Figure 1 ( A ) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators ( DFP , DFX or DFO ) . β - actin was used as loading control . Bafilomycin ( - ) : without Bafilomycin , Bafilomycin ( + ) : with 100 mM of Bafilomycin . ( B ) Dual excitation ( 550 / 488 nm ) of mt - mKeima transfected - Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment . Scale bar : 10 μm . ( C ) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt - mKeima at 633 / 488 nm for untreated ( left panel ) and DFP - treated ( 24 h , right panel ) Huh7 cells . ( D ) Untreated Huh7 cells or those treated with DFP ( 10 μM ) , DFX ( 10 μM ) or DFO ( 10 μM ) were quantified for mitophagy using mt - mKeima transfection and FACS analysis ( n = 4 , biological replicates ) . A high signal ( 633 / 488 ) area / cell area indicates the proportion of cells undergoing mitophagy . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 . ( E ) Electron microscopic images of untreated ( left panel ) and DFP - treated ( right panel ) Huh7 cells . Arrows indicate mitophagosome - like structures . Scale bar : 500 nm . The number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) between cells treated with DFP ( 1 . 0 mM ) and those without DFP . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 . ( F ) Quantification of mitophagy using mt - mKeima transfection and FACS analysis for Huh7 cells treated with DFP ( 0 , 0 . 01 . 0 . 5 or 1 . 0 mM as indicated ) in the absence or presence of bafilomycin ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 . ( G ) Immunoblots for Parkin , PINK1 , phosphorylated p - 62 ( p - p62 ) , HSP60 , LC3 , p62 / SQSTM , and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells . HSP60 and β - actin were used as loading controls for mitochondria and whole cell lysates , respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin.(B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm.(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.(E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.(F) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.(G) Immunoblots for Parkin, PINK1, phosphorylated p-62 (p-p62), HSP60, LC3, p62/SQSTM, and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells. HSP60 and β-actin were used as loading controls for mitochondria and whole cell lysates, respectively.


Figure 2 ( A ) Cytoplasmic and mitochondrial iron content was measured by a ferrozine - based assay for Huh7 cells with DFP , DFX or DFO treatment ( 0 . 01 mM ) for 24 h ( n = 5 , biological replicates ) . The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) for 24 h . ( n = 5 , biological replicates ) The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 . ( C ) Immunoblots ( left panel ) for mitochondrial ferritin ( FTMT ) , frataxin , mitoferrin , heat shock protein 60 ( HSP60 ) , ferritin heavy chain ( FTH1 ) and transferrin receptor ( TfR ) and β - actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) . HSP60 and β - actin were used as loading controls for mitochondrial and whole cell lysates , respectively . The expression level of FTMT was normalized to HSP60 ( n = 4 , biological replicates , right panel ) . The central horizontal bar and the error bards indicate mean ± SD . * : P < 0 . 05 . ( D ) The mRNA expression of FTMT in Huh7 cells was quantified by real - time RT - PCR before and after DFP ( 1 . 0 mM ) treatment ( n = 4 , biological replicates ) . The expression of FTMT mRNA level was normalized to GAPDH mRNA . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * * : P < 0 . 01 . ( E ) The mRNA expression of FTMT in Huh7 cells was quantified by real - time RT - PCR before DFP ( 1 . 0 mM ) treatment and 3 , 6 , 12 , and 24 h after DFP treatment ( n = 5 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 , * * * : P < 0 . 001 . ( F ) DNA - binding activity of six transcription factors ( specific protein 1 [ SP1 ] , cAMP response element binding protein [ CREB ] , Ying yang 1 [ YY1 ] , GATA2 , forkhead box protein A1 [ FoxA1 ] and CCAAT - enhancer - binding protein β [ C / EBPβ ] ) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP ( 1 . 0 mM ) treatment in Huh7 cells ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * : P < 0 . 05 . ( G ) The DNA - binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM ) as indicated in Huh7 cells ( n = 4 , biological replicates ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( H ) FTMT mRNA expression was quantified for Huh7 cells by real - time RT - PCR before and after DFP ( 1 . 0 mM ) treatment with or without SP1 knockdown by siRNA ( n = 5 , biological replicates ) . mRNA expression of FTMT level was normalized to GAPDH . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 . ( I ) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP ( 1 . 0 mM ) treatment . β - actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions , respectively . ( J ) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP ( 0 . 1 mM ) treatment . β - actin was used as the loading control . ( K ) Immunoblots for FTMT , HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without HIF1α or SP1 knockdown by siRNA . HSP60 and β - actin were used as loading controls for the mitochondrial fraction and whole cell lysates , respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Cytoplasmic and mitochondrial iron content was measured by a ferrozine-based assay for Huh7 cells with DFP, DFX or DFO treatment (0.01 mM) for 24 h (n=5, biological replicates). The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate. The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.(B) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated) for 24 h. (n=5, biological replicates) The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.(C) Immunoblots (left panel) for mitochondrial ferritin (FTMT), frataxin, mitoferrin, heat shock protein 60 (HSP60), ferritin heavy chain (FTH1) and transferrin receptor (TfR) and β-actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated). HSP60 and β-actin were used as loading controls for mitochondrial and whole cell lysates, respectively. The expression level of FTMT was normalized to HSP60 (n=4, biological replicates, right panel). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05.(D) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before and after DFP (1.0 mM) treatment (n=4, biological replicates). The expression of FTMT mRNA level was normalized to GAPDH mRNA. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.(E) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before DFP (1.0 mM) treatment and 3, 6, 12, and 24 h after DFP treatment (n=5, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01, ***: P<0.001.(F) DNA-binding activity of six transcription factors (specific protein 1 [SP1], cAMP response element binding protein [CREB], Ying yang 1 [YY1], GATA2, forkhead box protein A1 [FoxA1] and CCAAT-enhancer-binding protein β [ C/EBPβ]) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP (1.0 mM) treatment in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. *: P<0.05.(G) The DNA-binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM) as indicated in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.(H) FTMT mRNA expression was quantified for Huh7 cells by real-time RT-PCR before and after DFP (1.0 mM) treatment with or without SP1 knockdown by siRNA (n=5, biological replicates). mRNA expression of FTMT level was normalized to GAPDH. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.(I) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP (1.0 mM) treatment. β-actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions, respectively.(J) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP (0.1 mM) treatment. β-actin was used as the loading control.(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.


Figure 3 ( A ) Immunoblots for mitochondrial ferritin ( FTMT ) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells ( upper panels ) and the quantification of mitophagy using mt - mKeima transfection and FACS analysis ( n = 4 , biological replicates ) ( lower panels ) before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . NC : negative control siRNA , * * : P < 0 . 01 . ( B ) Immunofluorescence staining for LC3 ( red ) , the mitochondrial marker Tom20 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 . 0 mM ) treatment with or without FTMT knockdown by siRNA . FTMT colocalize with LC3 at the mitochondria ( white puncta indicated by arrows ) . Boxed areas are enlarged below . Scale bar : 5 μm ( C ) Immunoblots for mitochondrial ferritin ( FTMT ) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA - resistant FTMT ( left panel ) . HSP60 as the loading control . Those cells were subjected to the quantification of mitophagy using mt - mKeima transfection and FACS analysis before and after DFP ( 1 . 0 mM ) treatment ( n = 5 , biological replicates ) ( right panel ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * * : P < 0 . 001 . ( D ) Super resolution images of immunofluorescence staining for LC3 ( red ) , FTMT ( green ) and Tom20 ( blue ) in FTMT knockdown - Huh7 cells transfected with FTMT - GFP or FTMT siRNA - resistant FTMT - GFP before and after DFP ( 1 . 0 mM ) treatment . White arrows indicate colocalization of LC3 , FTMT , and Tom20 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunoblots for mitochondrial ferritin (FTMT) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells (upper panels) and the quantification of mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates) (lower panels) before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.(B) Immunofluorescence staining for LC3 (red), the mitochondrial marker Tom20 (green) and FTMT (blue) in Huh7 cells before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. FTMT colocalize with LC3 at the mitochondria (white puncta indicated by arrows). Boxed areas are enlarged below. Scale bar: 5 μm(C) Immunoblots for mitochondrial ferritin (FTMT) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA-resistant FTMT (left panel). HSP60 as the loading control. Those cells were subjected to the quantification of mitophagy using mt-mKeima transfection and FACS analysis before and after DFP (1.0 mM) treatment (n=5, biological replicates) (right panel). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. ***: P<0.001.(D) Super resolution images of immunofluorescence staining for LC3 (red), FTMT (green) and Tom20 (blue) in FTMT knockdown-Huh7 cells transfected with FTMT-GFP or FTMT siRNA-resistant FTMT-GFP before and after DFP (1.0 mM) treatment. White arrows indicate colocalization of LC3, FTMT, and Tom20.


Figure 4 ( A ) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti - FTMT - coupled magnetic beads and immunoblotted with the indicated antibodies ( anti - NCOA4 or anti - LC3 ) . ( B ) GST pulldown assay using recombinant GST - NCOA4 mixed with recombinant His - FTMT . Bound FTMT was analyzed by immunoblotting using either anti - FTMT or anti - His antibodies . ( C ) Immunofluorescence staining for Tom20 ( red ) , NCOA4 ( green ) and FTMT ( blue ) in Huh7 cells before and after DFP ( 1 mM ) treatment . Boxed areas are enlarged on the right . The white puncta indicated by white arrows show the colocalization of FTMT , NCOA4 and Tom20 . Scale bar : 10 μm and 5 μm in enlarged images . ( D ) Super - resolution images of immunofluorescence staining for Tom20 ( blue ) , NCOA4 ( red ) and FTMT ( green ) in Huh7 cells after DFP ( 1 mM ) treatment , using the single - molecule fluorescence microscope . The white arrows indicate colocalization of these proteins . ( E ) Immunofluorescence staining for Tom20 ( blue ) , FTMT - GFP ( green ) , recombinant FTMTR82A - GFP ( green ) , NCOA4 - mCherry ( red ) and NCOA4I489A / W497A - mCherry ( red ) in Huh7 cells . Boxed areas are enlarged on the right . The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20 , FTMT - GFP and NCOA4 - mCherry . The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT - GFP and NCOA4 - mCherry . Scale bar : 10 μm and 5 μm in enlarged images . ( F ) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies . Co - immunoprecipitation revealed a specific interaction of NCOA4 - mCherry with FTMT - GFP or FTMTD57A - GFP , which has a substitution of alanine ( A ) for aspartic acid ( D ) at position 57 in the FTMT mitochondrial leader sequence ( lanes 1 and 3 ) . ( G ) Following DFP ( 1 . 0 mM ) or ammonium iron sulfate ( FAS ) treatment as indicated , extracted proteins from Huh7 cells expressing FTMT - GFP and NCOA4 - mCherry were mixed with anti - GFP - coupled magnetic beads and immunoblotted with anti - mCherry and anti - GFP antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3).(B) GST pulldown assay using recombinant GST-NCOA4 mixed with recombinant His-FTMT. Bound FTMT was analyzed by immunoblotting using either anti-FTMT or anti-His antibodies.(C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images.(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.(E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images.(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.


Figure 5 ( A ) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria . The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC - 1 for 1 h at 30 ° C and subjected to flow cytometric analysis ( n = 4 , biological replicates ) by excitation with a blue laser ( 488 nm ) with simultaneous emission assessment using a 585 / 42 detector for JC - 1 red and a 530 / 30 for JC - 1 green . The trapezoid areas show the population of depolarized mitochondria . Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . * * : P < 0 . 01 . ( B ) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer ( n = 4 , technical replicates ) . Huh7 cells were incubated with 1 mM of DFP for different lengths of time ( 0 , 3 , 6 , 18 , and 24 h ) . Oxygen consumption was measured , and the respiration rate was analyzed by injecting of 1μM oligomycin , 1 μM carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) and 10 μM rotenone / antimycin A . OCR : oxygen consumption rate . The central point and the error bards indicate mean ± SD . ( C ) Immunofluorescence staining for FTMT - GFP ( green ) and LC3 ( red ) in Huh7 cells transfected with FTMT - GFP . The proportion of colocalization was quantified for four randomly selected areas ( technical replicates ) using ImageJ software version 1 . 46 ( NIH , Bethesda , MD ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * * : P < 0 . 01 . ( D ) Immunofluorescence staining for Ferritin - GFP heavy chain ( FTH1 ) ( green ) and LC3 ( red ) in Huh7 cells transfected with FTH1 - GFP . The proportion of colocalization was quantified as described in ( C ) . * : P < 0 . 05 , * * : P < 0 . 01 . ( E ) Immunoblots for the precursor form and processed form of FTMT - GFP , endogenous FTMT , NCOA4 , LC3 , and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT - GFP with or without digestion with proteinase K in the presence or absence of DFP . mtHSP70 was used as the loading control . ( F ) Immunoblots for FTMT , VDAC , and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment . The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment . mtHSP70 was used as the non - membrane - integral protein control and VDAC as the membrane - integral protein control . mtHSP70 at the right - hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.(C) Immunofluorescence staining for FTMT-GFP (green) and LC3 (red) in Huh7 cells transfected with FTMT-GFP. The proportion of colocalization was quantified for four randomly selected areas (technical replicates) using ImageJ software version 1.46 (NIH, Bethesda, MD). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.(D) Immunofluorescence staining for Ferritin-GFP heavy chain (FTH1) (green) and LC3 (red) in Huh7 cells transfected with FTH1-GFP. The proportion of colocalization was quantified as described in (C). *: P<0.05, **: P<0.01.(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.


Figure 6 ( A ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , and LC3 ( green ) in Huh7 cells untreated or treated with DFP ( 1 mM ) for 24 h , or with CCCP ( 30 μM ) for 6 h . Boxed areas are enlarged on the right . Scale bar : 10 μm , and 5 μm in enlarged images . ( B ) Immunofluorescence staining for the polarized mitochondria ( red ) , Tom20 ( blue ) , LC3 ( blue ) , and FTMTD57A ( green ) using Huh7 cells expressing FTMTD57A . Boxed areas are enlarged on the right . The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria , Tom20 and FTMTD57A or the colocalization of the polarized mitochondria , FTMTD57A , and LC3 . Scale bar : 10 μm , and 5 μm in enlarged images . ( C ) STAM mice were fed a high - fat diet and orally administered 0 . 0375 mg / g body weight of DFP , 0 . 075 mg / g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age ( n = 6 ) . Arrows indicate liver tumors . Scale bar : 1 . 0 cm . The number and maximum size of liver tumors were compared among the three groups . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( D ) Histology of the nontumorous liver ( H & E and Sirius red stain , original magnification x40 ) and superoxide anion production in the livers of STAM mice fed a high - fat diet with or without DFP ( 0 . 075 mg / g ) administration . Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion . Scale bar : 100 μm . ( E ) Histology of the hepatocellular carcinoma using H & E staining in STAM mice fed a high - fat diet . The boxed area is enlarged on the right ( x40 in left panel and x400 in right panel ) . The tumor has increased cell density , an increased ratio of nucleus to cytoplasm , and thickened trabeculae Scale bar : 100 μm . ( F ) Electron microscopic images of livers in control ( C57BL / 6 ) and DMBA + HFD mice that were orally administered 0 . 075 mg / g of DFP or distilled water . Scale bars : 2 . 0 μm . The boxed areas ( x 1 , 500 ) are enlarged below ( x6 , 000 on left and x10 , 000 on right ) . The white arrow indicates mitophagosome - like structures . The mean number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) among the four groups ( control , DMBA + HFD mice treated with 0 , 0 . 0375 or 0 . 075 mg / g of DFP ) ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.(B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images.(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.(E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm.(F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


Figure 7 ( A ) Immunoblots for FTMT , frataxin , mitoferrin , HSP60 , FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control ( C57BL / 6J ) mice and STAM mice treated with DFP ( 0 , 0 . 0375 , or 0 . 075 mg / g body weight ) ( n = 6 ) . The expression of FTMT , frataxin , mitoferrin , FTH1 , and TfR were normalized to HSP60 or β - actin , respectively . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . ( B ) The mean number of mitophagosome - like structures / 100 μm2 from four randomly selected areas in the livers of DFP ( 0 . 075 mg / g body weight ) - administered mice were compared between control and FTMT siRNA - treated groups for STAM mice ( n = 6 ) and DMBA + HFD mice ( n = 6 ) . The central horizontal bar and the error bards indicate mean ± SD . Two sample t test was used for statistical analysis . NC : negative control siRNA , * : P < 0 . 05 . ( C ) Frozen liver sections of DFP ( 0 . 075 mg / g body weight ) - administered mice ( STAM mice and DMBA + HFD ) with or without FTMT knockdown were stained with dihydroethidium . Red fluorescence indicates the ROS production . Scale bar : 100 μm . siNC : negative control siRNA . ( D , E ) STAM mice ( D ) were fed a high - fat diet without DFP treatment or orally administered with 0 . 075 mg / g body weight of DFP from the age of 4 weeks , injected with FTMT siRNA ( 10 μl / g body weight ) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice , and followed until 16 weeks of age ( n = 6 ) . DMBA + HFD mice ( E ) were fed a high - fat diet , treated with DFP and siRNA in the same manner with STAM mice , and followed until 30 weeks of age ( n = 6 ) . The number and maximum size of liver tumors were compared among the three groups ( control without DFP , negative control siRNA , and FTMT siRNA with DFP ) . The central horizontal bar and the error bards indicate mean ± SD . The Tukey ' s honestly significant test was used for statistical analysis . * : P < 0 . 05 , * * : P < 0 . 01 . Representative liver images are shown in the upper panel . Scale bar : 1 cm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05. (C) Frozen liver sections of DFP (0.075 mg/g body weight)-administered mice (STAM mice and DMBA + HFD) with or without FTMT knockdown were stained with dihydroethidium. Red fluorescence indicates the ROS production. Scale bar: 100 μm. siNC: negative control siRNA.(D, E) STAM mice (D) were fed a high-fat diet without DFP treatment or orally administered with 0.075 mg/g body weight of DFP from the age of 4 weeks, injected with FTMT siRNA (10 μl/g body weight) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice, and followed until 16 weeks of age (n=6). DMBA + HFD mice (E) were fed a high-fat diet, treated with DFP and siRNA in the same manner with STAM mice, and followed until 30 weeks of age (n=6). The number and maximum size of liver tumors were compared among the three groups (control without DFP, negative control siRNA, and FTMT siRNA with DFP). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. Representative liver images are shown in the upper panel. Scale bar: 1 cm.


Figure 1 ( C - F ) C . Total fresh weight , D . Total dry weight , E . Primary root length and F . Lateral root density of 21 - day - old , non - colonized ( Mock ) and SA190 - colonized plants upon growth for 16 days on ½ MS or ½ MS + 25 % PEG . Boxplot shows data from three independent biological replicates ( technical replicates n = 18 - 24 ) where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * * * p < 0 . 001 ) . ( G , H ) G . Growth and H . Survival percentage of mock - and SA190 - colonized plants in jiffy pots grown upon watering for 2 weeks , no watering for 3 weeks and after one week of re - watering . All plots represent the mean of 3 biological replicates ( technical replicates n = 36 ) . Whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * * * p < 0 . 001 ) . J . Confocal microscopy showing the colonization of SA190 : GFP on five days - old seedlings grown on ½ MS and 25 % PEG . Green signal corresponds to GFP - tagged SA190 while in magenta are plant cell wall stained with propidium iodide ( PI ) . Bar indicates 100 nm . K . SA190 colonization levels in Arabidopsis under normal ( ½ MS ) and drought proxy ( ½ MS + 25 % PEG ) conditions as quantified by colony forming units of bacteria per mg of plant fresh weight . Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C-F) C. Total fresh weight, D. Total dry weight, E. Primary root length and F. Lateral root density of 21-day-old, non-colonized (Mock) and SA190-colonized plants upon growth for 16 days on ½ MS or ½ MS + 25% PEG. Boxplot shows data from three independent biological replicates (technical replicates n = 18-24) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).(G, H) G. Growth and H. Survival percentage of mock- and SA190-colonized plants in jiffy pots grown upon watering for 2 weeks, no watering for 3 weeks and after one week of re-watering. All plots represent the mean of 3 biological replicates (technical replicates n = 36). Whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).J. Confocal microscopy showing the colonization of SA190:GFP on five days-old seedlings grown on ½ MS and 25% PEG. Green signal corresponds to GFP-tagged SA190 while in magenta are plant cell wall stained with propidium iodide (PI). Bar indicates 100 nm. K. SA190 colonization levels in Arabidopsis under normal (½ MS) and drought proxy (½ MS + 25% PEG) conditions as quantified by colony forming units of bacteria per mg of plant fresh weight. Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).


Figure 2F - G . Primary root length and Lateral root density of 21 - day - old , mock - and SA190 - colonized Col - 0 , aba2 - 1 and qpyr / pyl plants upon growth for 16 days on ½ MS + 25 % PEG . Data information : Boxplot shows data from three independent biological replicates ( technical replicates n = 36 ) where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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Figure 2F-G. Primary root length and Lateral root density of 21-day-old, mock- and SA190-colonized Col-0, aba2-1 and qpyr/pyl plants upon growth for 16 days on ½ MS + 25% PEG. Data information: Boxplot shows data from three independent biological replicates (technical replicates n = 36) where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).


Figure 3B . Relative gene expression of aquaporin PIP and TIP genes by qRT - PCR analysis normalized to tubulin levels in 21 - days old mock - and SA190 - colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25 % PEG . Data information : Values represent the means of three biological experiments . Error bars indicate SE . All plots represent the mean of 3 biological replicates ( biological replicates n = 3 ) . Asterisks indicate a statistical difference based on the Student ' s t - test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . C . Relative gene expression of aquaporin PIP and TIP genes by qRT - PCR analysis in Col - 0 , aba2 - 1 and qpyr / pyl mutants normalized to tubulin levels in 21 - days old mock - and SA190 - colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25 % PEG . Data information : Values represent the means of three biological experiments . Error bars indicate SE . All plots represent the mean of 3 biological replicates ( biological replicates n = 3 ) . Asterisks indicate a statistical difference based on the Student ' s t - test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).C. Relative gene expression of aquaporin PIP and TIP genes by qRT-PCR analysis in Col-0, aba2-1 and qpyr/pyl mutants normalized to tubulin levels in 21-days old mock- and SA190-colonized roots of Arabidopsis grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).


Figure 4Relative enrichment of the H3K4me3 mark at the indicated gene loci of selected PIPs and TIPs of non - colonized ( Mock ) and SA190 - colonized ( SA190 ) plants in Col - 0 and aba2 - 1 mutant as determined by chromatin immunoprecipitation - qPCR ( ChIP - qPCR ) . The regions targeted for amplification are labelled as R1 , R2 and R3 . R1 encompasses 5 ' - UTR , R2 is close to the transcription start site ( TSS ) while R3 is outside the gene body . Amplification values were normalized to input and H3 and region 3 ( R3 ) of non - colonized ( Mock ) Col - 0 plants . The plots represent the means of 2 biological replicates . Error bars represent SE . ( ns ) non - significant Asterisks indicate a statistical difference based on 2 - way ANOVA ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 for p value differences between the conditions when compared to Col - 0 Mock using Dunnett ' s multiple comparison test ) .

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Figure 4Relative enrichment of the H3K4me3 mark at the indicated gene loci of selected PIPs and TIPs of non-colonized (Mock) and SA190-colonized (SA190) plants in Col-0 and aba2-1 mutant as determined by chromatin immunoprecipitation-qPCR (ChIP-qPCR). The regions targeted for amplification are labelled as R1, R2 and R3. R1 encompasses 5'-UTR, R2 is close to the transcription start site (TSS) while R3 is outside the gene body. Amplification values were normalized to input and H3 and region 3 (R3) of non-colonized (Mock) Col-0 plants. The plots represent the means of 2 biological replicates. Error bars represent SE. (ns) non-significant Asterisks indicate a statistical difference based on 2-way ANOVA (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for p value differences between the conditions when compared to Col-0 Mock using Dunnett's multiple comparison test).


Figure 5A - B . Total fresh weight of 21 days old non - colonized - ( Mock ) and SA190 - colonized ( SA190 ) Col - 0 , tip2 ; 1 and qpip plants grown for 16 days under normal ( ½ MS ) or drought stress proxy conditions ( ½ MS + 25 % PEG ) . Data information : All plots represent the mean of 3 biological replicates . Error bars represent SE . Boxplot shows data from three independent biological replicates ( technical replicates n = ( A ) 16 - 20 , ( B ) 8 - 20 where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) . Asterisks indicate a statistical difference based on the Student ' s t - test * * * p < 0 . 001 ) . G . Transpiration rate of mock - or SA190 - colonized 21 days old plants after transfer of 5 - day - old seedlings from ½ MS to ½ MS + / - 25 % PEG . Transpiration was quantified as rate of water loss by calculating the changes in fresh weight for 30 min with reading at every 5 min interval . H - I . Comparison of stomatal aperture of SA190 - and non - colonized 21 - days old plants grown for 16 days under non - stress ( ½ MS ) or drought stress proxy ( ½ MS + 25 % PEG ) conditions . Scale bar = 20 µm . Data information : All plots represent the mean of 3 biological replicates . Error bars represent SE . Boxplot shows data from three independent biological replicates ( technical replicates n = ( I ) 94 - 104 , where whiskers represents minimum and maximum values , " + " indicates mean and horizontal line represents median . Asterisks indicate the statistical differences based on the Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) . Asterisks indicate a statistical difference based on the Student ' s t - test * * * p < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-B. Total fresh weight of 21 days old non-colonized- (Mock) and SA190-colonized (SA190) Col-0, tip2;1 and qpip plants grown for 16 days under normal (½ MS) or drought stress proxy conditions (½ MS + 25% PEG). Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (A) 16-20, (B) 8-20 where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).G. Transpiration rate of mock- or SA190- colonized 21 days old plants after transfer of 5-day-old seedlings from ½ MS to ½ MS +/- 25 % PEG. Transpiration was quantified as rate of water loss by calculating the changes in fresh weight for 30 min with reading at every 5 min interval.H-I. Comparison of stomatal aperture of SA190- and non-colonized 21-days old plants grown for 16 days under non-stress (½ MS) or drought stress proxy (½ MS + 25% PEG) conditions. Scale bar = 20 µm. Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (I) 94-104, where whiskers represents minimum and maximum values, "+" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).


Figure 1Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in leptoteneDouble immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in zygotene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in , early pachytene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in mid pachytene Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in diplotene Arrows in ( I - L ) indicate the position of some chromocenters . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . DAPI staining of the chromatin ( blue ) is shown for some spermatocytes . Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in diakinesis Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . O , P Double immunolabeling of PDS5A ( green ) and kinetochores revealed with an ACA serum ( red ) in a metaphase I spermatocyte . Data information : DAPI staining of the chromatin ( blue ) is shown for some spermatocytes . Q , R Double immunolabeling of PDS5A ( green ) and REC8 ( pseudocolored in purple ) in a metaphase I spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . DAPI staining of the chromatin ( blue ) is shown for some spermatocytes . S , T Double immunolabeling of PDS5A ( green ) and kinetochores ( ACA , red ) in a metaphase II spermatocyte . Data information : DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptoteneDouble immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in zygotene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in , early pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in mid pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diakinesis Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Figure 2Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages . Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Immunolabeling of PDS5B ( green ) and REC8 ( pseudocolored in blue onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Immunolabeling of PDS5B ( green ) and SYCP3 ( red ) and TRF1 ( pseudocolored in yellow onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Immunolabeling of PDS5B ( green ) and ACA ( pseudocolored in purple onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) on diplotene spread nuclei . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated . Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) on diakinesis spread nuclei . Data information : Sex chromosomes ( X , Y ) and bivalents ( XY ) are indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Immunolabeling of PDS5B (green) and REC8 (pseudocolored in blue onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Immunolabeling of PDS5B (green) and SYCP3 (red) and TRF1 (pseudocolored in yellow onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Immunolabeling of PDS5B (green) and ACA (pseudocolored in purple onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.Double immunolabeling of PDS5B (green) and SYCP3 (red) on diakinesis spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Figure 3A - F Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in a metaphase I spermatocyte ( A - B ) and selected bivalents ( C - F ) . The sex bivalent ( XY ) is indicated . G - J Double immunolabeling of PDS5B ( green ) and kinetochores ( ACA , red ) , and staining of the chromatin ( DAPI ) in a metaphase II spermatocyte .

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Figure 3A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated.G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Figure 4Double immunolabeling of PDS5A ( green , A - E ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . Double immunolabeling of PDS5B ( green , F - J ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . K - M Preparations from the same mice were immunolabeled with the testis - specific histone variant H1t ( green ) and SYCP3 ( red ) . N Scatter dot - plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype . Bars indicate mean and standard error . Statistical significance was assessed using an unpaired two - tailed T - test function ( * * * * , p < 0 . 0001 ) .

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Figure 4Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).


Figure 5A - H Double immunolabeling of SYCP1 ( green ) and SYCP3 ( red ) in wild - type ( A - D ) and Pds5AB cKO ( E - H ) spread spermatocytes at the indicated stages . I - L Double immunolabeling of HORMAD2 ( pseudocolored in white ) and SYCP3 ( red ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Figure 5A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages. I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 6Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC3 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and the cohesin subunits REC8 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and the cohesin subunits SMC1β ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors Sororin ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and the cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 7Double immunolabeling of SYCP3 ( red ) and either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages . Double immunolabeling of SYCP3 ( red ) and RAD51 ( green in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . I Scatter dot - plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype . Bars indicate mean and standard error and statistical significance was assessed with an unpaired two - tailed T - test function ( * * * * , p < 0 . 0001 ) . J - L Double immunolabeling of MLH1 ( pseudocolored in white ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.Double immunolabeling of SYCP3 (red) and RAD51 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 8Double immunolabeling of SYCP3 ( red ) and TRF1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and RAP1 ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . I Selected zygotene - like and pachytene - like bivalents presenting altered TRF1 distribution as ( from left to right ) ( i ) telomere stretches , ( ii ) multiple telomere , ( iii ) distant telomeres and ( iv ) telomere - less . Arrowheads in ( I ) indicate the position of altered telomeres . J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages ( n = 1646 telomeres in zygotene - like spermatocytes and 738 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . K , L As in ( I , J ) , respectively , but for RAP1 staining ( n = 1376 telomeres in zygotene - like spermatocytes and 328 in pachytene - like ones ) . Arrowheads in ( K ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . M - P Double immunolabeling of SUN1 ( green ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . 0 , 1 and 2 in ( N and O ) indicate the number of chromosome ends labelled by SUN1 in a given bivalent . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Q As in ( I ) , but for SUN1 staining . Arrowheads in ( Q ) indicate the position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . R Selected zygotene - like and early pachytene - like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 ( red ) and SUN1 ( green ) , and their respective percentage of appearance . S Quantification of telomeres with a regular or altered disposition of SUN1 ( n = 715 telomeres in zygotene - like spermatocytes and 410 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 9A - C Immunolabeling of SYCP3 ( red ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . D 300 % magnification of selected zygotene - like and pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals . Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytesDouble immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less . White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in ( T ) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm . Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at the indicated stages . Asterisks indicate the position of the enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres and telomere - less .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytesDouble immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in (T) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less.


Figure 10Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice M - P ) . The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in ( F indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 μm . Double immunolabeling of TRF1 ( green ) and SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice The first three columns correspond to z - projections of 15 focal planes throughout the Top , Equator and Bottom regions of the nucleus . The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column ( Z - Projection ) . White arrowheads in N ) indicate the presence of TRF1 signals non - attached to the NE . Scale bar , 10 μm . Q - T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches ( Q ) , multiple telomere ( R ) , distant telomere ( S ) and telomere - less ( T ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in N) indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.Q-T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches (Q), multiple telomere (R), distant telomere (S) and telomere-less (T).


Figure 1D . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) , gasdermin D ( GSDMD ) and GAPDH ( loading control ) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Data are from one three independent experiments ( DE . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in D . Data information : Each symbol represents an independent biological replicate ( E ) . * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( E ) . Data are from three independent experiments E ; mean and s . e . m . in EG . IncuCyte live - imaging analysis of WT and Casp11 - / - BMDM viability after infection with M . catarrhalis as in D , or transfection with LPS . Data information : Data are pooled from two ( G ) independent experiments mean and s . e . m . in G ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Data are from one three independent experiments (DE. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D. Data information: Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from three independent experiments E; mean and s.e.m. in EG. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).


Figure 2A . Immunoblot analysis of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) and gasdermin D ( GSDMD ) in WT , Nlrp3 - / - , Casp11 - / - and Aim2 - / - BMDMs left untreated ( Med . ) or 5 h after transfection with 5 µg of LOS from M . catarrhalis ( O35E ) or 5 µg of LPS from E . coli . Data information : Data are from three independent experiments ( A , B . Release of IL - 1β , IL - 18 , TNF and LDH from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B , NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test ( B , Data are pooled from three independent experiments ( B , mean and s . e . m . in B ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli. Data information: Data are from three independent experiments (A,B. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B, NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, Data are pooled from three independent experiments (B, mean and s.e.m. in B,


Figure 3F . Transcript abundance for genes encoding GTPases in WT and Ifnar1 - / - BMDMs infected with M . catarrhalis as in C . Transcript abundance is provided in counts per million ( CPM ) . Data information : Data are pooled from three independent experimentsG . qRT - PCR analysis of genes encoding the family of guanylate binding proteins ( GBP1 - 11 ) in WT and Ifnar1 - / - BMDMs left untreated ( Med . ) or following treated as in C . Data information : Each symbol represents an independent biological replicate G ) . NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test G ) ) . Data are from one experiment representative of four ( G ) independent experiments ( mean and s . e . m . in G ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM). Data information: Data are pooled from three independent experimentsG. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C. Data information: Each symbol represents an independent biological replicate G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test G)). Data are from one experiment representative of four (G) independent experiments (mean and s.e.m. in G).


Figure 4A . Confocal microscopy analysis of intracellular M . catarrhalis ( green ) and GBP1 , GBP2 , GBP3 , GBP5 or GBP7 ( red ) in WT BMDMs left untreated ( Med . ) or assessed 12 h after infection with M . catarrhalis ( Ne11 , MOI 20 ) . B . Quantitation of GBP1 - , GBP2 - , GBP3 - , GBP5 - and GBP7 - postive M . catarrhalis in WT BMDMs as treated in A . Data information : Arrowheads indicate bacteria colocalised with GBPs . To quantify the proportion of GBP - positive bacteria in a field of view , we used the single red channel ( anti - GBP staining ) and compared this to the green channel ( anti - M . catarrhalis staining ) . The number of GBP - positive M . catarrhalis was divided by the total number of M . catarrhalis counted to obtain the proportion of GBP - positive bacteria ( B ) . A total of 100 BMDMs were analysed to quantify the proportion of GBP - positive bacteria per cell using confocal microscopy ( B ) Data are from three independent experiments ( A , or are pooled from two ( B ) independent experiments Scale bars , 5 µm ( A , F . Recovery of M . catarrhalis ( as colony - forming units ( CFU ) from primary WT , Gbpchr3 - KO and Ifnar1 - / - lung fibroblasts 4 , 8 and 12 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Each symbol represents an independent biological replicate F ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons test Data are from three independent experiments F ; mean and s . e . m . G . Quantitation of intracellular bacteria by Transmission electron microscopy ( TEM ) in WT , Gbpchr3 - KO and Ifnar1 - / - BMDMs infected with M . catarrhalis ( Ne11 , MOI 50 ) for 12 h . Each symbol represents an independent experiment ( E and F ) . Data information : A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy ( G ) . Data are from one experiment representative of two ( G ) independent experiments mean and s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A. Data information: Arrowheads indicate bacteria colocalised with GBPs. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) Data are from three independent experiments (A, or are pooled from two (B) independent experiments Scale bars, 5 µm (A,F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Each symbol represents an independent biological replicate F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test Data are from three independent experiments F; mean and s.e.m.G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F). Data information: A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) independent experiments mean and s.e.m.