EMBO/SourceData huggingface dataset:
Figure 5HERC3 knockdown inhibits breast tumorigenesis. Luciferase-harboring MDA-MB-231 cells expressing shHERC3 or shCtrl were subcutaneously injected into 5-week-old nude mice. Four weeks after injection, mice were monitored by bioluminescence using Xenogen IVIS imaging system. A, representative tumors expressing luciferase are indicated by radiance bar (p/sec/cm2/sr). B, quantitation of bioluminescence data. Data are shown as mean ±SEM; n = 6 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01, ***P < 0.001.J, K shHERC3 attenuates breast cancer lung metastasis. Mice were injected with control or HERC3-deficient MDA-MB-231 cells. J, Bright-field images (upper panel, scale bars, 2 mm) and H&E staining (lower panel, scale bars, 50 μm) of the lungs from mice. K, number of lung metastasis nodules. Data are shown as mean ±SEM; n = 5 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.RNAi-resistant HERC3 and unphosphorylatable YAP-S381A can counteract the tumor suppressive effect of HERC3 depletion. HERC3-deficient cells with or without ectopic expression of YAP-S381A, RNAi-resistant variants of HERC3-WT or HERC3-CA and control MDA-MB-231 cells were performed subcutaneously injection as described in Panel A. L, tumors were dissected and photographed at four weeks after cell implantation. M, volume from all tumors was recorded.
Figure 6HERC3 is highly expressed in breast cancer specimens. A, representative images of IHC staining of HERC3 in tumor samples of breast cancer specimens and their paired adjacent normal tissues. Scale bars, 50 µm.D HERC3 is correlated with poor prognosis in breast cancer patients. Kaplan-Meier curves for overall survival of breast cancer patients according to the HERC3 expression level (upper left panel) (http://kmplot.com/analysis/index.php?p=service&cancer=breast). HERC3 is negatively correlated with the overall survival (upper right panel), the distant metastasis free survival (lower left panel) and the relapse free survival (lower right panel) in breast cancer patients. Microarray analysis was performed to examine the prognostic potential of HERC3 in breast cancer using the PrognoScan database with one cohort (GSE7390) of 198 breast cancer specimens (http://www.prognoscan.org). E HERC3 expression is positively correlated with TAZ or active YAP expression in breast cancer patients. Representative images of IHC staining of HERC3, TAZ and active YAP in tumor samples of breast cancer patients
Figure 2(A) Sequential COX-SDH histochemical staining in muscle biopsies of Pa2, Pa3-1, Pa4 and Pa6 (as indicated), indicating a mosaic pattern of COX deficiency. Scale bar represents 100 µm.(C) Long-range PCR assay to detect mtDNA deletions in TOP3A patient muscle (lanes 2-5). Samples from unaffected individuals (lanes 6 and 7) and a single-deletion mitochondrial disease patient (lane 8) are used as negative and positive controls, respectively. 'M' indicates marker.
Figure 3(A-B) Analysis of mtDNA structure in muscle from individuals with TOP3A-related mitochondrial disease using Southern blotting following restriction with BamHI (A) or PvuII (B). The black bar indicates the probe (nt. 16,262 - 128).mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (C) Western blot of TOP3A protein expression. Cells were untransfected (lane 1) or transfected with TOP3A siRNA (lanes 2-11), then induced to express WT TOP3A (lane 3) or pathological variants of TOP3A (lanes 4-11). Note that TOP3A Ser810* does not carry a C-terminal HA tag. β-actin is used as a loading control.mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (D) mtDNA copy number for TOP3A rescue cells as in (C) determined using qPCR. Error bars represent SEM, n = 3 (Ala95Val) or n = 4 (all other samples), where each data point represents the mean of 3 technical triplicates. Significance values are shown for one-way ANOVA compared to untreated wild-type cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (E) Southern blot of mtDNA structure from cells as in (C). The probe (black bar, nt. 16,262 - 128) detects both full-length (FL) mtDNA and 7S DNA. The presence of hemicatenated mtDNAs is indicated. (F) Southern blot of uncut mtDNA to visualise different topological forms, from cells as in (C). Control DNA treated with BamHI (lane 1) or E. coli topoisomerase I (lane 2) act as markers for linear and relaxed monomeric mtDNA, respectively. Diagrams indicate the migration of different mtDNA forms.
Figure 4(A) Purification of TOP3A variants. Each variant (3 pmol) was separated on a 4-20% stain-free Criterion TGX SDS-PAGE gel (Bio-Rad) and imaged using a stain-free imager.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.(K) Quantification of DNA binding activity as in (B-J), expressed as the proportion of bound DNA. Data represents mean values with at least 3 independent experiments per data point. Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01, ****p<0.0001.
Figure 5(A-I) Relaxation of negatively supercoiled pUC19 substrate by recombinant TOP3A variants. 'M' indicates marker.(J) Quantification of DNA relaxation data as in (A-I). Data represents the percentage of substrate released from negatively supercoiled form, expressed as the mean of 3 independent experiments (except WT, for which n = 4). Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. ****p<0.0001.
Figure 6(B-J) ssDNA decatenation assays using TOP3A variants, using substrates constructed as in (A).(K) Quantification of ssDNA decatenation activity as in (B-J). Data represents the percentage of decatenated substrate expressed as mean values, ±SEM, from independent experiments, n = 3 (WT, p.Ala95Val, p.Met100Val, p.Arg558Trp), n = 4 (p.Ala176Val), n = 5 (p.Met575Val), n = 2 (p.Leu37Val). Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01.(M) Quantifications of ssDNA decatenation assays as in (L). Data represents the mean percentage of decatenated substrate from 3 independent experiments. Significance values are shown for one-way ANOVA compared to the WT protein. *p<0.05, **p<0.01, ***p<0.001.
Figure 1HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs in primary (I) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs secondary (J) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.
Figure 2(C) Representative flow cytometric analyses of EdU incorporation and DNA content in HSCs after culture under pH 7.4 and pH 6.9 conditions for 40 h. Percentage of cells in G1, S and G2/M phase of cell cycle under pH 7.4 or pH 6.9 conditions. (n=4 biological replicates, ***p < 0.001; using two-tailed unpaired Student's t-test)(E) Relative levels of expression of the Hif1α transcriptional targets Pdk-1 and Vegfa (n=4 biological replicates, *p < 0.05; using two-tailed unpaired Student's t-test).(F) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7.4 or pH 6.9 conditions (Scale bar = 5μm)(H) Representative ultra-structural transmission electron microscopy images of untreated and cultured HSCs under pH 7.4 or pH 6.9 conditions. Data represent n=20 cells from 2 biological replicates. Arrows label mitochondria, while arrowheads label vacuolar structures (Scale bar = 1μm).
Figure 3(B) Hierarchical clustering and heatmap representing global visualization of all the DEGs in HSCs present at pH 6.9 or pH 7.4 for 40 h (n=6 biological replicates). Results were visualized using Treeview.
Figure 4(C) Relative levels of spermidine and spermine in pH 6.9 and pH 7.4 conditions with respect to untreated samples. n=3 biological replicates. *p < 0.05 and exact p values are mentioned between groups.(E) Effect of DFMO treatment on pHi of HSCs., n= 5 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.(G) Pictures of HSCs after culture at pH 7.4 or pH 6.9 and in presence or absence of DFMO and SPN for 40 h. Data are representative of 6 biological replicates. Scale bar = 100μm (H) Cell volume of HSCs cultured at pH 7.4 in the presence of DFMO or in combination with SPN. Data are from >80 cells per group, representative of 3 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.(I,J) Reconstitution level of donor derived -Ly5.2+ cells, -lineage distribution in peripheral blood and BM in primary (I) and secondary (J) recipients from 100 HSCs cultured at pH 7.4 in presence of DFMO or in combination of SPN. Data in figure I,J are from n= 9-12 recipient mice from 2 independent biological transplantation experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed unpaired Student's t-test.(K) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO (Scale bar = 5μm). Bar graphs represent quantification of LAMP2 levels in cells. Data are from > 50 cell representing 3 biological replicates. ***p < 0.001, ****p < 0.0001 using two-tailed unpaired Student's t-test.(L) Reconstitution level of donor derived Ly5.2+ cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO. n= 7-9 mice from 2 biological transplantation experiments for murine HSC data. For human HSPC transplants, n= 6 recipient per condition from 3 biological transplantation experiments. *p < 0.05, or exact p value mentioned between groups. Statistic significance was calculated using two-tailed unpaired Student's t-test.
Figure 1(B) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control.(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).(D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4.(E) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used.(G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used.(I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml).(K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.
Figure 1(A) Western blot analysis of the immunoproteasome subunits LMP2, LMP7 and β-actin (loading control) in three PolgA+/+ and three PolgAmut/mut cell lines.(B) RT-qPCR analysis of immunoproteasome subunits Psmb8, Psmb9, Psmb10 in three different PolgA+/+ and three PolgAmut/mut cell lines.(C) Activity assay for the catalytic subunits of the proteasome using the pan activity-based probe (ABP) MV151, the β1/LMP2 specific LW124, and the β5/LMP7 specific MVB127 ABPs in three PolgA+/+ and three PolgAmut/mut cell lines. Resolution of β5/LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels. Total protein staining was used as a loading control.
Figure 2(D) representative Western blots for the analysis of the cGAS/STING signaling pathway in PolgA+/+ and PolgAmut/mut cells with β-actin serving as loading control.(E) Secreted IFNβ in supernatants from three distinct PolgA+/+ and PolgAmut/mut cell lines. The 15.81 pg/ml detected by ELISA in PolgAmut/mut cell supernatants corresponds to 18.97 IU/ml recombinant mouse (rm) IFN-β according to the R&D Systems cytokine conversion table(F) RT-qPCR analysis of Stat1 in three different PolgA+/+ and three PolgAmut/mut cell lines.
Figure 3(A) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells after incubation with either PolgA+/+ or PolgAmut/mut cell-conditioned medium for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control set to 1 in PolgA+/+ incubated with either PolgA+/+ or PolgAmut/mut cell-conditioned medium from three different cell lines.(B) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) upon siRNA-mediated silencing of cGAS or STING in PolgAmut/mut cells compared to untransfected (Ctrl) and non-sense scrambled transfection control (NC) in three independent experiments.(E) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) in PolgA+/+ cells transfected with mouse wildtype mtDNA, mtDNA pretreated with DNase I or only DNase I for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in PolgA+/+ cells. Values are given as fold over PolgA+/+ control with the mean of normalized PolgA+/+ signal normalized to 1. (F) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells transfected with cGAMP for 24 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control (Ctrl) set to 1.(G) Protein expression of LMP2, LMP7 and β-actin (loading control) in wildtype (WT), cGAS KO or STINGgt/gt losss of function MEFs that had been transfected with 200 ng/ml mtDNA for 48 h.
Figure 5(B) UTY-assay of CD8+ reporter T cells upon co-culture with different ratios of PolgA+/+ or PolgAmut/mut cells. Data are combined results of three independent experiments and normalized to the signal of maximum induction of PolgA+/+ cells in co-culture with CD8+ reporter T cells.(E) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either WT or LMP7 KO mice and transfected with mtDNA. Splenocytes were isolated from 4 mice each in two different preparations.
Figure 6(A) Western blot analysis of immunoproteasome subunits LMP2, LMP7, and of STAT1 in pmAT2 cells transfected with mtDNA or mtDNA pretreated with DNase I for 48 h or treated with IFNβ for 24 h. β-actin was used as a loading control. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in pmAT2 from three independent pmAT2 preparations.(C) Representative Western blot analysis of phLF for LMP2, LMP7, STAT1, and β-actin (loading control) upon transfection of mtDNA or mtDNA pretreated with DNase I for 24 h, 48 h or 72 h.(G) Western blot analysis of STING, pTBK1, TBK1, pIRF3, IRF3, pIκBα, IκBα, pP65, P65 and β-actin (loading control) in phLFs transfected with mtDNA for 2 h, 6 h, or 24 h, respectively.
Figure 7(B) Flow cytometric detection of Influenza-M1/HLA-A2 complex in phLF upon transfection with mtDNA, mtDNA pretreated with DNase I, or treatment with IFNβ (n=3 independent experiments).(D) CD8+ T cell activation assay of Influenza-M1/HLA-A2 restricted CD8+ T cells upon co-culture with mtDNA-transfected phLFs loaded with M1 peptide or upon treatment with IFNβ or IFNγ displaying secretion of IL-2 from three different biological experiments.
Figure 8(A) Western blot analysis of the immunoproteasome subunits LMP2 and LMP7 in wild type (WT), cGAS KO and STINGgt/gt loss of function MEFs transfected with 1 μg/ml HT-DNA or plasmid-DNA for 48 h. β-actin was used as a loading control. Densitometric analysis of LMP2 and LMP7 expression normalized to β-actin loading control in wild type (WT) controls (ctrl) from three independent experiments.(B) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either wildtype (WT) or LMP7 KO mice and transfected with htDNA. Splenocytes were isolated from 4 mice each in two different preparations.(C) Western blot analysis of immunoproteasome subunits LMP2 and LMP7 in wild type (WT), heterozygous STAT1 mutated (STAT1*+/-) and homozygous STAT1 mutated (STAT1*-/-) skin fibroblasts transfected with 1 μg/ml HT-DNA or pretreated HT-DNA with DNase I for 48 h.
Figure 9(D-E) Representative immunofluorescence detection of LMP2 (red), CD8+ (green) and PD-1(red) in control and IPF lungs, arrows indicate double positive CD8+ PD-1+ cells, magnifications 10X, 20X and 40X correspond to a scale bar of 100 µm. Arrows indicate epithelial cells (E) or macrophages (M) and cells stained positive for CD8.(F) Flow cytometric analysis of CD3, CD8, and CCR7 expression on peripheral blood mononuclear cells (PBMCs) of control (n=21) and IPF (n=66) patients. Data are presented as gated % of parent. Data were analyzed with two-tailed unpaired Student's t-test. Asterisks indicate significance as *P<0.05.
Figure 1B. Normalized number of filaments nucleated over time, from SPIN90-Arp2/3 exposed to 2 μΜ G-actin (15% labeled with Alexa488) and 1 μΜ profilin, with 0 or 0.5 µM of GST-VCA from different NPFs. Solid lines are exponential fits, yielding nucleation rates knuc=(1.06±0.03)х10-3 s-1 without VCA, and knuc=(3.23±0.08)х10-3, (2.58±0.05)х10-3, and (2.28±0.03)х10-3 s-1 with VCA from N-WASP, WASP and WASH, respectively.
Figure 2Dissociation of SPIN90-Arp2/3-nucleated filaments. (B) The fraction of filaments still attached to the surface, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.Dissociation of SPIN90-Arp2/3-nucleated filaments. (C) Detachment rates, determined by exponential fits of survival curves (as in B) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.10±0.01, 0.65±0.36 and 0.82±0.14 µM, for N-WASP, WASH and WASP, respectively; and a maximum detachment rate of 0.033± 0.001, 0.031± 0.006, 0.010± 0.001 s-1 for WASH, N-WASP and WASP, respectively).Dissociation of Arp2/3-mediated branches. (E) The fraction of remaining branches, versus time, for different concentrations of GST-NWASP-VCA. Black lines are exponential fits.Dissociation of SPIN90-Arp2/3-nucleated filaments. (A) Schematic of the detachment experiment. Actin filaments were first nucleated from a SPIN90-Arp2/3-decorated surface, and elongated. Buffer with different concentrations of VCA was flowed in, and filament detachment events were monitored over time. Dissociation of Arp2/3-mediated branches. (F) Debranching rates, determined by exponential fits of survival curves (as in E) as a function of the concentration of VCA motifs from different NPFs. The data are fitted by a Michaelis-Menten equation (resulting in an apparent KD of 0.57±0.35, 0.81±0.36 and 0.53±0.23 µM, for WASH, N-WASP and WASP, respectively; and a maximum detachment rate of 0.020± 0.004, 0.012± 0.002, 0.005± 0.001 s-1 for WASH, N-WASP and WASP, respectively).
Figure 3A. The fraction of filaments still attached to the surface by SPIN90-Arp2/3, over time, when exposed to different concentrations of GMF. For each experiment, n=36-43 filaments. Black lines are exponential fits. Each experiment was repeated 3 times (technical replicates), yielding similar results.B. Detachment rates, determined by exponential fits of survival curves shown in A, as a function of the concentration of GMF. The error bars are determined by fitting the edges of the 95% confidence intervals (not shown, for readability) of the survival curves. The data are fitted by a Michaelis-Menten equation, yielding an apparent dissociation constant KD=70±23 nM and a maximum detachment rate of 0.017±0.001s-1.
Figure 4Assessing the presence of Arp2/3 on SPIN90 after filament departure. (B-C) The fraction of SPIN90 that re-nucleated a filament 500 seconds after the detachment of the initial filament, was quantified for different conditions.GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (G) Immuno-blots from pull-down assays where GST beads, decorated with GST or GST-SPIN90-Cter (the functional domain of SPIN90), were incubated with 0.4 μΜ Arp2/3 and gradient concentrations of GMF or VCA for 1 hour at room temperature. After the unbound protein was washed out, the amount of Arp2/3 attached to the beads was detected by Anti-ArpC2 antibody. Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST-SPIN90 (Appendix Figure S2).GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (H-I) Quantification of the amount of ArpC2 remaining on the beads, in different conditions.
Figure 5A-C. Survival fractions for branches exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, A), with 1 µM VCA (blue curves, B), or with 1 µM GMF (red curves, C). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.D. Summary of the debranching rates, resulting from the exponential fits in (A-C).E-G. Survival fractions for SPIN90-Arp2/3-nucleated filaments exposed to different forces in the microfluidics chamber, while being exposed to 0.15µM G-actin, alone (gray curves, E), with 0.1 µM VCA (blue curves, F), or with 0.1 µM GMF (red curves, G). The shaded areas represent 95% confidence intervals. Black lines are exponential fits.H. Summary of the detachment rates for SPIN90-Arp2/3-nucleated filaments, resulting from the exponential fits in (E-G).