Journal: bioRxiv

Article Title: SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos, and background subtraction

doi: 10.1101/2022.01.04.474883

Figure Lengend Snippet: 60 planes 3D volumes of live RPE1 cells double stained with PKMR for Mitochondria ( a , e ) and with Tubulin Tracker TM Deep Red for Microtubules ( b , f ) were acquired within 2.2 s per stack using lattice light-sheet microscopy. MIP of representative raw (see Material and Methods section) images for Mitochondria and Microtubules, respectively, before ( a , b ) and after ( e, f ) SPITFIR(e) 4D denoising and 3D deconvolution (3D Gaussian PSF, σ xy = 1.5 pixels and σ z = 1.0 pixel). Insets are zoomed area illustrating SPITFIR(e) improvement in spatial resolution and SNR. 3D angular views and MIP of composite images before ( c , d ) and after ( g , h ) SPITFIR(e) treatment (Red for Microtubules; Green for Mitochondria). 4D denoising SPITFIR(e) image improvement shown on a single x-y plane ( i ) zoomed from the inset indicated in ( d, h ). Raw image is indicated as a blue lined sector in the upper part and in the left circle, beneath; 4D denoised image is similarly indicated in magenta, while the 4D denoised + 3D deconvolved image is in yellow. Intensity line profiles were measured as indicated in the 3 circles and plotted for each processing step and both Mitochondria and Microtubules at the lower part of ( i ). j , Workflow for image restoration extracted from the global flow chart in . Scale bars are indicated in the bottom right corner ( a-h ). Related videos are shown as Supplementary movies S1 and S2.

Article Snippet: The hTERT-immortalized RPE1 cells (Human Retinal Pigment Epithelial Cell) were purchased from ATCC (CRL-4000).

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos, and background subtraction

doi: 10.1101/2022.01.04.474883

Figure Lengend Snippet: a , Image depicting mCherry-LifeAct in RPE1 cells (scale bar: 5 μ m). b , Results with SPITFIR(e) with different levels of sparsity (“weak” ( ρ = 0.9), “moderate ( ρ = 0.6), “high” ( ρ = 0.1), automatic selection of the regularization parameter and different strategies with SPITFIR(e): 2D Deconvolution, 3D Decovolution, 3D Denoising + 3D Deconvolution, 3D Denoising + 2D deconvolution ( σ xy = 1.5 pixels and σ z = 0.5 pixel).

Article Snippet: The hTERT-immortalized RPE1 cells (Human Retinal Pigment Epithelial Cell) were purchased from ATCC (CRL-4000).

Techniques: Selection

Journal: bioRxiv

Article Title: SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos, and background subtraction

doi: 10.1101/2022.01.04.474883

Figure Lengend Snippet: Live RPE1 cells double stained with PKMO (mitochondria) and Tubulin TrackerTM Deep Red (microtubules) were imaged by STED nanoscopy before and after being processed by SPITFIR(e) ( a ). Partial overview of 2D STED raw data of RPE1 cells for mitochondria, microtubules and their superimposed images are shown from left to right ( a , upper panel). 2D STED images were improved by SPITFIR(e) using two different 2D Gaussian model σ xy = 1.0 pixel ( a , middle panel) and σ xy = 1.5 pixels ( a , lower panel). Insets (1 and 2) show zoomed area where SPITFIR(e) image quality improvement is illustrated by comparison of two processing conditions (middle and lower panel) with the same area in the raw STED image. Inset( b ) shows a magnified composite image where the 2D STED raw part is indicated as a blue lined rectangle and 2D denoised + 2D deconvolved parts are indicated in red ( σ xy = 1.0 pixel) and green ( σ xy = 1.5 pixels). ( b ) Normalized intensity line profiles were measured in the insets ( b ) for cristae regions. Yellow line indicated in the inset ( b5 ) serve to identify fluorescence profiles. Pixel size is equal to 25 nm. Scale bars: 1 μ m in ( a ) and ( b ). Line profiles of individual cristae (n=75, from 8 different acquisitions) were fitted using a Gaussian model. Full width half maximum (FWHM) was estimated on raw STED and 2D denoised + 2D deconvolved with σ xy = 1.0 pixel ( c ). SPITFIR(e) improvement is shown separately for all the line profiles analyzed and the line profiles from bigger cristae (FWHM raw = 60 nm), indication of lower SNR. Statistics analysis is including in ( d ). It indicates the improvement of average resolution (unpaired two-sample t -test) and variance of cristae resolution ( F -test).

Article Snippet: The hTERT-immortalized RPE1 cells (Human Retinal Pigment Epithelial Cell) were purchased from ATCC (CRL-4000).

Techniques: Staining, Comparison, Fluorescence

Journal: Progress in retinal and eye research

Article Title: Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases

doi: 10.1016/j.preteyeres.2017.01.004

Figure Lengend Snippet: Status of registered clinical trials using cell-based therapies for AMD and RP.

Article Snippet: Cell Cure Neurosciences Ltd. , Advanced dry AMD , Phase I/II - Recruiting , Safety and efficacy , Subretinal transplantation of human embryonic stem cell derived retinal pigmented epithelial (OpRegen) cells , Israel , 5-Oct-16 , NCT02286089.

Techniques: Clinical Proteomics, Transplantation Assay, Derivative Assay, Injection, Membrane, Saline

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet: Characterization of structural requirements and functional properties of TAZ and YAP nuclear import (A) Domain organization of TAZ with regions important for nucleocytoplasmic shuttling highlighted. Lats-phosphorylation sites are shown as yellow circles with labels, hydrophobic motifs FLxx[I,V,L,M] as blue lines. CC: coiled-coil region; NES: nuclear export signal; NLS: nuclear localization signal; PBM: PDZ domain binding motif; TAD: transactivation domain; TBD: TEAD-binding domain; WW: WW domain; WW-NLS: globular NLS composed of the WW domain. (B) Localization of diffusion-limited 5C constructs in pig proximal tubular epithelial LLC-PK1 cells, also see Figure S1 . 5C-R5A-NES: shuttling control comprising a variant of the SV40 NLS and the HIV Ref NES. WT: 5C-TAZ 4SA or 5C-YAP 5SA; ΔNLS: constructs with deletion of residues 321–345 (TAZ) or 413–427 (YAP); ΔPBM: constructs missing the last 5 aa (the PBM); control: 5C control. Left: Representative fluorescence microscopy images of transfected cells, without (top) and with 4h LMB treatment (bottom). The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of the 5C-constructs upon LMB treatment for the indicated times. Median nucleocytoplasmic fluorescence ratios (median N/C of >100 cells) were determined for individual constructs as described previously and data were fitted to mono-exponential growth curves with plateau. Number of repeats: 4. (C) Localization of 5C-NLS constructs. Left: Representative fluorescence microscopy images of transfected LLC-PK1 cells. The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of 5C, 5C-TAZ NLS, 5C-YAP-NLS, and 5C-R5A in LLC-PK1 and HEK cells. (D) The TAZ NLS inhibits the nuclear uptake of full-length TAZ. Cells were transfected with constant amounts of 5C or 5C-TAZ 4SA in combination with mCherry-TAZ NLS at ratios 1:0, 1:1, 1:2, 1:3, and 1:4. mCherry encoding vector was added to keep the total amounts of DNA constant. The increase in nuclear accumulation of 5C constructs after 6h LMB treatment, relative to no LMB addition is shown as ΔN/C (LMB). Number of repeats: 8. Also see Figure S2 A. (E) Localization of 1C constructs in LLC-PK1 and RPE-1 cells. (F and G) Interaction between TAZ and IPO7. mCitrine (1C) and 1C-TAZ constructs were expressed in HEK cells. IPO7 (F) or Citrine-constructs (G) were immunoprecipitated and analyzed by western blotting using GFP- and IPO7 specific antibodies. Red asterisks indicate the position of relevant bands. (H) Effect of ivermectin (iver; 25 μM) on the nuclear accumulation of endogenous TAZ/YAP and MRTF upon treatment with low calcium medium (LCM). Response is given as the percentage of uninhibited N/C increase at 60 min (TAZ/YAP) or 20 min (MRTF) LCM treatment. (I) Nuclear accumulation of 5C-TAZ NLS and 5C-SV40 NLS in the presence of indicated concentrations of ivermectin. Also see Figure S2 B. (J) Effect of ivermectin on the nuclear accumulation of shuttling 5C-constructs induced by LMB treatment. ΔN/C (LMB) are calculated as in (D). (K) Nuclear import of TAZ 4SA is Ran-independent. Cells were co-transfected with indicated constructs and non-related (siNR) or Ran-specific (siRan) siRNA and treated with LMB for 4h. (L) Nuclear import of TAZ 4SA is ATP-independent. Left: Representative fluorescence microscopy images showing the effect of 2-deoxyglucose and azide on the cellular distribution of KPNA2. The scale bar represents 50 μm. Right: cells transfected with indicated constructs and treated for 4h with or without LMB, in the presence or absence of 2-deoxyglucose and azide. Means ± SD are depicted, and number of repeats are indicated. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001; one-way ANOVA and Tukey-Kramer test.

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Functional Assay, Phospho-proteomics, Binding Assay, Diffusion-based Assay, Construct, Control, Variant Assay, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet:

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Recombinant, Transfection, Luciferase, Reporter Assay, Control, Plasmid Preparation, Software