pcrispaint gfp2 puror  (Addgene inc)

Journal: bioRxiv

Article Title: K-Ras controls asymmetric cell divisions from the primary cilium

doi: 10.64898/2026.03.05.709789

Figure Lengend Snippet: (A) Confocal images of C2C12 cells grown in high serum for 72 h and immunolabelled for Pax7 in the nucleus and ciliary marker Arl13B. Phalloidin staining visualizes cell outlines. Scale bar = 20 µm. (B) Quantification of ciliation using data as in (A), N = 3 independent biological repeats with a total of n = 800 cells. Means ± SD are plotted. Statistical analysis was done with the unpaired t-test. ( C-F ) Modelled (curves) and measured (points) of the population evolution of MuSC, TAC and differentiated C2C12 cells (Diff) after serum switching at day 0 to trigger differentiation. Experimental samples were treated at day -1 with non-targeting siRNA (siCTRL, C), siRNA targeting KRAS4A/4B (si-pan KRAS, D), NRAS (E) or HRAS (F). Plots to the right show predicted percentage of ciliated cells over time with the line indicating day 3 in differentiating low serum. (G) Confocal imaging-based quantification of ciliation of C2C12 transfected with the indicated siRNA (100 nM) and grown in low serum for 72 h. N = 3, n > 500. Means ± SD are plotted. Statistical analysis was done with one-way ANOVA and the Dunnett’s T3 multiple comparisons test. (H) Flow cytometric quantification of MyHC terminal differentiation marker expression in C2C12 treated as in (G), N = 3. Means ± SD are plotted. Statistical analysis was done with one-way ANOVA and Dunnett’s T3 multiple comparisons test.

Article Snippet: To visualize K-Ras with STED microscopy, C2C12 cells expressing GFP2-K-Ras were fixed with 4 % (w/v) paraformaldehyde and immunolabelled with ChromoTek GFP-booster ATTO647N.

Techniques: Marker, Staining, Imaging, Transfection, Expressing

Journal: bioRxiv

Article Title: K-Ras controls asymmetric cell divisions from the primary cilium

doi: 10.64898/2026.03.05.709789

Figure Lengend Snippet: ( A-C ) Confocal images of C2C12 cells grown in high serum for 48 h after transfection with GFP2-K-Ras (A, n = 73), GFP2-N-Ras (B, n = 48) or GFP2-H-Ras (C, n = 47) and immunolabeled for ciliary marker Arl13B (arrow). Bottom , images show orthogonal views. Scale bar = 10 µm. (D) Quantification of Ras signal in the primary cilium (PC) as compared to the plasma membrane (PM) using data as in (A-C), N = 3. Means ± SD are plotted. Statistical comparison was done against K-Ras with the Kruskal-Wallis test and Dunn’s post hoc test. (E) Confocal images of C2C12 cells treated with PDE6D-targeting siRNA (100 nM), transfected with GFP2-K-Ras and cultured thereafter for 48 h in high serum. Scale bar = 10 µm. (F) Quantification of K-Ras signal in the primary cilium (PC) as compared to the plasma membrane (PM) using data as in (E), N = 3; siCTRL, n = 30; si PDE6D , n = 30. Means ± SD are plotted. Statistical analysis was done with the Mann-Whitney test. (G) Confocal images of C2C12-mEos3.2-KRAS knock-in cells cultured in high serum for 24 h directly after FACS-sorting and immunolabelled for ciliary marker Arl13B and centriolar marker γ-tubulin. Scale bar = 10 µm.

Article Snippet: To visualize K-Ras with STED microscopy, C2C12 cells expressing GFP2-K-Ras were fixed with 4 % (w/v) paraformaldehyde and immunolabelled with ChromoTek GFP-booster ATTO647N.

Techniques: Transfection, Immunolabeling, Marker, Clinical Proteomics, Membrane, Comparison, Cell Culture, MANN-WHITNEY, Knock-In

Journal: bioRxiv

Article Title: K-Ras controls asymmetric cell divisions from the primary cilium

doi: 10.64898/2026.03.05.709789

Figure Lengend Snippet: (A) Confocal images of C2C12 cells grown in high serum for 24 h after transfection with mEGFP-K-Ras. Images were acquired before (-1 s) and after (1 – 4 s) photobleaching at 0 s. Images represent the 10 µm × 10 µm photobleached region. FRAP was monitored at the ciliary tip (arrowhead) and wider base (arrow). Scale bar = 4 µm. (B) Recovery curves for tip and base using data as in (A). Mean ± SEM of fluorescence intensities from all cells per time point, N = 3, n = 23 per condition. ( C , D ) Analysis of individual FRAP curves summarized in (B) yielded diffusion coefficient (C; N = 3, n = 15) and mobile fraction (D; N = 3, n = 23) at ciliary tip and base. Means ± SD are plotted. Statistical analysis was done with the Mann-Whitney test. ( E, F ) STED images of C2C12 cells grown in high serum for 48 h after transfection with GFP2-K-Ras and immunolabeled for GFP using an ATTO647N-conjugated nanobody and ciliary membrane marker Arl13B or centriolar marker γ-tubulin. Position of the cilium (arrowhead) relative to the centrioles (arrow) is indicated. Scale bar = 2 µm. ( G, H ) Confocal images of C2C12 cells grown in high serum for 48 h. Cells were not transfected with K-Ras constructs, but with GFP2-B-Raf (G) and immunolabelled for acetylated α-tubulin marking the cilium (G; N = 3, n = 50) or immunolabelled for endogenous phospho-S217/S221-MEK1/2 and acetylated α-tubulin to mark the cilium (H; N = 3, n = 50). Scale bar = 2 µm.

Article Snippet: To visualize K-Ras with STED microscopy, C2C12 cells expressing GFP2-K-Ras were fixed with 4 % (w/v) paraformaldehyde and immunolabelled with ChromoTek GFP-booster ATTO647N.

Techniques: Transfection, Fluorescence, Diffusion-based Assay, MANN-WHITNEY, Immunolabeling, Membrane, Marker, Construct

Journal: bioRxiv

Article Title: K-Ras controls asymmetric cell divisions from the primary cilium

doi: 10.64898/2026.03.05.709789

Figure Lengend Snippet: (A) BRET titration curve of Rluc8-PDE6D and GFP2-K-Ras-SI as compared to GFP2-K-Ras expressed in HEK cells, N = 3. Means ± SD are plotted. Statistical analysis was performed with the extra sum-of-squares F-test. ( B, C ) Confocal images of C2C12 cells grown in low serum for 72 h after transfection with mEGFP-K-Ras (top) or mEGFP-K-Ras-SI (bottom) and immunolabelled for ciliary markers (arrows) Arl13B (B) or acetylated α-tubulin (C). The mCherry-PDE6D construct was co-expressed at 1:1 DNA ratio with K-Ras variant constructs (C). Scale = 10 µm. (D) Quantification of Ras signal in the primary cilium (PC) as compared to the plasma membrane (PM) per cell using data as in (B,C), N = 3, mEGFP-K-Ras, n = 31; mEGFP-K-Ras-SI, n = 31. Means ± SD are plotted. Statistical analysis was done with the Mann-Whitney test. (E) Confocal imaging-based quantification of ciliation of C2C12 transfected with the indicated mEGFP-tagged K-Ras constructs and cultured for 72 h in low serum. The mCherry-PDE6D construct was co-expressed at 1:1 DNA ratio with K-Ras variant constructs as indicated, N = 3, n ≥ 200 per condition. Means ± SD are plotted. Statistical analysis was done with one-way ANOVA and Dunnett’s T3 multiple comparisons test. (F) Flow cytometric quantification of MyHC terminal differentiation marker expression in C2C12 cells grown in low serum for 72 h after transfection with mEGFP-tagged K-Ras variant constructs alone or co-transfected with mCherry-PDE6D as in (E), N = 3. Means ± SD are plotted. Statistical analysis was done with one-way ANOVA and Dunnett’s T3 multiple comparisons test.

Article Snippet: To visualize K-Ras with STED microscopy, C2C12 cells expressing GFP2-K-Ras were fixed with 4 % (w/v) paraformaldehyde and immunolabelled with ChromoTek GFP-booster ATTO647N.

Techniques: Titration, Transfection, Construct, Variant Assay, Clinical Proteomics, Membrane, MANN-WHITNEY, Imaging, Cell Culture, Marker, Expressing