Journal: ACS Nano

Article Title: Quantitative Stimulated Emission Depletion (STED) Microscopy with DNA-Fluorophore Labels

doi: 10.1021/acsnano.5c21411

Figure Lengend Snippet: Quantitative STED microscopy of EGFR dimerization in CHO cells. (A) Schematic illustration of EGFR-ALFA-mEGFP fusion protein (left) and canonical model of EGFR dimerization (right). (B) Two-color confocal and STED microscopy of EGFR labeled with NB@GFP-STAR 635P (magenta) and NB@ALFA-4xP3 (green) in a resting cell (i) and an EGF-treated cell (ii) with magnified views (below). Scale bars are 4 μm (confocal overview), 2 μm (STED overview), 500 nm (magnified STED images). Images are displayed with identical dynamic range for each color channel and acquisition mode. (C) Intensity distributions for (i) resting and (ii) EGF-stimulated cells, with Gaussian fits and small icons showing contributions to the individual peaks (see Methods). (iii) Corrected EGFR dimer fraction in EGF-stimulated cells (see Methods) and standard error indicated as a whisker.

Article Snippet: Cells were washed three times with PBS and incubated with an antibody incubation buffer containing FluoTag-x4 anti-GFP Abberior STAR RED nanobodies (NanoTag Biotechnologies, Germany) and alpaca anti-ALFA nanobodies modified with the custom-designed 4xP3 sequence (Massive Photonics, Germany) ( Table S1 ) in a 1:100 dilution for 1 h at room temperature.

Techniques: Microscopy, Labeling, Whisker Assay

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) Timeline of shRNA infection and analysis of mRNA and protein expression. B) Western blot shows expression of eIF3B protein in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. C) mRNA expression of Eif3b in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. D) Flow cytometry results show percent of cells labeled with CD61-PE antibody compared to isotype control at a range of differentiation timepoints following stable infection with the indicated shRNAs. Error bars show standard deviation for all replicates at each time point. E) mRNA expression of Itgb3 in Ocy454 cells at differentiation day 10 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. F) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA. G) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: shRNA, Infection, Expressing, Western Blot, Flow Cytometry, Labeling, Control, Standard Deviation, Fluorescence, Stable Transfection

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-D) mRNA expression of targeted genes at differentiation day 7 in Ocy454 cells stably expressing the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05 compared to FLuc. E-H) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample. I) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Fluorescence, Control, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) mRNA expression of Astn1 in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. B) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Astn1-targeting shRNA for one representative sample. C) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc. D-L) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Infection, Fluorescence, Control, Stable Transfection, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-G) mRNA expression of osteocyte maturity genes at differentiation day 10 in Ocy454 cells stably expressing the indicated shRNAs. *p<0.05 compared to FLuc. H) Alpha-tubulin immunofluorescence or isotype control immunofluorescence (red) and DAPI (blue) in Ocy454 cells without shRNA infection. Scale bars = 20 μm. I) Ocy454 cells labeled with phalloidin (green) and DAPI (blue) following stable expression of the indicated shRNAs. Scale bars = 20 μm. J-K) Cell Profiler measurements of form factor and perimeter. Each plotted point represents metrics of one cell. Lines designate the median and interquartile range of n=47-158 cells. *p<0.05.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Immunofluorescence, Control, shRNA, Infection, Labeling