gui-based image analysis program Search Results


sd 900 imagery  (Canon inc)
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enterprise guide software version 6.1  (SAS institute)
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Enterprise Guide Software Version 6.1, supplied by SAS institute, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gui-based software usvseg  (MathWorks Inc)
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Gui Based Software Usvseg, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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matlab-based gui programme fiesta  (MathWorks Inc)
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computer program sas 9.4  (SAS institute)
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Computer Program Sas 9.4, supplied by SAS institute, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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matlab-based program tracas  (MathWorks Inc)
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pspcas9 bb 2a gfp grna rtn4  (Addgene inc)
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Addgene inc pspcas9 bb 2a gfp grna rtn4
Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
Pspcas9 Bb 2a Gfp Grna Rtn4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gui-based image analysis program  (MathWorks Inc)
90
MathWorks Inc gui-based image analysis program
Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
Gui Based Image Analysis Program, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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graphic user interface (gui)-based image analysis program  (MathWorks Inc)
90
MathWorks Inc graphic user interface (gui)-based image analysis program
Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
Graphic User Interface (Gui) Based Image Analysis Program, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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programa computacional sas  (SAS institute)
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SAS institute programa computacional sas
Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
Programa Computacional Sas, supplied by SAS institute, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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7.5 software  (SPSS Inc)
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SPSS Inc 7.5 software
Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
7.5 Software, supplied by SPSS Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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r statistical computing environment 3.4.4 gui 1.70 el capitan build (7507)  (RStudio)
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Figure 1. <t>RTN4</t> hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).
R Statistical Computing Environment 3.4.4 Gui 1.70 El Capitan Build (7507), supplied by RStudio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 1. RTN4 hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).

Journal: Cell reports

Article Title: Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

doi: 10.1016/j.celrep.2024.114357

Figure Lengend Snippet: Figure 1. RTN4 hinders neuronal outgrowth/regeneration in human iPSC-derived cortical neurons (A) Representative image of differentiated human iPSC-derived cortical neurons (iNeurons; day 21) stained by synaptophysin 1 (green, a presynaptic vesicle marker), microtubule-associated protein 2 (MAP2; magenta, a neuronal marker), and Hoechst 33258 (blue, a nucleus label). (B and C) Micrographs of (B) immunolabeled endogenous RTN4 in fixed iNeurons (day 21) with sub-diffraction-limit resolution and (C) of exogenous RTN4a (RTN4a-Halo::TMR, yellow) co-stained with a plasma membrane marker (Cellbright, magenta). (D) Normalized area covered by neurites during outgrowth of the WT and a pool of stably RTN4a-overexpressing (OE) or RTN4 knockout (KO) iNeurons. Inset: corresponding growth rate extracted from a linear fit. Note the extensive stable RTN4a-Halo expression throughout RTN4a OE iNeurons. See also Figure S3C. (E and F) Micrographs of iNeurons with exogenously introduced RTN4a-Halo (J646 labeled, orange) at the indicated differentiation stage. (E) Lentiviral particles of RTN4a-Halo were introduced 48 h pre differentiation (shown are images from differentiation days 3, 5, and 7 in the same field). (F) As in (E), but RTN4a-Halo was introduced post differentiation (day 14). Note the compromised neurite outgrowth in RTN4a-OE cells and detectable distribution of RTN4a in neurites when iNeurons possess neurites. (G) Representative time-lapse images of neurite regeneration following a mechanical injury. (H) Neurite regeneration rate in WT and RTN4 KO iNeurons. Shown are means ± SEM from three independent experiments. *p < 0.05 (Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hu_Rtn4_genotying_f 50-AGTATTCAGCATTGTGAGCGTA-30 This study N/A COS7 Rtn4 KO_genotyping-f 50-GAGTGGGTTTAAAATGTGGG-30 This study N/A COS7 Rtn4 KO_genotyping-r 50-GTATATCCTAAAGCTGATGGTCAC-30 This study N/A Recombinant DNA pSpCas9(BB)-2A-GFP Addgene RRID: Addgene_48138 pSpCas9(BB)-2A-GFP_gRNA RTN4 This study N/A pLV_Exp_Puro-TRE3G_XbaI_Hu Rtn3A-TEVHaloTag This study N/A pcDNA3-ER-GCaMP3 Addgene RRID: Addgene_64854 pFLAG_ER mCherry gift from D.Ron40 N/A Halo-Sec61b gift from J. Bewersdorf47 N/A pmPA-GFP-ER-5 Addgene RRID: Addgene_57132 pMD1g/pRRE Addgene RRID: Addgene_12251 pRSV-Rev Addgene RRID: Addgene_12253 pDM2.G Addgene RRID: Addgene_12259 mCherry_SBP_RTN4A gift from G.G.Farı́as19 N/A ER-Crowding_FRET1_CRH2_KDEL_pCDNA3-1(+) gift from D. Ron31 N/A pLV_Rtn4-SNAP This study N/A pLV_TRE3G_hRTN4a-Halo This study N/A D4ER gift from D. Ron41 N/A SNAP-Sec61b This study N/A mEmerald-Calnexin Addgene RRID: Addgene_54021 mcherry_RTN4a L.M.Westrate44 N/A pCMV_mCherry_SBP This study N/A RTN2C-mEmerald-N1 This study N/A pLV_RTN4c-SNAP This study N/A pCMV_mCherry-SBP-RTN2C This study N/A pCMV_mCherry-SBP-Sec61b This study N/A Software and algorithms Prism9 Graph Pad https://www.graphpad.com/; RRID: SCR_002798 Fiji (ImageJ) NIH, USA https://fiji.sc/; RRID: SCR_002285 Leica Application Suite X Leica https://www.leica-microsystems.com/ products/microscope-software/p/ leica-las-x-ls/; RRID: SCR_013673 MATLAB MathWorks http://www.mathworks.com/products/ matlab/; RRID: SCR_001622 IncuCyte S3 Live Cell Analysis System Sartorius https://www.sartorius.com/en/ products/live-cell-imaging-analysis/ live-cell-analysis-instruments/s3-livecell-analysis-instrument#id-797316; RRID: SCR_023147 Other The custom code for analysing CPAC measurements This study https://doi.org/10.5281/zenodo.11206308 The custom code for the finite volume simulation of concentration profiles spreading over network structures This study https://doi.org/10.5281/zenodo.11206293 14 Cell Reports --, 114357, --, 2024 OPEN ACCESS OPEN ACCESS

Techniques: Derivative Assay, Staining, Marker, Immunolabeling, Clinical Proteomics, Membrane, Stable Transfection, Knock-Out, Expressing, Labeling

Figure 2. RTN4 narrows ER tubules (A) Fluorescence micrographs of endogenous RTN4 (immunolabeled, green) and an exogenously expressed ER luminal marker (mCherryER, magenta) in fixed COS-7 cells, shown along with a line-scan analysis of the fluorescence intensity along peripheral ER tubules (dashed line in the inset). (B) Colocalization analysis of the ER luminal marker (mCherryER) vs. overexpressed ER membrane proteins (SNAP-Sec61b, RTN4a-SNAP, or RTN4c-SNAP) (n = 15). Note decreased colocalization in RTN4 overexpression, reflecting luminal exclusion (Pearson correlation coefficients, mean ± SD, SNAP-Sec61b: 0.750 ± 0.089, RTN4a-SNAP: 0.493 ± 0.145, RTN4c-SNAP; 0.425 ± 0.157). Note the incomplete colocalization observed in SNAP-Sec61b-OE cells due to endogenous RTN4 anisotropic distribution, as shown in (A). (C) A representative micrograph with ER tubules and sheets used for ER radius estimation in COS-7 WT (51.3 ± 11.6 nm) and RTN4a-OE cells (0.2 mg; 47.2 ± 10.9 nm, 0.4 mg; 41.4 ± 11.1 nm). Note a decrease of the ER tubule radius with increasing amounts of RTN4a. (D) Measurements as in (C) of COS-7 WT (51.7 ± 14.0 nm) and RTN4 KO (57.5 ± 12.4 nm) and SH-SY5Y WT (51.4 ± 9.0 nm) and RTN4 KO (65.0 ± 14.5 nm) cells. COS-7 WT, n = 47; COS-7 RTN4 KO, n = 26; SH-SY5Y WT, n = 30; SH-SY5Y RTN4 KO, n = 26. (C and D) The values are means ± SD. Note that two ER membrane markers, calnexin and Sec61b, yield the same values for WT samples. (E) Representative TEM images (left) were segmented. ER tubules are depicted in magenta (right). Measured ER tubule radius is shown for COS-7 WT (37.3 ± 8.8 nm, n = 50), RTN4a-OE (28.8 ± 7.5 nm, n = 48), and RTN4 KO (42.8 ± 8.2 nm, n = 56) cells. The values are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001; n.s., not significant.

Journal: Cell reports

Article Title: Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

doi: 10.1016/j.celrep.2024.114357

Figure Lengend Snippet: Figure 2. RTN4 narrows ER tubules (A) Fluorescence micrographs of endogenous RTN4 (immunolabeled, green) and an exogenously expressed ER luminal marker (mCherryER, magenta) in fixed COS-7 cells, shown along with a line-scan analysis of the fluorescence intensity along peripheral ER tubules (dashed line in the inset). (B) Colocalization analysis of the ER luminal marker (mCherryER) vs. overexpressed ER membrane proteins (SNAP-Sec61b, RTN4a-SNAP, or RTN4c-SNAP) (n = 15). Note decreased colocalization in RTN4 overexpression, reflecting luminal exclusion (Pearson correlation coefficients, mean ± SD, SNAP-Sec61b: 0.750 ± 0.089, RTN4a-SNAP: 0.493 ± 0.145, RTN4c-SNAP; 0.425 ± 0.157). Note the incomplete colocalization observed in SNAP-Sec61b-OE cells due to endogenous RTN4 anisotropic distribution, as shown in (A). (C) A representative micrograph with ER tubules and sheets used for ER radius estimation in COS-7 WT (51.3 ± 11.6 nm) and RTN4a-OE cells (0.2 mg; 47.2 ± 10.9 nm, 0.4 mg; 41.4 ± 11.1 nm). Note a decrease of the ER tubule radius with increasing amounts of RTN4a. (D) Measurements as in (C) of COS-7 WT (51.7 ± 14.0 nm) and RTN4 KO (57.5 ± 12.4 nm) and SH-SY5Y WT (51.4 ± 9.0 nm) and RTN4 KO (65.0 ± 14.5 nm) cells. COS-7 WT, n = 47; COS-7 RTN4 KO, n = 26; SH-SY5Y WT, n = 30; SH-SY5Y RTN4 KO, n = 26. (C and D) The values are means ± SD. Note that two ER membrane markers, calnexin and Sec61b, yield the same values for WT samples. (E) Representative TEM images (left) were segmented. ER tubules are depicted in magenta (right). Measured ER tubule radius is shown for COS-7 WT (37.3 ± 8.8 nm, n = 50), RTN4a-OE (28.8 ± 7.5 nm, n = 48), and RTN4 KO (42.8 ± 8.2 nm, n = 56) cells. The values are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001; n.s., not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hu_Rtn4_genotying_f 50-AGTATTCAGCATTGTGAGCGTA-30 This study N/A COS7 Rtn4 KO_genotyping-f 50-GAGTGGGTTTAAAATGTGGG-30 This study N/A COS7 Rtn4 KO_genotyping-r 50-GTATATCCTAAAGCTGATGGTCAC-30 This study N/A Recombinant DNA pSpCas9(BB)-2A-GFP Addgene RRID: Addgene_48138 pSpCas9(BB)-2A-GFP_gRNA RTN4 This study N/A pLV_Exp_Puro-TRE3G_XbaI_Hu Rtn3A-TEVHaloTag This study N/A pcDNA3-ER-GCaMP3 Addgene RRID: Addgene_64854 pFLAG_ER mCherry gift from D.Ron40 N/A Halo-Sec61b gift from J. Bewersdorf47 N/A pmPA-GFP-ER-5 Addgene RRID: Addgene_57132 pMD1g/pRRE Addgene RRID: Addgene_12251 pRSV-Rev Addgene RRID: Addgene_12253 pDM2.G Addgene RRID: Addgene_12259 mCherry_SBP_RTN4A gift from G.G.Farı́as19 N/A ER-Crowding_FRET1_CRH2_KDEL_pCDNA3-1(+) gift from D. Ron31 N/A pLV_Rtn4-SNAP This study N/A pLV_TRE3G_hRTN4a-Halo This study N/A D4ER gift from D. Ron41 N/A SNAP-Sec61b This study N/A mEmerald-Calnexin Addgene RRID: Addgene_54021 mcherry_RTN4a L.M.Westrate44 N/A pCMV_mCherry_SBP This study N/A RTN2C-mEmerald-N1 This study N/A pLV_RTN4c-SNAP This study N/A pCMV_mCherry-SBP-RTN2C This study N/A pCMV_mCherry-SBP-Sec61b This study N/A Software and algorithms Prism9 Graph Pad https://www.graphpad.com/; RRID: SCR_002798 Fiji (ImageJ) NIH, USA https://fiji.sc/; RRID: SCR_002285 Leica Application Suite X Leica https://www.leica-microsystems.com/ products/microscope-software/p/ leica-las-x-ls/; RRID: SCR_013673 MATLAB MathWorks http://www.mathworks.com/products/ matlab/; RRID: SCR_001622 IncuCyte S3 Live Cell Analysis System Sartorius https://www.sartorius.com/en/ products/live-cell-imaging-analysis/ live-cell-analysis-instruments/s3-livecell-analysis-instrument#id-797316; RRID: SCR_023147 Other The custom code for analysing CPAC measurements This study https://doi.org/10.5281/zenodo.11206308 The custom code for the finite volume simulation of concentration profiles spreading over network structures This study https://doi.org/10.5281/zenodo.11206293 14 Cell Reports --, 114357, --, 2024 OPEN ACCESS OPEN ACCESS

Techniques: Fluorescence, Immunolabeling, Marker, Membrane, Over Expression

Figure 4. ER morphoregulation by RTN4 modulates ER luminal transport rates (A) Simulations of spreading from a continuously photoactivated region on a regular lattice of tubules in a cellular geometry. The rightmost image shows a concentration profile with added synthetic noise, analyzed as for experimental CPAC data. Rings at different distances from the photoactivated region are shown in transparent color. Example wedge regions used to track signal intensity are shown for each ring. The plot shows simulated noisy signal intensity over time in each of the wedge regions (matching color). Vertical dashed lines indicate the arrival half-time (t1/2) for the two nearest regions. (B) Simulated median arrival t1/2 averaged over all regions at a given distance from the photoactivated center in cell geometries (n = 8). Concentration profiles spread by diffusion alone (red) are compared against those on an active network with randomly oriented flows in each edge (blue), of velocity v = 20 mm/s, and persisting over t = 0.1 s. Results are plotted on log-log axes to emphasize scaling behavior (black dashed line, t1/2 R2; green dashed line, t1/2 R shown as guides to the eye). Note that active flows result in a super-diffusive (sub-quadratic) scaling of signal arrival times versus distance. Inset: the same plot on linear axes. Error bars show standard deviation of the median, obtained by bootstrapping at the level of individual simulated cells. (C) Representative images of continuous photoactivation chase (CPAC) in COS-7 cells transiently expressing RTN3A-HaloTag (RTN3A-Halo) labeled by tetra- methyl rhodamine (50 nM, 2 h) and photoactivatable-Janelia Fluor 646 (paJ646; 200 nM, 2 h). A white box denotes the area of paJ646 photoactivation by laser illumination (405 nm). Traces of photoactivated signal intensity are shown for each of the marked wedge regions. (D) Median half-time of photoactivated signal rise (arrival t1/2, exemplified by vertical dashed lines in (C) for paGFPER (blue) or an ER membrane marker (RTN3A- Halo::paJ646, red) at various distances from the activation spot. Note that measurements for membrane protein are well matched by simulations of diffusive spread with D = 1.8 mm2/s. Luminal protein measurements show transport with super-diffusional scaling and cannot be fit by higher diffusion coefficients (compared to simulations up to D = 6 mm2/s, green lines). (E and F) paGFPER arrival t1/2 measurements as in (D) in mCherryER (control, RTN4 KO) or mCherry-RTN4a OE (RTN4 OE) COS-7 cells (n = 15 each) (E) and in SH- SY5Y cells (F; P derived from Kolmogorov-Smirnov test). Bars in (F) indicate median. (G) Fluorescence lifetime imaging microscopy (FLIM) measurements of an ER-localized molecular crowding probe in cells as in (F) (n = 34, 19, and 42, respectively).

Journal: Cell reports

Article Title: Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

doi: 10.1016/j.celrep.2024.114357

Figure Lengend Snippet: Figure 4. ER morphoregulation by RTN4 modulates ER luminal transport rates (A) Simulations of spreading from a continuously photoactivated region on a regular lattice of tubules in a cellular geometry. The rightmost image shows a concentration profile with added synthetic noise, analyzed as for experimental CPAC data. Rings at different distances from the photoactivated region are shown in transparent color. Example wedge regions used to track signal intensity are shown for each ring. The plot shows simulated noisy signal intensity over time in each of the wedge regions (matching color). Vertical dashed lines indicate the arrival half-time (t1/2) for the two nearest regions. (B) Simulated median arrival t1/2 averaged over all regions at a given distance from the photoactivated center in cell geometries (n = 8). Concentration profiles spread by diffusion alone (red) are compared against those on an active network with randomly oriented flows in each edge (blue), of velocity v = 20 mm/s, and persisting over t = 0.1 s. Results are plotted on log-log axes to emphasize scaling behavior (black dashed line, t1/2 R2; green dashed line, t1/2 R shown as guides to the eye). Note that active flows result in a super-diffusive (sub-quadratic) scaling of signal arrival times versus distance. Inset: the same plot on linear axes. Error bars show standard deviation of the median, obtained by bootstrapping at the level of individual simulated cells. (C) Representative images of continuous photoactivation chase (CPAC) in COS-7 cells transiently expressing RTN3A-HaloTag (RTN3A-Halo) labeled by tetra- methyl rhodamine (50 nM, 2 h) and photoactivatable-Janelia Fluor 646 (paJ646; 200 nM, 2 h). A white box denotes the area of paJ646 photoactivation by laser illumination (405 nm). Traces of photoactivated signal intensity are shown for each of the marked wedge regions. (D) Median half-time of photoactivated signal rise (arrival t1/2, exemplified by vertical dashed lines in (C) for paGFPER (blue) or an ER membrane marker (RTN3A- Halo::paJ646, red) at various distances from the activation spot. Note that measurements for membrane protein are well matched by simulations of diffusive spread with D = 1.8 mm2/s. Luminal protein measurements show transport with super-diffusional scaling and cannot be fit by higher diffusion coefficients (compared to simulations up to D = 6 mm2/s, green lines). (E and F) paGFPER arrival t1/2 measurements as in (D) in mCherryER (control, RTN4 KO) or mCherry-RTN4a OE (RTN4 OE) COS-7 cells (n = 15 each) (E) and in SH- SY5Y cells (F; P derived from Kolmogorov-Smirnov test). Bars in (F) indicate median. (G) Fluorescence lifetime imaging microscopy (FLIM) measurements of an ER-localized molecular crowding probe in cells as in (F) (n = 34, 19, and 42, respectively).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hu_Rtn4_genotying_f 50-AGTATTCAGCATTGTGAGCGTA-30 This study N/A COS7 Rtn4 KO_genotyping-f 50-GAGTGGGTTTAAAATGTGGG-30 This study N/A COS7 Rtn4 KO_genotyping-r 50-GTATATCCTAAAGCTGATGGTCAC-30 This study N/A Recombinant DNA pSpCas9(BB)-2A-GFP Addgene RRID: Addgene_48138 pSpCas9(BB)-2A-GFP_gRNA RTN4 This study N/A pLV_Exp_Puro-TRE3G_XbaI_Hu Rtn3A-TEVHaloTag This study N/A pcDNA3-ER-GCaMP3 Addgene RRID: Addgene_64854 pFLAG_ER mCherry gift from D.Ron40 N/A Halo-Sec61b gift from J. Bewersdorf47 N/A pmPA-GFP-ER-5 Addgene RRID: Addgene_57132 pMD1g/pRRE Addgene RRID: Addgene_12251 pRSV-Rev Addgene RRID: Addgene_12253 pDM2.G Addgene RRID: Addgene_12259 mCherry_SBP_RTN4A gift from G.G.Farı́as19 N/A ER-Crowding_FRET1_CRH2_KDEL_pCDNA3-1(+) gift from D. Ron31 N/A pLV_Rtn4-SNAP This study N/A pLV_TRE3G_hRTN4a-Halo This study N/A D4ER gift from D. Ron41 N/A SNAP-Sec61b This study N/A mEmerald-Calnexin Addgene RRID: Addgene_54021 mcherry_RTN4a L.M.Westrate44 N/A pCMV_mCherry_SBP This study N/A RTN2C-mEmerald-N1 This study N/A pLV_RTN4c-SNAP This study N/A pCMV_mCherry-SBP-RTN2C This study N/A pCMV_mCherry-SBP-Sec61b This study N/A Software and algorithms Prism9 Graph Pad https://www.graphpad.com/; RRID: SCR_002798 Fiji (ImageJ) NIH, USA https://fiji.sc/; RRID: SCR_002285 Leica Application Suite X Leica https://www.leica-microsystems.com/ products/microscope-software/p/ leica-las-x-ls/; RRID: SCR_013673 MATLAB MathWorks http://www.mathworks.com/products/ matlab/; RRID: SCR_001622 IncuCyte S3 Live Cell Analysis System Sartorius https://www.sartorius.com/en/ products/live-cell-imaging-analysis/ live-cell-analysis-instruments/s3-livecell-analysis-instrument#id-797316; RRID: SCR_023147 Other The custom code for analysing CPAC measurements This study https://doi.org/10.5281/zenodo.11206308 The custom code for the finite volume simulation of concentration profiles spreading over network structures This study https://doi.org/10.5281/zenodo.11206293 14 Cell Reports --, 114357, --, 2024 OPEN ACCESS OPEN ACCESS

Techniques: Concentration Assay, Diffusion-based Assay, Standard Deviation, Expressing, Labeling, Membrane, Marker, Activation Assay, Control, Derivative Assay, Fluorescence, Imaging, Microscopy

Figure 5. RTN4 ER morphoregulatory/transport effect curbs ER Ca2+ release capacity (A) Physical simulation of the dependence between ER Ca2+ release capacity and luminal transport, incorporating equilibrated binding to Ca2+ buffer proteins, local ER release, diffusive luminal transport of free/buffered Ca2+, and luminal flow, with plots of total Ca2+ released over time. Note that release capacity de- creases both as a result of halting active flows and from the direct decrease in flow rate due to tubule narrowing. (B) Schematic of light-induced ER Ca2+ release and monitoring assay. (C) Representative fluorescence intensity image series of GCaMP3ER at photo-uncaging regions of COS-7 cells preloaded with caged IP3 (3 mM, 3 h, uncaging by continuous 405-nm laser illumination). (D) Traces of GCaMP3ER signal as in (C) in WT or RTN4a-OE SH-SY5Y cells, shown along with the integrated ER Ca2+ released during the uncaging period (the area under the curve; WT, n = 17; RTN4a OE, n = 20). Shown are means ± SEM from samples in three independent experiments. ****p < 0.001 (one-way ANOVA). (E) As in (D) for WT and RTN4 KO SH-SY5Y cells (WT, n = 15; RTN4 KO, n = 23). *p < 0.05 (Student’s t test). (D and E) Note the equivalent dynamic range in the WT. (F) FLIM measurements of ER Ca2+ using the D4ER probe in cells as in (D) and (E). Note that the lifetime is inversely proportional to [Ca2+]ER.

Journal: Cell reports

Article Title: Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

doi: 10.1016/j.celrep.2024.114357

Figure Lengend Snippet: Figure 5. RTN4 ER morphoregulatory/transport effect curbs ER Ca2+ release capacity (A) Physical simulation of the dependence between ER Ca2+ release capacity and luminal transport, incorporating equilibrated binding to Ca2+ buffer proteins, local ER release, diffusive luminal transport of free/buffered Ca2+, and luminal flow, with plots of total Ca2+ released over time. Note that release capacity de- creases both as a result of halting active flows and from the direct decrease in flow rate due to tubule narrowing. (B) Schematic of light-induced ER Ca2+ release and monitoring assay. (C) Representative fluorescence intensity image series of GCaMP3ER at photo-uncaging regions of COS-7 cells preloaded with caged IP3 (3 mM, 3 h, uncaging by continuous 405-nm laser illumination). (D) Traces of GCaMP3ER signal as in (C) in WT or RTN4a-OE SH-SY5Y cells, shown along with the integrated ER Ca2+ released during the uncaging period (the area under the curve; WT, n = 17; RTN4a OE, n = 20). Shown are means ± SEM from samples in three independent experiments. ****p < 0.001 (one-way ANOVA). (E) As in (D) for WT and RTN4 KO SH-SY5Y cells (WT, n = 15; RTN4 KO, n = 23). *p < 0.05 (Student’s t test). (D and E) Note the equivalent dynamic range in the WT. (F) FLIM measurements of ER Ca2+ using the D4ER probe in cells as in (D) and (E). Note that the lifetime is inversely proportional to [Ca2+]ER.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hu_Rtn4_genotying_f 50-AGTATTCAGCATTGTGAGCGTA-30 This study N/A COS7 Rtn4 KO_genotyping-f 50-GAGTGGGTTTAAAATGTGGG-30 This study N/A COS7 Rtn4 KO_genotyping-r 50-GTATATCCTAAAGCTGATGGTCAC-30 This study N/A Recombinant DNA pSpCas9(BB)-2A-GFP Addgene RRID: Addgene_48138 pSpCas9(BB)-2A-GFP_gRNA RTN4 This study N/A pLV_Exp_Puro-TRE3G_XbaI_Hu Rtn3A-TEVHaloTag This study N/A pcDNA3-ER-GCaMP3 Addgene RRID: Addgene_64854 pFLAG_ER mCherry gift from D.Ron40 N/A Halo-Sec61b gift from J. Bewersdorf47 N/A pmPA-GFP-ER-5 Addgene RRID: Addgene_57132 pMD1g/pRRE Addgene RRID: Addgene_12251 pRSV-Rev Addgene RRID: Addgene_12253 pDM2.G Addgene RRID: Addgene_12259 mCherry_SBP_RTN4A gift from G.G.Farı́as19 N/A ER-Crowding_FRET1_CRH2_KDEL_pCDNA3-1(+) gift from D. Ron31 N/A pLV_Rtn4-SNAP This study N/A pLV_TRE3G_hRTN4a-Halo This study N/A D4ER gift from D. Ron41 N/A SNAP-Sec61b This study N/A mEmerald-Calnexin Addgene RRID: Addgene_54021 mcherry_RTN4a L.M.Westrate44 N/A pCMV_mCherry_SBP This study N/A RTN2C-mEmerald-N1 This study N/A pLV_RTN4c-SNAP This study N/A pCMV_mCherry-SBP-RTN2C This study N/A pCMV_mCherry-SBP-Sec61b This study N/A Software and algorithms Prism9 Graph Pad https://www.graphpad.com/; RRID: SCR_002798 Fiji (ImageJ) NIH, USA https://fiji.sc/; RRID: SCR_002285 Leica Application Suite X Leica https://www.leica-microsystems.com/ products/microscope-software/p/ leica-las-x-ls/; RRID: SCR_013673 MATLAB MathWorks http://www.mathworks.com/products/ matlab/; RRID: SCR_001622 IncuCyte S3 Live Cell Analysis System Sartorius https://www.sartorius.com/en/ products/live-cell-imaging-analysis/ live-cell-analysis-instruments/s3-livecell-analysis-instrument#id-797316; RRID: SCR_023147 Other The custom code for analysing CPAC measurements This study https://doi.org/10.5281/zenodo.11206308 The custom code for the finite volume simulation of concentration profiles spreading over network structures This study https://doi.org/10.5281/zenodo.11206293 14 Cell Reports --, 114357, --, 2024 OPEN ACCESS OPEN ACCESS

Techniques: Binding Assay