Figure S4.
Characterization of IQGAP1-expressing NIH-3T3 cells. (A) Blot confirming two clonal IQGAP1 knockout cell lines. Blot was probed for IQGAP1 (1:1,000; BD610612) and α-tubulin (1:2,500; ab18251) as a loading control, with appropriate secondary antibodies (1:5000 IR-dye-680 for IQGAP1 and IR-dye-800 for α-tubulin). (B) Blot of NIH-3T3 cell extracts indicating expression of IQGAP1 plasmids (primary: 1:1,000, BD610612; secondary: 1:5,000, IR-dye-680). Ponceau stained membrane used as loading control. −, (mock) no vector. +, cells transfected with IQGAP1 plasmid. (C) TIRF images of IQGAP1 knockout cells probed for IQGAP1 (primary: 1:250, BD610612; secondary: 1:1,000, 488- Alexa Fluor 488 donkey anti-mouse) and SiR-SNAP labeling transfected with mock or SNAP-IQGAP1 plasmid. (D) Quantification of transfection efficiency from images in C (n = 11 fields of view [FOVs] per condition from three different coverslips). Shaded dots were grouped by each coverslip. Scale, 100 µm. Error bars, SE. Statistics, Student’s t test: (a) P ≤ 0.05 compared with mock. (E) Images showing GFP and DIC signals to determine the transfection efficiency of GFP-actin plasmids by cells also transfected with plasmids harboring each IQGAP1 (untagged, SNAP-tagged, or SNAP-IQGAP1(BAD)), or with no second plasmid (mock and Endogenous [WT]), as indicated. (F) Quantification of transfection efficiency from images in E (n = 60 FOVs [dots] analyzed per condition, pooled from three coverslips). Scale, 100 µm. Error bars, SE. Statistics, ANOVA: ns, P ≥ 0.05 comparing all conditions. (G and H) Morphology on micropatterns of NIH-3T3 cells expressing the various IQGAP1 plasmids in E and H associated quantification. Scale, 10 μm. Dots represent measurements from individual cells (n = 27–35 cells per condition). Error bars, SD. Statistics, ANOVA: (a) P ≤ 0.05 compared with endogenous MmIQGAP1; (b) P ≤ 0.05 compared with mock (IQGAP1 knockout cells); (c) P ≤ 0.05 compared with IQGAP1 knockout cells expressing SNAP-IQGAP1 on a plasmid; ns, P ≥ 0.05 comparing treatments under the line. (I and J) Maximum intensity images of microtubules from cells in G and J associated quantification (total α-tubulin signal). Statistics, ANOVA: comparisons as in H. Source data are available for this figure: SourceData FS4.

Characterization of IQGAP1-expressing NIH-3T3 cells. (A) Blot confirming two clonal IQGAP1 knockout cell lines. Blot was probed for IQGAP1 (1:1,000; BD610612) and α-tubulin (1:2,500; ab18251) as a loading control, with appropriate secondary antibodies (1:5000 IR-dye-680 for IQGAP1 and IR-dye-800 for α-tubulin). (B) Blot of NIH-3T3 cell extracts indicating expression of IQGAP1 plasmids (primary: 1:1,000, BD610612; secondary: 1:5,000, IR-dye-680). Ponceau stained membrane used as loading control. −, (mock) no vector. +, cells transfected with IQGAP1 plasmid. (C) TIRF images of IQGAP1 knockout cells probed for IQGAP1 (primary: 1:250, BD610612; secondary: 1:1,000, 488- Alexa Fluor 488 donkey anti-mouse) and SiR-SNAP labeling transfected with mock or SNAP-IQGAP1 plasmid. (D) Quantification of transfection efficiency from images in C (n = 11 fields of view [FOVs] per condition from three different coverslips). Shaded dots were grouped by each coverslip. Scale, 100 µm. Error bars, SE. Statistics, Student’s t test: (a) P ≤ 0.05 compared with mock. (E) Images showing GFP and DIC signals to determine the transfection efficiency of GFP-actin plasmids by cells also transfected with plasmids harboring each IQGAP1 (untagged, SNAP-tagged, or SNAP-IQGAP1(BAD)), or with no second plasmid (mock and Endogenous [WT]), as indicated. (F) Quantification of transfection efficiency from images in E (n = 60 FOVs [dots] analyzed per condition, pooled from three coverslips). Scale, 100 µm. Error bars, SE. Statistics, ANOVA: ns, P ≥ 0.05 comparing all conditions. (G and H) Morphology on micropatterns of NIH-3T3 cells expressing the various IQGAP1 plasmids in E and H associated quantification. Scale, 10 μm. Dots represent measurements from individual cells (n = 27–35 cells per condition). Error bars, SD. Statistics, ANOVA: (a) P ≤ 0.05 compared with endogenous MmIQGAP1; (b) P ≤ 0.05 compared with mock (IQGAP1 knockout cells); (c) P ≤ 0.05 compared with IQGAP1 knockout cells expressing SNAP-IQGAP1 on a plasmid; ns, P ≥ 0.05 comparing treatments under the line. (I and J) Maximum intensity images of microtubules from cells in G and J associated quantification (total α-tubulin signal). Statistics, ANOVA: comparisons as in H. Source data are available for this figure: SourceData FS4.

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