EMBO/SourceData huggingface dataset:
Figure 2Left panel: EdU incorporation in siRNA-transfected Capan-1 cells (72 h post-transfection), n = 6 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: siTBL1#1 8.24 × 10−4 (*), 5.43 × 10−4 (#), 7.93 × 10−6 ($); siTBL1 #2 1.66 × 10−11 (*), 1.27 × 10−11 (#), 8.79 × 10−13 ($), one-way ANOVA with Bonferroni post-test. Right panel: EdU incorporation of Panc02 cells with stable expression of shRNA; n = 6 cell culture wells per group; significantly different (P < 0.05) from control (shNC) #P = 0.0398; Welch's t-test.Cellgrowth of siRNA-transfected Capan-1 or Panc8680 cells; n = 3 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: Capan-1 cells 48 h time-point: 0.0408 (*) for siTBL1 #1 / 0.0464 for siTBL1 #2, Capan-1 cells 72 h time-point: 8.60 × 10−5 (*), 1.82 × 10−3 (#), 3.24 × 10−5 ($) for siTBL1 #1 / 2.39 × 10−10 (*), 3.34 × 10−9 (#), 1.08 × 10−10 ($) for siTBL1 #2; Panc8680 72 h time-point: 1.55 × 10−4 (*), 2.73 × 10−5 ($); two-way ANOVA with Bonferroni post-test.Uptake of radio-labeled 3H-2-deoxyglucose in siRNA-transfected Capan-1 cells (48 h post-transfection); n = 6 cell culture wells per group; statistically different (P < 0.05) from control (siNC #2) $P = 0.03; Welch's t-test.Extracellular flux measurement of siRNA-transfected Capan-1 cells with glycolysis stress test assay (48 h post-transfection); 2-DG: 2-deoxyglucose; ECAR: extracellular acidification rate. n = 6-8 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2) $. Upper symbols: statistics for siTBL#1, P-values: 2.18 × 10−3 (*) time-point 8, 9.83 × 10−3 (*) time-point 9, 0.0102 (*) time-point 10, 0.044 (*) time-point 11; 2.42 × 10−6 (#) time-point 8, 1.47 × 10−4 (#) time-point 9, 3.17 × 10−4 (#) time-point 10, 0.0277 (#) time-point 11; 9.44 × 10−7 ($) time-point 8, 1.23 × 10−6 ($) time-point 9, 1.33 × 10−6 ($) time-point 10, 7.14 × 10−5 ($) time-point 11. Lower symbols: statistics for siTBL#2, P-values: 3.94 × 10−3 (*) time-point 5, 0.0143 (*) time-point 6, 0.0342 (*) time-point 7, 1.45 × 10−3 (*) time-point 8, 1.27 × 10−3 (*) time-point 9, 2.38 × 10−3 (*) time-point 10; 0.01 (#) time-point 5, 0.0362 (#) time-point 6, 1.22 × 10−6 (#) time-point 8, 1.28 × 10−5 (#) time-point 9, 5.84 × 10−5 (#) time-point 10; 0.0118 ($) time-point 5, 0.0208 ($) time-point 6, 0.0291 ($) time-point 7, 4.52 × 10−7 ($) time-point 8, 6.04 × 10−8 ($) time-point 9, 1.43 × 10−7 ($) time-point 10, 4.83 × 10−3 ($) time-point 11. Two-way ANOVA with Bonferroni post-test.
Figure 3Gene expression microarray from Capan-1 cells transfected with siRNA. Heatmap shows genes annotated to KEGG pathway hsa04110 "Cell Cycle" and/or gene ontology cluster GO:0007049 "Cell Cycle".Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice.Chromatin immunoprecipitation from Capan-1 cells with or without siRNA-mediated knockdown of TBL1. IgG and primers for PIK3CA coding region served as a negative control; histone H3 served as a positive control.Proliferation time course assay in control and TBL1 shRNA-transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K. Cells were stained with crystal violet, and absorbance was measured at 595 nm. n = 9 cell culture wells per group; statistically significant at P < 0.05 for indicated comparisons: shNC pBabe-puro vs. shNC pBabe-PI3K (*), shTBL1 pBabe-puro vs. sbTBL1 pBabe-PI3K (#), shNC pBabe-puro vs. shTBL1 pBabe-puro ($), shNC pBabe-PI3K vs. shTBL1 pBabe-PI3K (†); P-values: day 2: 2.01 × 10−3 (#), 4.11 × 10−9 ($), 5.77 × 10−3 (†); day 3: 4.50 × 10−5 (*), 1.01 × 10−9 (#), 1.71 × 10−30 ($), 1.22 × 10−6 (†); day 4: 5.61 × 10−22 (*), 1.10 × 10−45 (#), 6.65 × 10−62 ($); two-way ANOVA with Bonferroni post-test.Matrigel invasion assay in same cells as in (D). Invading cells per microscopy field were counted. n = 6 microscopy fields per group; statistically significant at P < 0.05; P-values: 6.64 × 10−4 (*), 1.35 × 10−7 (#), 6.94 × 10−5 ($); two-way ANOVA with Bonferroni post-test.mRNA expression of PIK3CA in human patient samples; significant difference (P < 0.05) between tumor and normal tissue *P = 2.13 × 10−4 determined by paired two-tailed Student's t-test, n = 18 patients.Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue (normalized to 18S rRNA and relative to a pooled organ donor sample); n = 17 patients; P-value determined by testing for slope of regression line ≠ 0.Survival analysis in 105 patients with PDAC: High PIK3CA expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.
Figure 4A-D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6/N mice. Six days later, tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA. (A-C): Data plotted as mean ± SEM; n = 4 animals in control (shNC) group and n = 3 animals in knockdown (shTBL1) group; significantly different (P < 0.05) between control (shNC) and knockdown (shTBL1) *. (A) Tumor volume determined by transcutaneous measurement with digital calipers; P-values: 9.21 × 10−3 at day 15, 6.02 × 10−4 at day 18, 2.49 × 10−4 at day 20; two-way ANOVA with Bonferroni post-test. (B) Luminescence of tumors after intraperitoneal injection of firefly d-luciferin; P = 0.0133 at day 20; two-way ANOVA with Bonferroni post-test. (C) Tumor mass at necropsy; P = 0.0299, one-sided Welch's t-test. (D) Luminescence images of one representative mouse in each group.E-G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection; n = 4 animals in shNC+GEM group, n = 5 animals in all other groups; significantly different (P < 0.05) between groups: shNC vs. shNC+GEM (*), shNC vs. shTBL1 (#), shNC vs. shTBL1+GEM ($), shNC+GEM vs. shTBL1 (%), shNC+GEM vs. shTBL+GEM (†); shTBL1 vs. shTBL1+GEM (‡); two-way ANOVA with Bonferroni post-test. (E) Tumor volume determined by transcutaneous measurement with digital calipers. P-values: day 17: 8.48 × 10−3 (#), 1.07 × 10−3 ($), day 20: 3.07 × 10−3 (*), 9.37 × 10−6 (#), 1.56 × 10−8 ($), 0.0239 (†). (F) Tumor volume relative to the volume at day 7 post-implantation; P-values: day 17: 3.94 × 10−3 ($), day 20: 1.09 × 10−3 (*), 1.39 × 10−7 ($), 0.01195 (%), 3.12 × 10−6 (‡). (G) Area under curve for relative tumor volume; P = 0.0195 (‡).
Figure 5TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.
Figure 1DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C).DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent DU145 (E) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image.
Figure 2(A) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1 and β-catenin with cell confluency. E-cadherin was used as a positive control. Tubulin was used as a loading control., immunofluorescent labelling of UBTD1 and E-cadherin (B) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.immunofluorescent labelling of UBTD1 and β-catenin (C) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.Immunoblots of UBTD1, β-catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). EGFR, RhoGDI and Lamin A/C were used as controls for the membrane (M), cytosol (C) and nucleus (N) compartments, respectively.Co-immunoprecipitation between endogenous β-catenin and UBTD1 was performed in confluent DU145 cells. IgG isotype was used as a negative control.(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.(H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image.
Figure 3Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.
Figure 4(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control.(B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control.(C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.(D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control.(E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion.Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool).(H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.
Figure 5DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Quantification of CTGF1, CYR61 and BIRC5 mRNA levels.(C) Immunoblots of YAP in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1 (+). Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Immunoblot of GFP shows the level of UBTD1 transfection. CHC was used as a loading control.(D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image.(E) Immunoblots of p-YAP and YAP in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Immunoblot of p-YAP reflects the activity of the Hippo pathway. Immunoblot of UBTD1 and LATS show siRNA knock-down efficiency. Actin was used as a loading control.Representative confocal immunofluorescence images (F) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (G) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool).(H) Immunoblots of YAP in DU145 control (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Immunoblots of UBTD1, ROCK1 and ROCK2 show the level of siRNA knock-down efficiency. Actin was used as a loading control.Representative confocal immunofluorescence images (I) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.quantification (J) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).(K) Quantification of CTGF1, CYR61, UBTD1 and BIRC5 mRNA in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).
Figure 6DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (A) Quantification of EMT marker genes (vimentin, slug, snail, fibronectin, α-SMA, E-cadherin) and UBTD1.(B) Immunoblots of EMT markers (vimentin, E-cadherin, slug). Immunoblot of UBTD1 shows siRNA knock-down efficiency. Hsp90 and tubulin were used as loading controls.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (C) Quantification of DU145 cell migration in Transwell chamber inserts.DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (D) Quantification of DU145 cell invasion in Transwell chamber inserts.(E) Invasive properties of human prostate tumoroids depleted for UBTD1.(F) Quantification of UBTD1 mRNA levels in prostate tumor (n=70) and adjacent normal tissues (n=47).(G) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues (n=8).(H) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues (n=6).(I) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients (56 patients) are correlated with overall survival (OS). OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value.(J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS.
Figure 1(C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.
Figure 2(A) Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ S. flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post infection (mpi) are shown.(B) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.(C) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.
Figure 3(A) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.(B) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.(C) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.