EMBO/SourceData huggingface dataset:
Figure 2(A-B) GFP-expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.(C) Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 minutes or FCCP (4 μM) for 5 minutes.(D) The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).(E) HeLa cells with stable PHB1 ablation were incubated with H2O2 (2 μM) and CsA (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS levels (DHE).(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa cells stably expressing 4mt-RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).
Figure 4(A) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from at least 14 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm.(D) Quantitative analysis of data from (C). Data are from at least six images from three independent experiments (mean ± SEM). * p <0.05, ** p < 0.01, **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. After cells were lysed, NLRP3 was immunoprecipitated by anti-NLRP3 antibody. mtDNA levels (D-loop) from immunoprecipitants were detected by qPCR in J774A.1 cells with or without Phb1 knockdown. Data are from five independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(F) J774A.1 cells with Phb1 knockdown were incubated with EtBr (150 ng/ml) for 3 days, followed by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB was used to detect the protein levels in whole cell lysates and culture medium.(G) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were treated with NAC (2 mM) for 8 hours, LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and in the culture medium.
Figure 5(A) HeLa cells were incubated with H2O2 (5 mM) for 20 minutes and then lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. WB was used to detect the SPG7 or AFG3L2 levels, respectively, in the precipitated products.(B) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.HeLa cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.
Figure 6(A) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co-localization between mitochondria (MitoTracker, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of the data from (A). Data are from at least 12 images from three independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were lysed, and DNA was extracted. qPCR was used to detect the mtDNA levels (D-loop) in the cytosol relative to the nuclear DNA levels (Tert) in the whole cell lysate. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. The culture medium was collected and ELISA assays were used to detect IL-1β levels in culture medium. Data are from four independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with Phb1 knockdown, Spg7 knockdown, or combined Phb1 and Spg7 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(F) J774A.1 cells with Phb1 knockdown, Afg3l2 knockdown, and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(G) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.(H) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.
Figure 1B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).C Bright field images of CTC-ITB-01 cells growing adherently. D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).
Figure 3Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. A CNAs generated from CTCs from the primary blood collection (upper panel) and CTC-ITB-01, indicated as "CL" (medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. B CNA comparison between early (p04) and late (p30) CTC-ITB-01 passages (upper and medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (in blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.
Figure 4A Western blot analysis of selected protein markers, including ERα, EGFR, ERBB2, EpCAM, K18, K19, K8, E-cadherin, N-cadherin, vimentin, CD44, CD24, SNAIL, SLUG, TWIST and α-tubulin (as a loading control) (n=3 replicates). CTC-ITB-01 was compared to more mesenchymal ER- Hs578t and epithelial ER+ MCF-7 breast cancer cell lines.B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.C PAM50 classifier results showing probabilities of pooled CTC-ITB-01 cells matching specific molecular breast cancer subtypes. Starting from lowest probability, CTC-ITB-01 was classified as 1.02% (s=2.5%) normal-like, 3.11% (s=6.6%) Basal-like, 13.11% (s=9.3%) ERBB2-positive, 16.77% (s=23.7%) luminal A, and 65.22% (s=16.6%) luminal B breast cancer subtype. Data was generated from n=3 replicates.D PAM50 classifier results showing probabilities of the non-adherent and adherent CTC-ITB-01 fractions matching molecular subtypes. Selected bars are not visible due to extremely low probability (close to zero). Both fractions show greatest alignment with a luminal B subtype. Starting from lowest probability, the adherent fraction of CTC-ITB-01 was classified as 0% (±0%) normal-like, 6.22% (±9.0%) Basal-like, 17.85% (±8.6%) ERBB2-positive, 0% (s=0%) luminal A, and 75.92% (s=0.7%) luminal B breast cancer subtype. The suspension cell fraction of CTC-ITB-01 was classified as 2.04% (s=3.5%) normal-like, 0% (s=0%) Basal-like, 17.32% (s=9.7%) ERBB2-positive, 33.5% (s=23.8%) luminal A, and 68.67% (s=18.6%) luminal B breast cancer subtype, respectively. Data was generated from n=3 replicates.(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.
Figure 5A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.
Figure 6A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8). C H&E staining of xenografted mouse mammary glands show formation of primary tumors.D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.
Figure 1Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.Western blotting of Myc-Egg expressed in OSCs. Anti-Myc (Myc) and anti-β-Tubulin (Tub) antibodies were used. Tub was detected as a loading control. Control: empty vector was used as a negative control.Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.Western blotting of Egg immunopurified from total OSC lysates before (-) and after (+) phosphatase treatment. Anti-Egg antibodies were used for both immunoprecipitation (IP) and western blotting.Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.Rescue assays. Myc-Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting (WB) was performed using anti-Egg and anti-Myc antibodies. RT-qPCR was used to quantify Stalker2 transposon expression level. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). n.s.: not significant.
Figure 2Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of fractionated OSC materials. Anti-Egg, anti-β-Tubulin (Tub), and anti-histone H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).Rescue assays. Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. Stalker2 transposon expression was quantified using RT-qPCR. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).Rescue assays. Myc-Egg WT, Myc-Egg K1085R, and Myc-SV40-NLS-K1085R were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of the Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).
Figure 3Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates.Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.
Figure 4Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used.Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.
Figure 5Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction.Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.
Figure 6Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input.Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs.Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions.ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments.Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.
Figure 6Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.
Figure 1B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty)C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).D Activity of Rhom7 with/without its 7th TMD and/or C-terminal domain. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, , and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).