Journal: Scientific Reports

Article Title: Selective nanosecond laser removal of retinal pigment epithelium for cell therapy

doi: 10.1038/s41598-024-69917-z

Figure Lengend Snippet: Histological and immunohistochemical analysis. ( a ) H&E staining image of control group without laser treatment showing the normal morphology of retinal cells and the architecture of retinal structure. All retinal tissues were clearly observed including ILM, GCL, IPL, INL, ONL, OPL, PL, RPE, choroid, and sclera. A single continuous RPE monolayer was detected. ( b ) H&E images without stem cells injection. RPE layers were significantly changed with discontinuities and regions of RPE loss were found in the RPE layer (yellow arrow). ( c ) H&E images of the tissues treated with laser followed by stem cell transplantation. Yellow arrows indicate the evidence of the transplanted hiPSC-RPE cells grafted in the subretinal space with high pigmentation (black color). ( d–o ) Immunofluorescent images of the tissues stained with two different antibodies: RPE65 and NuMa. RPE65-positive indicate the signal from RPE cells. Red fluorescent color shows NuMa positive hiPSC-RPE cells. Blue fluorescent color demonstrates the cell’s nuclei stained by DAPI. No NuMa-positive signal was detected on the control group and laser treatment group. RPE loss was detected on treated group (yellow arrows). The hiPSC-RPE cells replaced RPE death were observed on the stem cell transplantation groups (Figure o ). Red arrows show the growing of hiPSC-RPE cells at the RPE damaged areas.

Article Snippet: To visualize the grafted hiPSC-RPE cells, IHC was performed using anti-RPE-65 against rabbit RPE65 (MAB5428, MilliporeSigma, MA, USA) and anti-NuMA antibodies against human NuMA (ab84680, Abcam, MA, USA).

Techniques: Immunohistochemical staining, Staining, Control, Injection, Transplantation Assay

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Spindles in CENP-E (left) and Ndc80 (right) siRNA treated RPE1 cells stably expressing CENP-A-GFP and centrin1-GFP (red) and stained with SiR-tubulin dye (gray). Scale bar, 2 μm.

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques:

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Spindles in Kif18A (top left), Kif18A and Kif4A (top right), Kif18A and PRC1 (bottom left), and Kif18A, Kif4A and PRC1 (bottom right) siRNA treated RPE1 cells stably expressing CENP-A-GFP and centrin1-GFP (red) and stained with SiR-tubulin dye (gray). Scale bar, 2 μm.

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques:

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: A spindle in NuMA siRNA treated RPE1 cell stably expressing CENP-A-GFP and centrin1-GFP (red) and stained with SiR-tubulin dye (gray). Scale bar, 2 μm.

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques:

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Measurements of flux, spindle, and kinetochore parameters

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques:

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Bridging microtubules flux faster than kinetochore microtubules (A and B) Distance between kMT (A) and bMT (B) speckles from the corresponding pole over time in untreated cells. Colored lines show individual speckles. Black line, mean; gray area, SEM. (C) Change in speckle-pole distance over time for speckles within k-fibers and bridging fibers in untreated cells. Circles, mean; error bars, SEM. (D) Poleward velocity of the k-fiber and bridging fiber speckles. Each dot corresponds to an individual speckle. Black lines, mean; gray areas, SEM. (E) Poleward velocity of the k-fiber versus poleward velocity of the bridging fiber. Circles, mean; error bars, SEM. siRNA treatments are color-coded; see legend. Note that Ndc80-depleted and Ndc80-depleted and ZM447439-treated cells are shown as arrows because poleward velocity of k-fibers could not be assessed. Theoretical predictions (lines) for v 0 = 0.1–10 μm/min (pink), and for μ KC = 1–100 pNmin/μm (brown), x KC = 0 μm, and other parameters are as in Figure 1 B. (F) Scheme showing that a speckle within the bridging fiber fluxes faster than a speckle within the k-fiber. (G) Spindle in a cell treated with Ndc80 siRNA and ZM447439 inhibitor. Legend as in Figure 2 A. Scale bar, 2 μm. Statistical analysis conducted using t test. ∗ p = 0.01–0.05, ∗∗ p = 0.01–0.001, ∗∗∗ p = 0.001–0.0001, ∗∗∗∗ p < 0.0001; ns, p ≥ 0.05.

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques:

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Kinetochore alignment depends on the ratio of k-fiber to bridging fiber flux velocity (A) Spindles in untreated, Kif18A, Kif18A and Kif4A, Kif18A and PRC1, and Kif18A, Kif4A and PRC1 depleted cells (from left to right). Legend as in Figure 2 A. Scale bars, 2 μm. (B) Scheme shows that the distance from equator was measured as the distance between sister kinetochore midpoint and the equatorial plane. (C) Kinetochore distance from equator in untreated and siRNA-treated cells. Each treatment is compared with untreated cells. Black lines, mean; gray areas, SEM. (D) Experimental data for the kinetochore distance from equator versus ratio of k-fiber to bridging fiber flux velocity in untreated and siRNA-treated cells. Circles; mean. Error bars; SEM. Theoretical predictions for centering efficiency, described as x 2 = 2 DT , where T is centering time and is calculated from kinetochore distance from center and centering velocity, T = x KC / v KC , as a function of the ratio of k-fiber to bridging fiber flux velocities. D = 0.009 μm 2 /min and 0.1 μm 2 /min, obtained from the fit to the data by varying the model parameter motor velocity (pink curve) or the effective friction at the kinetochore (brown curve), respectively. Treatments in (C) and (D) are color-coded according to the legend at the bottom. Statistical analysis was conducted using the Mann-Whitney test; p values as in Figure 3 .

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques: MANN-WHITNEY

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet: Coupling between bridging and k-fibers controls k-fiber flux velocity (A) Fixed spindles in RPE1 cells stably expressing CENP-A-GFP and centrin1-GFP (red). Cells are untreated, Kif18A, Kif4A, and Kif18A and Kif4A depleted (from left to right), immunostained for endogenous PRC1 (AF-594, green) and stained with DAPI (blue). Images are sum intensity projections of five z-planes. Scale bars, 1 μm. Graphs show normalized pole-to-pole PRC1 intensity profiles of complete spindles for corresponding treatments. For individual cells see Figure S7 E. Colored line, mean. (B) Scheme shows that spindles with shorter (left) and longer (right) overlap regions have better (left) and worse (right) kinetochore alignment at the spindle equator, respectively. (C and D) K-fiber (C) and bridging fiber (D) flux velocity versus PRC1-labeled overlap length. Treatments are color-coded as shown in the legend below. Circles, mean; error bars, SEM. (E) Scheme of NuMA localization. (F) Montage over time demonstrating the movement of a speckle belonging to the k-fiber (left) and bridging fiber (right) in NuMA siRNA treatment. Legend as in Figure 2 B. (G) Change in speckle-pole distance over time for speckles within bridging and k-fibers in cells treated with NuMA siRNA. Circles, mean; error bars, SEM. (H) Poleward velocity of the speckles in NuMA siRNA-treated (red, k-fiber; blue, bridging fiber) and untreated (gray) cells. Black lines, mean; gray areas, SEM. Statistical analysis was conducted using t test; p values as in Figure 3 .

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques: Stable Transfection, Expressing, Staining, Labeling

Journal: Cell Reports

Article Title: Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

doi: 10.1016/j.celrep.2022.111169

Figure Lengend Snippet:

Article Snippet: Human NuMA siRNA , Santa Cruz Biotechnology , #Cat sc-43978.

Techniques: Recombinant, Modification, Saline, Transfection, Software