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. 2023 Mar 10;12:e84391. doi: 10.7554/eLife.84391

Figure 3. Inhibitors of F-actin but not of endocytosis reduce intercellular transfer of Cas9.

(A–D, F) Donor cells: HEK293T wild-type (WT) with stable overexpression of his tagged Cas9-GFP/gRNA (His-Cas9/gRNA), with stable overexpression of SBP tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA) or with stable overexpression of SBP-Cas9-GFP/gRNA and Myc-streptavidin-CD63-mCherry (Str-CD63/SBP-Cas9/gRNA). The recipient cell line was MDA-MB-231 with a reporter plasmid. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. (A) Nluc/Fluc activities were measured after donor cells and acceptor cells were co-cultured for 3 days with DMSO or different inhibitors. (B) The indicated genes were knocked down in recipient cells via siRNA and then co-cultured with donor cells for 3 days. siCtrl represents a negative control for the siRNA knockdown. CLTC, clathrin heavy chain; AP2B1, adaptor-related protein complex 2 subunit beta 1; CAV-1, caveolin 1; FLOT2, flotillin 2. (C) The donor cells and acceptor cells were co-cultured for 3 days with either DMSO, 40, 80, or 200 nM latrunculin A (LatA), or 1, 2.5, or 5 μM latrunculin B (LatB). The Nluc/Fluc signal detected after co-culture suggested that more than half of the cells remained viable during drug treatment. (D) The indicated genes were knocked down in recipient cells via shRNA that were then co-cultured with donor cells for 3 days. siCtrl represents negative control for the siRNA knockdown. (E) Donor cells: HEK293T with stable overexpression of SBP tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA). The recipient cell line was MDA-MB-231 with the reporter plasmid. The indicated genes were knocked down in donor cells via shRNA that were then co-cultured with recipient cells for 3 days. (F) Donor cells and acceptor cells were co-cultured for 4 days with either DMSO, 10, or 25 μM formin inhibitor, SMIFH2. The Nluc/Fluc signal detected after co-culture suggested that more than 70% of the cells remained viable during drug treatment. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. Data in this figure represent mean ± SEM, n ≥ 3. ****p<0.0001, one-way ANOVA.

Figure 3.

Figure 3—figure supplement 1. Endocytosis inhibitors and endocytosis protein knockdown block transferrin uptake.

Figure 3—figure supplement 1.

(A) MDA-MB-231 cells were incubated with indicated inhibitors for 48 hr. Cells were washed 2× and then incubated with pHrodo red transferrin conjugate and Hoechst 33342 for 20 min, washed 4×, and observed cells by confocal microscopy. Scale bar is 10 μm. (B) The integrated intensity per cell from (A) was quantified by ImageJ. n = 200 cells were captured. Data represent mean ± SEM, n ≥ 3. ****p<0.0001, one-way ANOVA. (C) MDA-MB-231 cells were incubated with indicated inhibitors for 48 hr and then incubated with Phrodo Green Zymosan Bioparticles and Hoechst 33342. Scale bar is 10 μm. (D) The integrated intensity per cell from (C) was quantified by ImageJ. n = 200 cells were captured. Data represent mean ± SEM, n ≥ 3. ***p<0.001, two-tailed t-test. (E, F) The endocytosis factors were knocked down in MDA-MB-231 cells for 48 hr and processed for visualization and quantification as in (A, B). Data represent mean ± SEM, n ≥ 3. ****p<0.0001, one-way ANOVA. Scale bar is 10 μm.
Figure 3—figure supplement 2. Knockdown validation.

Figure 3—figure supplement 2.

(A–D) The endocytosis factors were knocked down for 72 hr in MDA-MB-231 cells, and lysate samples were assessed by SDS-PAGE and immunoblotting. (E–G) Actin or Arp2/3 complex were knocked down in MDA-MB-231 cells. (H–J) Actin or Arp2/3 complex were knocked down in HEK293T cells.
Figure 3—figure supplement 2—source data 1. Uncropped Western blot images corresponding to Figure 3—figure supplement 2A.
Figure 3—figure supplement 2—source data 2. Uncropped Western blot images corresponding to Figure 3—figure supplement 2B.
Figure 3—figure supplement 2—source data 3. Uncropped Western blot images corresponding to Figure 3—figure supplement 2C.
Figure 3—figure supplement 2—source data 4. Uncropped Western blot images corresponding to Figure 3—figure supplement 2D.
Figure 3—figure supplement 2—source data 5. Uncropped Western blot images corresponding to Figure 3—figure supplement 2E and H.
Figure 3—figure supplement 2—source data 6. Uncropped Western blot images corresponding to Figure 3—figure supplement 2F and I.
Figure 3—figure supplement 2—source data 7. Uncropped Western blot images corresponding to Figure 3—figure supplement 2G and J.