( G ) BMDMs isolated from Phb1F / F mice Phb1MyeKO mice were treated with NAC ( 2 mM ) for 8 hours , LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates in culture medium .

O O O B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

(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.


( A ) HeLa were incubated with H2O2 ( 5 mM ) for 20 minutes then lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . WB was used to detect SPG7 or AFG3L2 , respectively , in precipitated products .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O

(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 were stimulated with LPS ( 200 ng / ml ) for 6 hours lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O

(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 with or without Phb1 knockdown were lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O

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 with or without Phb1 knockdown were lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O

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.


( A ) J774A . 1 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were fixed subjected to IF to assess co - localization between mitochondria ( MitoTracker , red ) mtDNA ( PicoGreen , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative of data from ( A ) . Data are from at least 12 from three independent experiments ( mean ± ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were lysed , was extracted . qPCR was used to detect mtDNA ( D - loop ) in cytosol relative to ( Tert ) in whole lysate . Data are from six independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . culture medium was collected ELISA assays were used to detect IL - 1β in culture medium . Data are from four independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 with Phb1 knockdown , Spg7 knockdown , or combined Phb1 Spg7 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates culture medium .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(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 with Phb1 knockdown , Afg3l2 knockdown , combined Phb1 Afg3l2 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates culture medium .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(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 were stimulated with LPS ( 200 ng / ml ) for 6 hours lysed . whole lysate was immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect CYPD VDAC in precipitated products .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O

(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 with or without Phb1 knockdown were lysed . whole lysates were immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect CYPD VDAC in precipitated products .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O

(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.


B Representative pictures of different CTCs from initial CellSearch ® of metastatic breast cancer who gave rise to breast CTC . detected display clear keratin DAPI , CD45 - negativity as well as lack of , or very weak ( 4 , 8 ) , ERBB2 expression . of small ( about 5 µm in diameter , 1 , 2 ) large ( larger than 10µm in diameter , 2 , 3 ) were detected . While some CTCs displayed dot - like perinuclear keratin signals ( 1 , 2 ) , majority showed diffuse keratin . Additionally , CTC clusters of 4 - 8 were present ( 5 , 6 ) . Some CTCs showed multiple / lobed nuclei ( 4 , 7 , 8 ) .

O O B-EXP_ASSAY O O O O O O O O O B-DISEASE I-DISEASE O O O O B-TISSUE O O O O O B-GENEPROD B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

B 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 of CTC - ITB - 01 growing adherently . D Bright field of CTC - ITB - 01 growing non - adherently ( relation between adherent non - adherent : 80 / 20 % ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 %).


Unsupervised hierarchical clustering ( Ward linkage with Euclidean ) of CNA profiles generated from tumors , vaginal metastasis , isolated single pooled CTCs from blood collection CTC - ITB - 01 ( early later passage ) leukocytes . " CL " represents , followed by passage analyzed ( p04 / p30 ) . " CTC " marks circulating isolated from initial blood draw , followed by 01 / 05 / 10 indicating single ( 01 ) or pooled ( 5 or 10 , respectively ) . grey bars to left signify chromosomes , starting from top with chromosome 1 . Red color represents chromosome loss , blue represents gain . copy number aberrations were calculated using ' s own germline extracted from leukocytes as reference to eliminate copy number variations ( CNVs ) .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O

Unsupervised hierarchical clustering (Ward linkage with Euclidean distance) of CNA profiles generated from patient primary tumors, the vaginal metastasis, isolated single and pooled CTCs from the primary blood collection and CTC-ITB-01 (early and later passage) and leukocytes. "CL" represents the cell line, followed by the passage analyzed (p04/p30). "CTC" marks circulating tumor cells isolated from the initial blood draw, followed by 01/05/10 indicating single (01) or pooled cells (5 or 10, respectively). Light and dark grey bars to the left signify chromosomes, starting from the top with chromosome 1. Red color represents chromosome loss, blue represents gain. All copy number aberrations were calculated using the patient's own germline DNA extracted from leukocytes as reference to eliminate copy number variations (CNVs).


Direct comparisons of CNA profiles . Percentages of gene copy number gains losses across chromosomes for different sample types . Copy number gains are displayed in green , losses in red . A CNAs generated from CTCs from blood collection ( upper panel ) CTC - ITB - 01 , indicated as " CL " ( medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact ( Joosse et al , 2018 ) are indicated in lowest panel ( blue ) . Increased blue color represents lower p - value therefore higher statistical significance .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. 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 losses across chromosomes for different sample types . Copy number gains are displayed in green , losses in red . B CNA comparison between early ( p04 ) late ( p30 ) CTC - ITB - 01 passages ( upper medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact ( Joosse et al , 2018 ) are indicated in lowest panel ( in blue ) . Increased blue color represents lower p - value therefore higher statistical significance .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A Western blot of selected markers , including ERα , EGFR , ERBB2 , EpCAM , K18 , K19 , K8 , E - cadherin , N - cadherin , vimentin , CD44 , CD24 , SNAIL , SLUG , TWIST α - tubulin ( as a loading control ) ( n = 3 replicates ) . CTC - ITB - 01 was compared to more mesenchymal ER - Hs578t epithelial ER + MCF - 7 breast cancer lines .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE B-GENEPROD O B-CELL_LINE B-CELL_TYPE B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE O O

A 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 of CTC - ITB - 01 for pan - keratin ( orange ) , ER ( green ) , DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 ( 1 ) ( 2 ) . MCF - 7 are depicted as reference .

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

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 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 , 65 . 22 % ( s = 16 . 6 % ) luminal B breast cancer subtype . Data was generated from n = 3 replicates .

O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O

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 non - adherent 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 , 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 , 75 . 92 % ( s = 0 . 7 % ) luminal B breast cancer subtype . suspension 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 , 68 . 67 % ( s = 18 . 6 % ) luminal B breast cancer subtype , respectively . Data was generated from n = 3 replicates .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O

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 via flow cytometry using ALDEFLUOR ™ . CTC - ITB - 01 is compared to A549 as recommended control . An internal ( DEAB ) control is also depicted . 77 . 92 % of gated CTC - ITB - 01 population are ALDH + .

O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O

(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+.


A ICC of CTC - ITB - 01 for ß - catenin ( green ) , E - cadherin ( orange ) , DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

A 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 of CTC - ITB - 01 for α - catenin ( green ) , visualization of actin filaments by phalloidin ( orange ) , nuclei by DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

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 adherent non - adherently growing CTC - ITB - 01 fractions in relation to samples from different cancer entities . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . adherent fraction is shown with a black dot , non - adherent fraction with a black triangle , mean score per cancer entity by a black . D Generic EMT score of adherent non - adherently growing CTC - ITB - 01 fractions in relation to established cancer lines . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . adherent fraction is shown with a black dot , non - adherent fraction with a black triangle , mean score per cancer entity by a black .

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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.


A 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 ± of measurements performed on multiple ( n = 8 ) .

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A 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 of xenografted mouse mammary show formation of tumors .

C H&E staining of xenografted mouse mammary glands show formation of primary tumors.


D Stereoscope image of a mammary of immunodeficient NSG mouse . Fluorescently tagged CTC - ITB - 01 in green .

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D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.


E for ER , confirms retained in vivo ER - positivity of formed tumors . Representative of ER in higher ( left panel ) lower ( right panel ) magnification of 2 different areas of a xenografted mammary .

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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 of indicated organs from engrafted mice . Organs include brain ( 1 ) , lung ( 2 ) , liver ( 3 ) bones ( 4 ) . G Scatter blot showing ex vivo radiance of metastatic organs in three different mice ( M1 , M2 , M3 ) , indicating highest in liver bone . Data represent means ± SD of measurements performed on metastatic organs derived from three different mice .

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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.


A Equal numbers of CTC - ITB - 01 were seeded on 6 - well culture plates grown in presence or absence of different 17 - β - estradiol concentrations as indicated . After 10 days were fixed stained with Coomassie blue ( right panel ) . Macroscopic photos are shown of fixed colonies from grown in absence or presence of indicated E2 concentrations ( right panel ) . Scale bar 200 µm .

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A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.


B Stable knockdown of ER - alpha within CTC - ITB - 01 was performed by using lentiviral transfer of non - targeted ( scramble ) or two different targeted shRNAs against ER - alpha . CTC - ITB - 01 were seeded on 6 - well plates . After 10 days were fixed stained ( left upper panel ) . Macroscopic photos are shown of fixed ( left lower panel ) , scale bar 200 µm . Western blots of lysates from knockdown of ER - alpha expression in CTC - ITB - 01 transduced with same amounts of lentiviral vectors were probed with indicated antibodies . extracts were obtained 72h after lentiviral transduction ( right panel ) . Where indicated , western blots were visualized at long exposure ( l . e . ) / or short exposure ( s . e . ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

B Stable knockdown of ER-alpha within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted (scramble) or two different targeted shRNAs against ER-alpha. CTC-ITB-01 cells were seeded on 6-well plates. After 10 days cells were fixed and stained (left upper panel). Macroscopic photos are shown of the fixed cells (left lower panel), scale bar 200 µm. Western blots of lysates from knockdown of ER-alpha expression in CTC-ITB-01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies. The extracts were obtained 72h after lentiviral transduction (right panel). Where indicated, western blots were visualized at long exposure (l.e.) and/or short exposure (s.e.).


C CTC - ITB - 01 MCF - 7 were grown under same culture conditions in presence or absence of E2 as indicated . 24h after E2 deprivation or E2 - treatment were harvested . Western blots of lysates were probed with antibodies targeting ER - alpha , FOXM1 , Bcl - 2 , PR - A , PR - B ID1 , actin was used as loading control , visualized at long exposure ( l . e . ) / or short exposure ( s . e . ) .

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C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).


( D ) Growth curves of CTC - ITB - 01 under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live imaging system . mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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(D) Growth curves of CTC-ITB-01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


E ) Growth curves of MCF - 7 under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live imaging system . mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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E) Growth curves of MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


( F ) Influence of Palbociclib on CTC - ITB - 01 growth after 300 hours Data was chosen from one representative time point during exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons compared to vehicle substance dimethyl sulfoxide ( DMSO ) - . mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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(F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


G ) Influence of Palbociclib on MCF - 7 after 80 hours . Data was chosen from one representative time point during exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons compared to vehicle substance dimethyl sulfoxide ( DMSO ) - . mean values from three technical triplicates ( one experiment ) with standard deviation are shown .

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G) Influence of Palbociclib on MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.


( H ) Effect of Palbociclib on growth of CTC - ITB - 01 Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine IC50 . mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than symbols are not displayed .

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(H) Effect of Palbociclib on the growth of CTC-ITB-01 cells Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


I ) Effect of Palbociclib on growth of MCF - 7 . Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine IC50 . mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than symbols are not displayed .

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I) Effect of Palbociclib on the growth of MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) western blotting . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . M . W . indicates molecular marker .

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Western 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 ) anti - β - Tubulin ( Tub ) antibodies were used . Tub was detected as a loading control . Control : empty vector was used as a negative control .

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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 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 .

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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 ( - ) after ( + ) phosphatase treatment . Anti - Egg antibodies were used for both immunoprecipitation ( IP ) western blotting .

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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 its K1085R S215 / T217 ( STAA ) mutants expressed in OSCs . Egg modifications identified in this study are summarized on right . P : phosphorylation . Ub : monoubiquitination .

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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 second from top : silver of purified Myc - Egg Myc - EGFP . Second from bottom : CBB of histone H3 . 1 . Bottom : 14C autoradiogram . EGFP : Myc - EGFP was used as a negative control . l . c . : chain of antibody . Time : reaction time ( min ) of samples .

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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 its K1085R STAA mutants were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting ( WB ) was performed using anti - Egg anti - Myc antibodies . RT - qPCR was used to quantify Stalker2 transposon expression level . 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 - ) . n . s . : not significant .

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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.


Immunofluorescence of OSCs using anti - Egg antibodies . Endogenous Egg is shown in red . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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Immunofluorescence 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 ) , anti - histone H3 antibodies were used . H3 Tub were detected as markers for ( Nuc ) ( Cyto ) fractions , respectively . Whole : whole OSC lysates .

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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 ) after ( siUbc2 ) Ubc2 depletion . Anti - Myc - antibody was used for western blotting . ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 43 ( siEGFP ) 0 . 78 ( siUbc2 ) .

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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 using anti - Flag antibody to identify Flag - Ubc2 ( green ) expressed in OSCs . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Immunofluorescence of Myc - Egg WT , Myc - SV40 - NLS - Egg , Myc - PKI - NES - Egg expressed in OSCs using anti - Myc antibody . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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 , Myc - PKI - NES - Egg expressed in OSCs . Anti - Myc antibody was used . ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 50 ( WT ) , 2 . 29 ( SV40 - NLS ) , 0 . 95 ( PKI - NES ) .

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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 , Myc - PKI - NES - Egg were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) anti - Myc ( right ) antibodies . Stalker2 transposon expression was quantified using RT - qPCR . 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 - ) .

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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 of purified Myc - Egg WT , Myc - Egg K1085R , Myc - SV40 - NLS - Egg WT , Myc - SV40 - NLS - Egg K1085R . Middle : CBB of histone H3 . 1 . Bottom : 14C autoradiogram . Myc - EGFP was used as a negative control ( EGFP : not shown ) .

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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 , Myc - SV40 - NLS - K1085R were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) anti - Myc ( right ) antibodies . RT - qPCR quantified expression level of Stalker2 transposon . 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 - ) .

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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).


Immunofluorescence of endogenous Egg ( red ) in OSCs using anti - Egg antibody before ( siEGFP ) after ( siWde ) Wde depletion . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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Immunofluorescence 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 ) after ( siWde ) Wde depletion . Anti - Egg , anti - Wde , anti - Tub , anti - H3 antibodies were used . H3 Tub were detected as markers for ( Nuc ) ( Cyto ) fractions , respectively . Whole : whole OSC lysates .

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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 of endogenous Egg ( red ) in OSCs upon Flag - Wde ( green ) co - expression . Anti - Egg anti - Flag antibodies were used . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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 of Myc - Egg WT ( red ) in OSCs upon Wde co - expression ( green ) . Anti - Myc anti - Flag antibodies were used . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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 ( - ) after Wde WT Wde ∆ CC co - expression . Anti - Myc , anti - Flag , anti - Tub , anti - H3 antibodies were used . H3 Tub were detected as markers for ( Nuc ) ( Cyto ) fractions , respectively . Whole : whole OSC lysates .

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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.


- interaction assays . Myc - Egg Flag - Wde WT its ∆ CC mutant were expressed in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) anti - Flag ( lower ) antibodies were used for western blotting .

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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 of Myc - Egg WT ( red ) in OSCs upon Flag - Wde WT ∆ CC mutant ( green ) co - expression . Anti - Myc anti - Flag antibodies were used . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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.


Immunofluorescence of Myc - Egg WT , Myc - Egg K987A , Myc - Egg R988A mutants ( red ) in OSCs before after Flag - Wde ( green ) co - expression . Anti - Myc anti - Flag antibodies were used . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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Immunofluorescence 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 its K987A R988A mutants . Flag - Wde : Flag - Wde was co - expressed . SV40 - NLS : SV40 - NLS was added to Myc - Egg . Anti - Myc anti - Flag antibodies were used .

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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 of Myc - Egg K987A Myc - Egg R988A mutants ( red ) in OSCs . Both mutants were fused to SV40 - NLS . Anti - Myc antibody was used for detection . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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.


- interaction assays . Myc - Egg WT , Myc - SV40 - NLS - Egg WT , Myc - SV40 - NLS - Egg K1085R mutant were expressed with Flag - Wde in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) anti - Flag ( lower ) antibodies were used for western blotting .

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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.


Rescue assays . Myc - Egg WT Myc - SV40 - NLS - Egg WT were expressed in OSCs upon endogenous Egg Wde depletion ( siEgg siWde ) . Western blotting was performed using anti - Egg ( left ) anti - Myc ( right ) antibodies . RT - qPCR quantified expression level of Stalker2 transposon . 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 - ) .

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Rescue 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 ) ( ) fractions before ( - ) after Flag - Wde co - expression . Anti - Myc anti - Flag antibodies were used . Histone H3 was detected as a marker of fraction .

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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 of H3K9me3 ( green ) , Myc - Egg WT ( red ) , Myc - SV40 - NLS - Egg WT ( red ) in OSCs before after Flag - Wde ( yellow ) co - expression . Anti - H3K9me3 , anti - Myc anti - Flag antibodies were used . DAPI ( blue ) shows nuclei . Scale bar : 10 μm .

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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 H3K9me3 signal intensities per related to Figure 5B ( p values on unpaired Welch ' s t - ) . Box plots show median ( ) 25th - 75th percentile ( box ) ± 1 . 5 interquartile range ( n > 25 for each box ) . Circles represent outliers .

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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.


Heat map of Egg ChIP - seq within a 10kb window centered on Egg peaks . Note that ChIP peaks was called using ChIPed Input samples Metaplots showing Egg ChIP - seq signals for genomic regions around Egg peaks . Displayed are fold - changes in Egg occupancy normalized to input .

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Heat 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 ) Wde - depleted ( siWde ) OSCs .

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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 .

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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 ) after ( siWde ) Wde depletion . qPCR was performed for Piwi target transposons ( Stalker2 , mdg1 , gypsy ) normalized to % input of a PIwi non - target transposon ( roo ) . Error bars represent SD from three independent experiments .

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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 , gypsy in control ( siEGFP ) Wde - depleted ( siWde ) OSCs . Input : input was used as a control . RPM : reads per million mapped reads .

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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.


His - tagged GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD SARS - CoV - 2 NTD were incubated with BHK 21 expressing eGFP - fused ACE2s . Anti - His / APC antibody was used to detect expressing His - tagged . stained with GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD SARS - CoV - 2 NTD are shown in green , orange , marine red , respectively . SARS - CoV - 2 NTD was used as negative control .

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His-tagged GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins were incubated with BHK 21 cells expressing eGFP-fused ACE2s. Anti-His/APC antibody was used to detect cells expressing His-tagged protein. Cells stained with the GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins are shown in green, orange, marine and red, respectively. The SARS-CoV-2 NTD was used as negative control.


mFc - tagged ACE2s were captured by anti - mIgG Fc antibodies immobilized on CM5 chip , sequentially tested binding with serially diluted SARS - CoV - 2 RBD , GX / P2V / 2017 RBD GD / 1 / 2019 RBD . raw fitted curves are displayed in dotted solid lines , respectively . binding affinity between RBDs ( SARS - CoV - 2 RBD , GX / P2V / 2017 RBD GD / 1 / 2019 RBD ) 13 ACE2s are shown . Mean ± SD represents mean standard deviation of three independent experiments .

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The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Hela - hACE2 untransfected HeLa are infected with pseudotyped SARS - CoV - 2 , GX / P2V / 2017 GD / 1 / 2019 , respectively . Green indicates Hela - hACE2 infected with pseudovirus . Untransfected HeLa is used as negative control . scale bar indicates 400 μm . Statistic for infectivity of three pseudoviruses . Data represent results of five replicates . data are presented as mean ± SD . * * * p < 0 . 001 ( Student ' s t - ) . ns , no significant differences .

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Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm. Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.


Flow cytometric of SARS - CoV - 2 RBD its mutants ( K417R , Q498H K417R - Q498H ) binding to human , mouse , rat European hedgehog ACE2s expressed on surface of BHK21 . SARS - CoV - 2 NTD was used as negative control .

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Flow 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.


mFc - tagged ACE2s from human , mouse , rat European hedgehog were captured by anti - mIgG Fc antibodies immobilized on CM5 chip , sequentially tested binding with serially diluted SARS - CoV - 2 RBD its mutants ( K417R , Q498H K417R - Q498H ) .

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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).


binding affinities of four ACE2s to SARS - CoV - 2 RBD its mutants are shown . Mean ± SD represents mean standard deviation of three independent experiments .

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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.


B Western blot ( probing with an anti - FLAG mAb ) to detect cleavage of artificial substrate by wild - type ( WT ) / inactive ( S201A ) GlpG encoded on pBAD33 with / without arabinose ( Ara . ) . rhomboid substrates that are uncleaved , by GlpG are marked by black , red , arrows , respectively . Controls , empty pBAD33 ( empty )

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B 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 to detect cleavage of artificial substrate by wild - type ( WT ) / modified ( S133A or H187A ) Rhom7 encoded on pBAD33 with / without arabinose ( Ara . ) . , rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively . Controls , empty pBAD33 ( empty ) wild - type S . sonnei ( Ss ) .

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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 / or C - terminal domain . rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , , blue arrows , respectively . Controls , empty pBAD33 ( empty ) wild - type S . sonnei ( Ss ) .

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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).


C Western blot of candidate substrates reproducibly by GlpG from screen . Data information : In ( C rhomboid substrates that are uncleaved , by GlpG are marked by black , red , arrows , respectively . Controls , inactive enzymes wild - type S . sonnei ( Ss ) .

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C Western blot analysis of candidate substrates reproducibly cleaved by GlpG from the screen. Data information: In (C rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).


D Western blot of candidate substrates reproducibly by Rhom7 from screen . Data information : In D ) rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , red , blue arrows , respectively . Controls , inactive enzymes wild - type S . sonnei ( Ss ) .

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D Western blot analysis of candidate substrates reproducibly cleaved by Rhom7 from the screen. Data information: In D) rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, red, and blue arrows, respectively. Controls, inactive enzymes and wild-type S. sonnei (Ss).


Western blot ( probing with an anti - FLAG mAb ) to detect cleavage of HybO with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . homboid substrates that are uncleaved , are marked by black , rrows , ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). homboid substrates that are uncleaved, are marked by black, rrows, ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


Western blot to detect cleavage of HybA with ( + ) / without ( - ) chromosomal ( native ) or pBAD33 - encoded rhomboids ( plasmid ) . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , are marked by black , red , blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


Western blot to detect cleavage of HybAG296F by endogenous or pBAD33 - encoded rhomboid . Wild - type S . sonnei , Ss . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , re marked by black , red , blue arrows , espectively . ild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, re marked by black, red, and blue arrows, espectively. ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


Western blot to detect cleavage of HybAP300A by endogenous or pBAD33 - encoded rhomboid . rhomboid substrates that are uncleaved , by GlpG , or by Rhom7 , are marked by black , red , blue arrows , respectively . Wild - type S . sonnei , Ss . Wild - type ( WT ) / inactive ( SAHA : alanine substitution of catalytic serine histidine residues ) enzymes were pBAD33 - encoded ( plasmid ) in S . sonneiΔglpGΔrhom7 .

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Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.


A two - hybrid with HybA / or HybB fused chromosomally with T25 T18 domains of B . pertussis CyaA , respectively in absence of endogenous CyaA . were grown on LB agar containing 20 µg / ml X - gal at 37 ° C for 14 hours in presence / absence of O2 . Scale bar , 1 cm .

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A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.


B Western blot to detect HybA cleavage in S . sonneiΔrhom7 by wild - type ( + ) or inactive ( S201A ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) with ( + ) / without ( - ) HybB . HybA was expressed from its native locus or a plasmid ( pHybA ) . HybA that is uncleaved or by GlpG is marked by black red arrows , respectively .

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B Western blot analysis to detect HybA cleavage in S. sonneiΔrhom7 by wild-type (+) or inactive (S201A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (-) HybB. HybA was expressed from its native locus or a plasmid (pHybA). HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.


C of of / uncleaved HybA in strains with ( + ) or without ( - ) HybB . mean ± S . D . of three experiments . * , p < 0 . 05 ; * * * * , p < 0 . 0001 ( One - way ANOVA ) .

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C Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (-) HybB. mean ± S.D. of three experiments. *, p<0.05; ****, p<0.0001 (One-way ANOVA).


D Western blot to detect GlpG - mediated cleavage of native tagged HybA at indicated times after blocking translation by addition of chloramphenicol at T0 in absence of HybB ( - ) . , HybA that is uncleaved or by GlpG is marked by black red arrows , respectively .

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D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.


E Western blot to detect GlpG - mediated cleavage of C - terminally sfCherry - 3xFLAG tagged HybA at times after blocking translation by addition of chloramphenicol at T0 in presence of HybB ( + ) . , HybA that is uncleaved or by GlpG is marked by black red arrows , respectively .

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E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.


F Growth of lacking hydrogenases ( ΔhyaA - F ΔhycE + / - ΔhybO - G ) or GlpG ( ΔglpG ) , or expressing uncleavable HybB ( hybAG296F ) in 5 %

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F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2


G H2 consumption by S . sonnei . were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0 . 5 % fumarate , 12 . 5 μg / ml nicotinic acid 0 . 2 % casamino in a sealed glass chromatography vial . headspace was purged with 10 % H2 / 90 % argon cultures were incubated at 37 ° C with shaking at 180 rpm for 9 hours . H2 in headspace was sampled measured by chromatography .

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G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.


H S . sonnei growth in 10 % H2 with plasmid expressed wild - type or non - functional ( pglpGSAHA ) GlpG .

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H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.


B Western blot ( probing with an anti - FLAG mAb ) to detect cleavage of FdoH cleavage in S . sonneiΔrhom7 with ( + ) / without ( - ) FdoI . rhomboid substrates that are uncleaved , by GlpG are marked by black , red arrows , respectively .

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B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.


C Western blot to detect cleavage of FdnH in S . sonneiΔglpG with ( + ) or without ( - ) chromosomal Rhom7 ( native ) , or wild - type ( + ) / inactive ( SAHA ) Rhom7 expressed from pBAD33 in S . sonneiΔglpGΔrhom7 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively .

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C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively.


E Western blot to detect cleavage of wild - type ( WT ) or uncleavable ( P300A ) HybA by active ( + ) or inactive ( S201A ) chromosomal GlpG in presence ( + ) or absence ( - ) of HybB . rhomboid substrates that are uncleaved , by GlpG are marked by black , red arrows , respectively .

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E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.


F Western blot to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdoH by active ( + ) or inactive ( S201A ) chromosomal GlpG in presence ( + ) or absence ( - ) of FdoI . rhomboid substrates that are uncleaved , by GlpG are marked by black , red arrows , respectively .

F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively.


G Western blot to detect cleavage of wild - type ( WT ) or modified ( P259A ) FdnH with active ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) or without ( - ) FdnI . rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively .

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G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.


A Western blot ( probing with an anti - V5 mAb ) to detect degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) or without ( - ) HybB . Rhomboid substrates that are uncleaved , by GlpG , , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, , are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


B Degradation of V5 - tagged HybA at times after blocking translation at T0 in presence ( + ) or absence ( - ) of HybB . Rhomboid substrates that are uncleaved , by GlpG , black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

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B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB. Rhomboid substrates that are uncleaved, cleaved by GlpG, black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


C Western blot of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 chromosomally expressing wild - type ( + ) or inactive ( S201A ) GlpG with ( + ) / without ( - ) FdoI . Rhomboid substrates that are uncleaved , by GlpG are marked by black , red , arrows , respectively .

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C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI. Rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively.


D Western blot of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( SAHA ) Rhom7 expressed from pBAD33 with ( + ) / without ( - ) FdnI . Rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively .

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D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.


Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdoH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed chromosomally ( native ) or from pUC19 ( plasmid ) without FdoI , + / - exposure to 400 µM CuCl2 for 30 min . : Rhomboid substrates that are uncleaved , by GlpG , are marked by black , red , arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


F Degradation of N - terminally V5 - tagged wild - type ( WT ) or modified ( P259A ) FdnH in S . sonneiΔrhom7 with wild - type ( + ) or inactive ( S201A , SAHA ) GlpG expressed from pUC19 ( plasmid ) without FdnI ( - ) , , + / - exposure to 400 µM CuCl2 for 30 min . Rhomboid substrates that are uncleaved , by Rhom7 , are marked by black , blue arrows , respectively . Degradation products post rhomboid cleavage are marked by green arrows .

O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.


A Western blot probing localisation status of plasmid - encoded N - terminally V5 - tagged wild - type ( WT ) or modified ( P300A ) HybA in S . sonneiΔrhom7ΔhybB with wild - type ( + ) or inactive ( SAHA ) GlpG expressed from pUC19 . Whole lysate ( Whole . ) , Sol ( Sol . ) , Mem ( detergent - solubilised , Mem . ) , Aggregate ( Agg . ) fractions are shown . HybA that is uncleaved , only by GlpG , further degraded post GlpG cleavage is marked by black , red , green arrows , respectively .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O

A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.


( A ) Representative flow cytometry plots ( left ) pooled data ( right ) of competition assays with sgControl sgRnf31 B16 - F10 ova after culture with indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * * * * p < 0 . 0001 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.


( B ) CRISPR / Cas9 screen showing depleted B16 - F10 genes after one week culture with TNF + IFN - γ ( both 10 ng / mL ) ; blue genes indicate overall genes with p < 0 . 001 , black genes indicate genes of interest with p < 0 . 05 . Significance assessed using MAGeCK .

O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.


( H ) Representative flow cytometry plots ( left ) pooled data ( right ) showing PI of sgControl or sgRNF31 A375 after culture with indicated recombinant cytokine ( s ) ( 10 ng / mL ) for 48 hours ; n . s . : no significance , * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - for n = 3 biological replicates . Pooled data are represented as mean ± .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.