Figure S5.
Localization and function of ARHGEF17 in relation to E-cadherin, roles of E-cadherin and ARHGEF17 in RhoA activation, and roles of RhoA and ARHGEF17 in spindle orientation, related to Fig. 7. (A) RhoA-specific GEFs identified by BioID analysis. ARHGEF17 is shown as an isolated candidate because it was detected in both E-cadherin replicates without a background signal in biotin (−) but was excluded by the filter due to high background intensity in untransfected cells. GO enrichment analysis of the protein–protein interaction network (STRING) is shown on the right. Note that ARHGEF17 is predicted to localize to the cleavage furrow and contractile ring, whereas ARHGEF11 is not. (B) Schematic of rat ARHGEF17. ABR, actin-binding region; DH, Dbl homology; PH, pleckstrin homology; WD40, WD40-repeat domain. (C) ARHGEF17 localizes to the cell cortex and AJs in cells with primordial or tubular BCs. Shown are representative confocal images of Can 10 cells expressing ARHGEF17-GFP, cultured for 2 days after transduction, and stained with DAPI, phalloidin, and anti-E-cadherin antibody. (D–F) Localization of ARHGEF17 at AJs depends on F-actin and E-cadherin. (D) Time-lapse montage of Can 10 cells expressing ARHGEF17-GFP and mScarlet–β-actin before and after treatment with LatA. Arrows indicate AJs positive for ARHGEF17. See also Video 2. (E) Representative confocal images of Can 10 cells expressing ARHGEF17-GFP after treatment with 10 µM LatA for 30 min, combined with control siRNA or E-cadherin siRNA, and stained with DAPI, phalloidin, and anti-E-cadherin antibody. The regions indicated by arrowheads are shown as zoomed images. (F) Quantification of ARHGEF17-GFP intensity at AJs relative to the cytoplasm following treatment with LatA and/or siRNA. Data are from three independent experiments (≥16 cells per condition). (G) E-cadherin is required for ARHGEF17 localization at AJs but not PM. Quantification of ARHGEF17-GFP intensity at AJs relative to the cytoplasm (left) or PM to the cytoplasm (right) in control siRNA- or E-cadherin siRNA #1–transfected cells. Data are from three independent experiments (≥22 cells per condition). (H) Decrease in RhoA activity upon E-cadherin depletion. Top: representative confocal images of dT-2×rGBD–expressing Can 10 cells transfected with E-cadherin siRNA. Arrows indicate BCs with the reduced intensity of active RhoA in E-cadherin knockdown cells. Bottom: ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in control and E-cadherin-knockdown cells. Values are from two independent experiments (≥47 cells per condition). (I) Validation of ARHGEF17 siRNA efficiency. Immunoblot analysis of ARHGEF17-GFP-expressing HEK293T cells transfected with rat ARHGEF17 siRNAs. MWs of marker proteins are indicated in kDa. (J) ARHGEF17 depletion does not affect protein levels of E-cadherin, N-cadherin, p120-catenin, and RhoA. Left: representative immunoblots from Can 10 cells transfected with ARHGEF17 siRNAs. Right: quantification of indicated protein levels normalized to α-tubulin. Data represent means ± SD from three independent experiments; values are normalized to control siRNA-transfected cells. (K and L) Restoration of Rho activity by rescue expression of ARHGEF17. (K) dT-2×rGBD–expressing Can 10 cells were transfected with ARHGEF17 siRNA #2 one day earlier, after which they were transduced with lentiviruses expressing either GFP, si#2-resistant ARHGEF17-GFP, or ARHGEF17ΔDH-GFP. Representative confocal images are shown. Yellow arrows indicate BCs in ARHGEF17-knockdown cells. Note that RhoA activity was only restored by the expression of siRNA-resistant ARHGEF17. (L) Ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in the cells shown in (K). Values are from two independent experiments (≥18 cells per condition). (M) ARHGEF17 regulates oriented cell division. Proportions of dividing cells expressing dT-2×rGBD that undergo symmetric vs. asymmetric BC inheritance were quantified following transfection with control siRNA or ARHGEF17 siRNA and time-lapse confocal imaging from two independent experiments (≥31 cells per condition). (N) Depletion of ARHGEF17 and RhoA alters mitotic spindle orientation. Combined scatter and box-and-whisker plots show spindle angle distributions relative to the BC. Data are from two independent experiments (≥28 cells per condition). (O) Quantitative analysis of E-cadherin localization at AJs. Ratios of E-cadherin intensity at AJs to cytoplasmic intensity are shown. Data are from two independent experiments (≥21 cells per condition). Scale bars, 1 µm (zoomed images in E), 5 µm (E), 10 µm (D and K), 20 µm (C and H). P values are indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData FS5. Refer to the image caption for details. Panel A shows a schematic and Gene Ontology enrichment plots identifying RhoA-specific guanine nucleotide exchange factors associated with E-cadherin and N-cadherin complexes. Panel B shows a domain structure schematic of the ARHGEF17 protein including ABR, DH, PH, and WD40 regions. Panel C shows confocal fluorescence micrographs of cells expressing ARHGEF17–GFP stained for E-cadherin, F-actin, and nuclei in primordial and tubular bile canaliculi structures. Panel D shows time-lapse fluorescence micrographs of ARHGEF17–GFP and mScarlet-actin localization during Latrunculin A treatment. Panel E shows confocal fluorescence micrographs and enlarged views of ARHGEF17–GFP localization after Latrunculin A treatment and E-cadherin knockdown. Panel F shows box-and-whisker plots quantifying ARHGEF17 localization intensity at adherens junctions relative to cytosol after drug treatment and siRNA depletion. Panel G shows box-and-whisker plots quantifying ARHGEF17 localization at adherens junctions and plasma membrane regions after E-cadherin depletion. Panel H shows fluorescence micrographs and box-and-whisker plots analyzing active RhoA biosensor localization after E-cadherin knockdown. Panel I shows immunoblot images validating ARHGEF17 siRNA efficiency in transfected HEK293T cells. Panel J shows immunoblot images and bar graphs analyzing E-cadherin, N-cadherin, p120-catenin, and RhoA protein levels after ARHGEF17 depletion. Panel K shows fluorescence micrographs of active RhoA biosensor localization rescued by siRNA-resistant ARHGEF17 expression constructs. Panel L shows box-and-whisker plots quantifying active RhoA biosensor intensity at bile canaliculi relative to cytosol under rescue conditions. Panel M shows a stacked bar graph quantifying symmetric and asymmetric bile canaliculi inheritance after ARHGEF17 depletion. Panel N shows scatter plots and box-and-whisker plots comparing spindle angle distributions after RhoA or ARHGEF17 depletion. Panel O shows box-and-whisker plots quantifying E-cadherin intensity ratios at adherens junctions relative to cytoplasm after ARHGEF17 depletion.

Localization and function of ARHGEF17 in relation to E-cadherin, roles of E-cadherin and ARHGEF17 in RhoA activation, and roles of RhoA and ARHGEF17 in spindle orientation, related to Fig. 7. (A) RhoA-specific GEFs identified by BioID analysis. ARHGEF17 is shown as an isolated candidate because it was detected in both E-cadherin replicates without a background signal in biotin (−) but was excluded by the filter due to high background intensity in untransfected cells. GO enrichment analysis of the protein–protein interaction network (STRING) is shown on the right. Note that ARHGEF17 is predicted to localize to the cleavage furrow and contractile ring, whereas ARHGEF11 is not. (B) Schematic of rat ARHGEF17. ABR, actin-binding region; DH, Dbl homology; PH, pleckstrin homology; WD40, WD40-repeat domain. (C) ARHGEF17 localizes to the cell cortex and AJs in cells with primordial or tubular BCs. Shown are representative confocal images of Can 10 cells expressing ARHGEF17-GFP, cultured for 2 days after transduction, and stained with DAPI, phalloidin, and anti-E-cadherin antibody. (D–F) Localization of ARHGEF17 at AJs depends on F-actin and E-cadherin. (D) Time-lapse montage of Can 10 cells expressing ARHGEF17-GFP and mScarlet–β-actin before and after treatment with LatA. Arrows indicate AJs positive for ARHGEF17. See also Video 2. (E) Representative confocal images of Can 10 cells expressing ARHGEF17-GFP after treatment with 10 µM LatA for 30 min, combined with control siRNA or E-cadherin siRNA, and stained with DAPI, phalloidin, and anti-E-cadherin antibody. The regions indicated by arrowheads are shown as zoomed images. (F) Quantification of ARHGEF17-GFP intensity at AJs relative to the cytoplasm following treatment with LatA and/or siRNA. Data are from three independent experiments (≥16 cells per condition). (G) E-cadherin is required for ARHGEF17 localization at AJs but not PM. Quantification of ARHGEF17-GFP intensity at AJs relative to the cytoplasm (left) or PM to the cytoplasm (right) in control siRNA- or E-cadherin siRNA #1–transfected cells. Data are from three independent experiments (≥22 cells per condition). (H) Decrease in RhoA activity upon E-cadherin depletion. Top: representative confocal images of dT-2×rGBD–expressing Can 10 cells transfected with E-cadherin siRNA. Arrows indicate BCs with the reduced intensity of active RhoA in E-cadherin knockdown cells. Bottom: ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in control and E-cadherin-knockdown cells. Values are from two independent experiments (≥47 cells per condition). (I) Validation of ARHGEF17 siRNA efficiency. Immunoblot analysis of ARHGEF17-GFP-expressing HEK293T cells transfected with rat ARHGEF17 siRNAs. MWs of marker proteins are indicated in kDa. (J) ARHGEF17 depletion does not affect protein levels of E-cadherin, N-cadherin, p120-catenin, and RhoA. Left: representative immunoblots from Can 10 cells transfected with ARHGEF17 siRNAs. Right: quantification of indicated protein levels normalized to α-tubulin. Data represent means ± SD from three independent experiments; values are normalized to control siRNA-transfected cells. (K and L) Restoration of Rho activity by rescue expression of ARHGEF17. (K) dT-2×rGBD–expressing Can 10 cells were transfected with ARHGEF17 siRNA #2 one day earlier, after which they were transduced with lentiviruses expressing either GFP, si#2-resistant ARHGEF17-GFP, or ARHGEF17ΔDH-GFP. Representative confocal images are shown. Yellow arrows indicate BCs in ARHGEF17-knockdown cells. Note that RhoA activity was only restored by the expression of siRNA-resistant ARHGEF17. (L) Ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in the cells shown in (K). Values are from two independent experiments (≥18 cells per condition). (M) ARHGEF17 regulates oriented cell division. Proportions of dividing cells expressing dT-2×rGBD that undergo symmetric vs. asymmetric BC inheritance were quantified following transfection with control siRNA or ARHGEF17 siRNA and time-lapse confocal imaging from two independent experiments (≥31 cells per condition). (N) Depletion of ARHGEF17 and RhoA alters mitotic spindle orientation. Combined scatter and box-and-whisker plots show spindle angle distributions relative to the BC. Data are from two independent experiments (≥28 cells per condition). (O) Quantitative analysis of E-cadherin localization at AJs. Ratios of E-cadherin intensity at AJs to cytoplasmic intensity are shown. Data are from two independent experiments (≥21 cells per condition). Scale bars, 1 µm (zoomed images in E), 5 µm (E), 10 µm (D and K), 20 µm (C and H). P values are indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData FS5.

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