only the Double UP plasmid express mNeon- green (Cre(-) cell). When limited amounts of Cre are co-transfected into about half the neurons, these neurons express mScarlet (mNeon-green is excised) and unlabeled CIP4 or FBP17 (Cre(+) cell). (b) Schematic of experimental design and quantitation procedure. (c, e, g) Representative images of empty Double UP (c), Double UP + CIP4 (e), and Double UP + FBP17 (g) four days after electroporation at E14.5 (E14.5◊E18.5). CP= cortical plate, SP/IZ= subplate/intermediate zone, SVZ= subventricular zone, ST= striatum, V= lateral ventricle. Scale bar 100µm. (d, f, h) Dot plots of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all labeled neurons in a single coronal brain section. Connected dots indicate measurements were made in the same coronal section. Black diamonds and bars represent cumulative mean ± SEM. Paired t-test (two-tailed). (d) 7 brains, mNeon: mean 295±45.0µm, 1316 cells, mScarlet: 310±44.4µm, 1055 cells. (f) 6 brains, Control: 294±23.0µm, 745 cells, CIP4: 404±9.8µm, 731 cells. (h) 10 brains, Control: 224±21.0µm, 2144 cells, FBP17: 305±20.5µm, 1828 cells. (b) Created in BioRender. English, L. (2024) BioRender.com/u35d157.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Over Expression, Migration, Plasmid Preparation, Construct, Transfection, Quantitation Assay, Electroporation, Labeling, Two Tailed Test, Control

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: CIP4, but not FBP17, overexpression inhibits migration by decreasing number and length of processes. (a, h, o) Representative images of migration after electroporation with Double UP (a) Double UP CIP4 (h) and Double UP FBP17 (o) two days after electroporation at E14.5 (E14.5◊E16.5). CP= cortical plate, SP/IZ= subplate/ intermediate zone, SVZ= subventricular zone, ST= striatum, V= lateral ventricle. Scale bar 100µm. (b, i, p) Dot plots of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all cortical neurons in a coronal brain section. Connected dots indicate measurements were made in the same coronal section. (c, c’, j, j’, q, q’) Maximum projection of green cells (c, j, q) and magenta cells (c’, j’, q’) from Double UP, Double UP CIP4, and Double UP FBP17 displayed with inverted contrast to emphasize morphology. Scale bar 50 µm. (d, k, r) Representative traces of electroporated neurons from each condition to emphasize morphology. (e, l, s) SuperPlots showing comparison of the number of processes from individual green and magenta cells. Each dot represents an individual cell. Triangles represent the mean number of cell processes per brain connected with a line to indicate means are from the same brain. (f, m, t) SuperPlots showing comparison of the length of longest processes from individual green and magenta cells. Each dot represents an individual cell. Triangles represent the mean number of cell processes for per brain connected with a line to indicate means are from the same brain. (g, h, u) Stacked bar graphs of cell polarity percentages, nonpolar= 0 processes, unipolar= 1 process, bipolar= 2 processes, multipolar= 3+ processes. Error bars show SEM. Paired t-test (two-tailed). (b) 6 brains, mNeon: 424±15.3µm, 693 cells, mScarlet: 427±16.2µm, 554 cells. (e) 5 brains, mNeon: 2.9±0.1 processes, 75 cells, mScarlet: 3.4±0.2 processes, 75 cells. (f) 5 brains, mNeon: 29 ±1.4µm, 72 cells, mScarlet: 27±1.4µm, 73 cells. (g) 5 brains, mNeon: nonpolar 0% of cells mean 0.0±0.0 cells, unipolar 6.7% of cells mean 1.0±0.3 cells, bipolar 39% of cells mean 5.8±1.0 cells, multipolar 55% of cells mean 8.2±0.8 cells, n=75 cells. mScarlet: nonpolar 1.3% of cells mean 0.2±0.2 cells, unipolar 4% of cells mean 0.6±0.4 cells, bipolar 28% of cells mean 4.2±1.5 cells, multipolar 67% of cells mean 10±1.8 cells, 75 cells. (i) 7 brains, Control: 386±27.4µm, 860 cells, CIP4: 407±24.4µm, 873 cells. (l) 7 brains, Control: 3.3±0.3 process, 105 cells, CIP4: 1.0±0.2 process, 105 cells. (m) 7 brains, Control: 25±1.2µm, 104 cells, CIP4: 17±1.6µm, 56 cells. (n) 7 brains, Control: nonpolar 0% of cells mean 0.0±0.0 cells, unipolar 11% of cells mean 1.7±0.6 cells, bipolar 18% of cells mean 2.7±0.9 cells, multipolar 70% of cells mean 11±1.3 cells, 105 cells. CIP4: nonpolar 41% of cells mean 6.2±1.6 cells, unipolar 21% of cells mean 3.1±0.6 cells, bipolar 22% of cells mean 3.3±0.9 cells, multipolar 15% of cells mean 2.3±1.2 of cells, 105 cells. (p) 9 brains, Control: 412±17.5µm, 1308 cells, FBP17: 415 ±18.6µm, 832 cells. (s) 7 brains, Control: 2.8±0.1 processes, 105 cells, FBP17: 2.9±0. processes, 105 cells. (t) 7 brains, Control: 21±1µm, 101 cells, FBP17: 19±1.2µm, 85 cells. (u) brain, Control: nonpolar 3.8% of cells mean 0.6±0.4 cells, unipolar 18% of cells mean 2.7±1.5 cells, bipolar 20% of cells mean 3.0±0.7 cells, multipolar 58% of cells mean 8.7±1.9 cells, 105 cells. FBP17: nonpolar 16% of cells mean 2.4±0.9 cells, unipolar 18% of cells mean 2.7±1.6 cells, bipolar 13% of cells mean 2.0±0.7 cells, multipolar 52% of cells mean 7.8±2.2 cells, 105 cells. All values mean±SEM.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Over Expression, Migration, Electroporation, Comparison, Two Tailed Test, Control

Journal: bioRxiv

Article Title: F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

doi: 10.1101/2024.10.25.620310

Figure Lengend Snippet: Knockdown of CIP4 or FBP17 decreases migration at E14.5+4 days. (a) Representation of the Double UP and pSico plasmid constructs used in these experiments. Neurons that are transfected with both the Double UP plasmid and a floxed, inverted PGK- puromycin cassette express only mNeon-green and no shRNA (Control cell). When limited amounts of Cre are co-transfected into about half the population of neurons, these neurons express mScarlet (mNeon is excised) and shRNA (PGK-puro cassette excised) targeting either CIP4 or FBP17 (Knockdown cell). (b) Representative image of Double UP + pSico scramble shRNA four days after electroporation at E14.5 (E14.5◊E18.5). Scale bar 100µm. (c) Dot plot of migration of green and magenta cells. Each dot represents cumulative mean distance from the top of the cortical plate for all cortical neurons in a coronal brain section. Connected dots indicate measurements were made in the same coronal section. (d) Western blot of HEK293 cells transfected with mouse CIP4 and post-Cre pSico plasmids expressing either scramble or one of two CIP4-targeted shRNAs. (e) Quantification of western blots in (d). (f, h) Representative images of Double UP + pSico CIP4 shRNA1 (f) and shRNA2 (h) four days after electroporation at E14.5 (E14.5◊E18.5). (g, i) Dot plots of migration of green and magenta cells for CIP4 shRNA1 (g) and shRNA2 (i). (j) Western blot of HEK293 cells transfected with mouse FBP17 and post-Cre pSico plasmids expressing either scramble or one of two FBP17-targeted shRNAs. (k) Quantification of western blots (j). (l, n) Representative image of Double UP + pSico FBP17 shRNA1 (l) and shRNA2 (n) four days after electroporation at E14.5 (E14.5◊E18.5). (m, o) Dot plots of migration of green and magenta cells for FBP17 shRNA1 (m) and shRNA2 (o). Black diamonds and bars represent cumulative mean ± SEM. Paired t-test (two-tailed). (c) 10 brains, Control: 214±25.0µm, 2453 cells, scramble: 227±28.0µm, 2142 cells. (g) 6 brains, Control: 324±27.0µm, 1669 cells, CIP4 shRNA1: 386±27.4µm, 1631 cells. (i) 5 brains, Control: 204±20.5µm, 921 cells, CIP4 shRNA2: 243±24.7µm, 798 cells. (m) 7 brains, Control: 254±22.8µm, 959 cells, FBP17 shRNA1: 352±29.1µm, 1095 cells. (o) 5 brains, Control: 182±35.8µm, 1269 cells, FBP17 shRNA2: 207±34.5µm, 1082 cells.

Article Snippet: Double UP mNeon to mScarlet is available through Addgene (#125134). pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573). pSico PGK Puro was a gift from Tyler Jacks (Addgene plasmid #11586).

Techniques: Knockdown, Migration, Plasmid Preparation, Construct, Transfection, shRNA, Control, Electroporation, Western Blot, Expressing, Two Tailed Test

Journal: Metabolic engineering

Article Title: Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites.

doi: 10.1016/j.ymben.2023.10.002

Figure Lengend Snippet: Fig. 2. Confocal microscopy of the yeast strain BJNBC-003 expressing Venus-NLS and mScarlet-I fluorescent proteins and stained with DAPI to detect DNA. A-D, Bar = 50 μm. E-H, from left to right, bright-field image showing the yeast cells, red fluorescence corresponding to mScarlet-I, yellow fluorescence corresponding to Venus and, blue fluorescence corresponding to DAPI. The mScarlet-I signal is distributed throughout the cell whereas the Venus signal is co-localized with DNA in the nucleus. Bar = 5 μm.

Article Snippet: The gene coding mScarlet-I was amplified from the plasmid Double UP mNeongreen to mScarlet (Addgene #125134) by PCR using primers P1214 and P1215 and cloned into the EcoRV linearized pHYX137 by IVA in E. coli.

Techniques: Confocal Microscopy, Expressing, Staining, Fluorescence