Figure 6 A ) Scatter plot comparing the transcriptome of the two lamellocyte clusters , deduced from the WI dataset . The x - axis is the average expression levels of the genes and the y - axis the log2 fold change ( LM - 1 / LM - 2 ) . The genes significantly enriched in the LM - 1 cluster are highlighted in red ( p < 10 - 6 ) , those significantly enriched in the LM - 2 cluster in orange ( p < 10 - 6 ) . C ) Expression levels ( gradient of color ) and the percentage of cells ( size of the dots ) for the top 10 markers of lamellocytes ( in black ) , of markers specific to LM - 1 compared to LM - 2 ( in red ) and specific to LM - 2 compared to LM - 1 ( in orange ) . Note that most markers enriched in LM - 1 compared to LM - 2 are exclusively expressed in LM - 1 while most markers enriched in LM - 2 compared to LM - 1 are also expressed in the plasmatocyte and crystal cell clusters . D ) Hemocytes from WL OregonR infested by wasp . The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea ( in gray , top panels ) and Shot ( in gray , lower panels ) . Phalloidin - FITC ( in green ) labels the actin filament particularly abundant in lamellocytes ( Tokusumi et al . , 2009 ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , FITC and the lamellocyte markers , the middle panels show the FITC alone and the right panels the lamellocyte markers alone . The scale bars represent 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6).  C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters.  D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.


Figure 8 A ) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the graph on the right panel , indicates the Log2 of the ratio between the expression level in WI versus NI . Positive values indicate an up - regulation upon wasp infestation and negative values a down - regulation ( n = 3 , pool of 10 larvae per replicate , mean + / - SD are represented on the graph ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ . B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) . D ) Expression levels during the infestation timeline as in ( B , C ) for the markers of proliferation . E ) Weighted dot plot representing the enrichment ( Log2 fold change ) of the markers in hemocytes originating from the lymph gland ( DotGFP - WI ) compared to embryonic hemocytes ( srpGFP - WI ) from WI larva . The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp ( hemo ) Gal4 respectively combined with a lineage tracing construct . The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue ( not significant ) to light blue ( p < 10 - 3 ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ , " * * * " = p < 0 . 001 ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 A) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the graph on the right panel, indicates the Log2 of the ratio between the expression level in WI versus NI. Positive values indicate an up-regulation upon wasp infestation and negative values a down-regulation (n=3, pool of 10 larvae per replicate, mean +/- SD are represented on the graph). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[.  B,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C). D) Expression levels during the infestation timeline as in (B,C) for the markers of proliferation.  E) Weighted dot plot representing the enrichment (Log2 fold change) of the markers in hemocytes originating from the lymph gland (DotGFP-WI) compared to embryonic hemocytes (srpGFP-WI) from WI larva. The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp(hemo)Gal4 respectively combined with a lineage tracing construct. The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue (not significant) to light blue (p<10-3). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[, "***" = p <0.001].


Figure 1Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( A ) Induced Cyp1a1 mRNA expression in response to different treatments ( n = 3 per group ) . Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( B ) Assessment of GFP + stem cell percentages ( n = 3 replicates per treatment ) . Bars with different letters are significantly different ( p < 0 . 05 ) . ( D ) Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type ( Lgr5 - GFPCreERT2 X Tomatof / f , GT ) and AhR knock out ( AhRf / f X Lgr5 - GFPCreERT2 X Tomatof / f , HGT ) mice . Top left panel shows representative cells from WT negative control animal ( no GFP or Tomato expression ) . Top right panel shows Tomato only control ( CDX2P - CreERT2 X Tomatof / f ) . Middle panels represent DMSO or TCDD treatment in AhR WT mice , respectively . Mice were gavaged every other day , 4 times total , with vehicle or TCDD ( 25 µg / kg bw ) and terminated 1 d later . Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse . ( E ) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD ( n = 4 ) . ( F ) Percentage of Lgr5 + stem cells ( n = 4 mice per group ) in ( D ) . Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type and AhR knock out mice . ( G ) Quantitative analysis of the percentage of Lgr5 + stem cells in ( D ) , n = 7 - 10 mice per group . Representative immunohistochemistry of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Representative violin plots of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Solid line indicates median , and dotted lines indicate 25th / 75th quartiles .

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Figure 1Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type and AhR knock out mice. (G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.Representative immunohistochemistry of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm.Representative violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.


Figure 2 ( A ) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5 - EGFP - IRES - CreERT2 mice . ( B ) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice ( Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ GC ] ) and KO mice ( AhRf / f X Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ HGC ] ) , treated with DMSO ( control ) or TCDD for 5 d . Data are from ≥ 4 mice , scale bar 200 µm . ( C , D ) The organoid forming efficiency ( C ) and diameter ( D ) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD ( 10 nM ) . n = 4 - 5 mice per group in ( C ) . Each dot represents an individual organoid and grey line indicates mean in ( D ) . Bars with different letters are significantly different ( p < 0 . 05 ) . Organoid growth from GFPlow progenitor cells . ( E ) Representative brightfield images of organoids . ( F ) Organoid forming efficiency and ( G ) diameter . n = 4 - 8 mice per group in ( F ) . Each dot represents an individual organoid and grey line indicates mean in ( G ) . Scale bar 200 μm . Bars with different letters are significantly different ( p < 0 . 05 ) . ( H , I ) ( H ) Lgr5 expression in organoids and ( I ) sorted stem cells in the absence or presence of TCDD treatment ( n ≥ 3 independent samples per group ) . ( J ) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d . Scale 200 µm .

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Figure 2(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids.(F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.


Figure 3 ( C ) Heatmap comparison of the top 25 upstream regulators between AhR KO ( KO / WT ) and TCDD ( TCDD / vehicle in WT mice ) treated cells .

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Figure 3(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.


Figure 4 ( A ) FoxM1 , Plk1 , and Cdc25B mRNA expression in stem and progenitor cells with and without AhR , n = 4 mice per group . ( B ) FoxM1 , Plk1 , and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD , n = 3 - 4 mice per group . Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD , n = 3 or 4 per group . A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control . Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts ( E ) treated with DMSO or TCDD , n = 3 or 4 per group . ( F ) FoxM1 luciferase activity determination for young adult mouse colonocytes ( YAMCs ) treated with DMSO or 10 nM TCDD for 1 d , n = 3 per group . ( G Expression of FoxM1 and PLK1 at mRNA level , , n > = 3 independent organoids per treatment . H ) Expression of FoxM1 and PLK1 at protein level , n > = 3 independent organoids per treatment . ( I ) Representative brightfield images of mouse colonic organoids generated from WT Lgr5 + - GFPhi stem cells treated with DMSO or 5µM FDI - 6 . Scale bar 200 µm . ( J , K ) Quantification of organoid forming efficiency ( J ) and organoid area ( K ) from WT colonic stem cells treated with DMSO or 5µM FDI - 6 . Data are represented as mean ± SEM in ( K ) . ( L ) Organoid expression of Lgr5 in response to FDI - 6 . Lgr5 expression level was dramatically suppressed by FDI - 6 in a dose - dependent manner , n > = 3 per treatment . ( M ) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI - 6 for 2 d . Scale 200 µm .

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Figure 4(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.(G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment.H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment.(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.


Figure 5 ( A and B ) Proliferating Lgr5 + stem cells marked by EdU in the distal colon from AhR WT ( GT ) and KO ( HGT ) mice . Arrowhead and star symbols denote proliferating stem cells ( GFP + EdU + ) and static stem cells ( GFP + EdU - ) , respectively , during a 2 h EdU pulse , n = 3 per treatment . ( C ) Total number of EdU + cells per crypt in the distal colon of AhR WT and KO ( HGT ) mice , n = 3 mice per treatment . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . ( D & E ) Crypt cell proliferative zone analysis in AhR WT and KO ( HGT ) mice , presented as the distance from the crypt base to the highest EdU + cell divided by total crypt length . Red lines between crosses show the measurement of distance . Each symbol represents one crypt , n = 3 mice per treatment , scale bar 20 µm . Grey line represents mean . ( F & AhR KO promoted cell proliferation 5 d post DSS treatment . AhR WT ( AhRf / f [ H ] , n = 4 ) and AhR KO ( AhRf / f X Villin - Cre [ HV ] , n = 4 ) mice were used . G ) AhR KO promoted cell proliferation 5 d post DSS treatment . Data represent mean ± SEM , quantified as the ratio of the EdU + area relative to the DAPI + area using KeyenceTM software . ( H ) Increased cell proliferation ( EdU + cells ) observed in AhR KO vs WT organoids derived from HGC and GC mice , respectively ( n = 6 from 3 mice per group ) . ( I ) Effect of AhR KO on cell cycle S and G2 - M phases ( n = 3 mice per group ) . ( J ) . FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation . Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton ( 50 mg / kg ) for 2 consecutive days , n = 80 ~ 100 crypts from 4 mice per group . Scale bar , 50 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . Bars with different letters are significantly different ( p < 0 . 05 ) .

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Figure 5(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).


Figure 6 ( B ) Representative colon images from AhR WT and AhR KO mice following AOM / DSS treatment . Scale bar , 9 mm . ( C ) H & E staining of representative colon tumor sections from AhR WT and KO mice , scale bar 200 μm . ( E , F ) ( E ) Adenoma and ( F ) adenocarcinoma multiplicity in AhR WT and KO mice . ( G ) Tumor volume in AhR KO and WT mice . Grey line indicates mean . ( H ) FoxM1 mRNA expression in normal ( uninvolved ) crypts adjacent to colonic tumors . Images were quantified and expressed as FoxM1 positive area per crypt ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . ( I ) FoxM1 mRNA expression in colon tumors . FoxM1 mRNA ISH area was averaged per tumor on each slide ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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Figure 6(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


Figure 7 ( A - B ) mRNA expression of human colonic stem cell markers LGR5 and OLFM4 , n = 5 or 6 independent human samples . Paired student t - test . ( C ) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD . Scale bar 50 µm . mRNA expression of target genes , n = 5 or 6 independent human samples . Paired student t - test . mRNA expression of target genes , n = 5 or 6 independent human samples . Paired student t - test . ( H ) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n = 6 independent samples . Total protein was used as a loading control . ( I ) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD . n = 3 - 4 per group . ( J ) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d . C56 and C61 designate different human subjects . ( K ) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon . Subsequent sequencing revealed indels at the expected locations . ( L ) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies ( n = 304 ) . AhR low ( n = 152 ) is defined as below its median expression ; AhR high ( n = 152 ) is defined as above its median expression . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . ( M ) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies ( n = 303 ) analyzed by Spearman rank correlation . ( N ) Representative brightfield images of human colonic organoids from sorted live cells at day 6 , scale bar 200 μm . ( O , P ) ( O ) Human organoid forming efficiency , and ( P ) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO , TCDD and CH223191 ( 10 μM ) for 6 d . n = 5 per group , 3 independent experiments . Grey line indicates mean in ( P ) . Treatments not sharing a common letter are significantly different , p < 0 . 05 . ( Q ) Representative brightfield images of human colonic organoids treated with DMSO or FDI - 6 . FoxM1 inhibition suppressed human colonic organoid growth in a dose - dependent manner , scale bar 200 µm .

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

Figure 7(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.


Figure 1Volcano plots of piliation , aggregation and adhesion mutation scores for all pilE mutations . Stop and synonymous mutations are highlighted in blue and orange respectively . Adhesion and aggregation mutation scores relative to piliation . The correlation appears as a red dotted line .

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Figure 1Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.


Figure 2Scanning electron microscopy of single bacteria on a cellulose filter . Scale bars : 500 nm . Two representative immunofluorescence labelling of TFP . Scale bar : 1 µm . Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody . Black dots indicate all the individual de novo generated PilE mutants that were characterized . Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region . Solid lines indicate 95 % confidence interval . Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants . Mean values ± SEM are indicated for each strain . N ≥ 3 independent experiments . Paired t - test . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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Figure 2Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).


Figure 3Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody . Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated ( magenta ) . In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling . Scale bar : 2 µm . Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported . Each dot represents values for a PilE point mutant relative to the SB strain . Hypo - aggregative mutants are highlighted in orange while hyper - aggregative mutants are in light blue . solid lines indicate the 95 % confidence interval . Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot . PilX levels were normalized using PilE as a reference and normalized to pilESB values . Mean ratio ± SEM is indicated . N = 3 independent experiments . Paired t - test .

O B-PANEL_START 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 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

Figure 3Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.


Figure 4Early adhesion of pilE mutants as a function of aggregation . Values were normalized to that of pilESB . Each dot represents a pilE mutant and the pilV , pilD , pilX and pilC1 reference mutants . Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple . A blown - up view of the zone of interest ( lower left quarter ) is presented on the right panel . The shaded area highlights the absence of significant adhesion . Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB . Piliation length of a selection of mutants determined by immunofluorescence . Average pilus length ± SEM is indicated . N = 6 experiments with at least 50 bacteria analyzed in each experiment . Paired t - test . Number of pili per bacteria ± SEM determined as in ( E ) . Paired t - test . p < 0 . 05 ( * ) .

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Figure 4Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).


Figure 5Time - lapse of a cell - adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes . Initiation of flow corresponds to t = 0 . Scale bar : 1 µm . Spinning - disk confocal immunofluorescence of pili stained with both SM1 ( magenta ) and F10 ( green ) . The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light . In each field of view , a substrate - and a cell - bound pilus are shown . Upper panels : z - projection from the stacks in the xy plane ; bottom panels : side view in the z plane . Scale bar : 2 µm . White arrows correspond to SM1 staining at the tip of pili . Quantification of adhesion after infection of HUVEC cells for 30 minutes , expressed as the number of adherent bacteria over the number of cell nuclei . Prior to infection , bacteria were incubated at 37 ° C with or without antibody or nanobody for 15 minutes . Mean values ± SEM are indicated for each strain . N = 5 independent experiments . Paired t - test . p < 0 . 05 ( * ) .

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Figure 5Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm.Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).


Figure 1Isolated sporozoites from the hemolymph ( HL ) and salivary glands ( SG ) of infected mosquitoes stained with Hoechst and SiR - tubulin . Note the similar staining of WT and α1 - tubulin ( - ) complemented lines . Scale bars : 5 µm . SiR - tubulin staining ( green , I and II ) and TEM ( III ) show that microtubules shrink after wild type sporozoite formation . During budding microtubules can be up to 15 µm long ( I ) , while after formation they measure on average only 6 µm ( graph ) . Inset in II shows sporozoite cross - section at the sporoblast membrane ; note the SiR - tubulin fluorescence ( green , arrowheads ) next to the nuclear Hoechst stain ( blue ) . ( III ) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule ( white line , length ) . Green arrowheads point to microtubules close to the sporoblast . Rh : rhoptry ; N : nucleus ; ER : endoplasmic reticulum ; Mi : mitochondrion ; Sb : sporoblast . * * * * indicate p < 0 . 0001 ; Kruskal - Wallis - test . Scale bars : I & II : 5 µm , magnification box in II : 1 µm , III : 1 µm , magnification box in III : 0 . 1 µm . Comparison of sporozoite development in oocysts of WT , α1 - tubulin ( - ) and complemented lines . Hoechst ( blue ) and SiR - tubulin ( green ) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls , while SiR tubulin staining is absent from α1 - tubulin ( - ) oocysts . mCherry stains the cytoplasm of the parasite . Scale bars : 5 µm . Transmission electron micrographs of developing sporozoites in WT and α1 - tubulin ( - ) parasite lines . Note the slender sporozoites in the WT oocysts , which are absent in the mutant . Sb : sporoblast ; scale bars : 5 µm . TEM cross sections of WT and α1 - tubulin ( - ) parasites showing the aberrant structure of the latter . Subpellicular and centriolar plaque ( spindle pole body ) associated ( hemispindle ) microtubules are readily recognizable in the WT sporozoites ( green arrowheads in insert ) but absent in the mutant , while the inner membrane complex is formed below the plasma membrane of both parasites ( white arrowheads ) . Note the different scale chosen to highlight the malformed shapes in the mutant . N : nucleus ; scale bars : 0 . 2 µm , magnification boxes : 0 . 1 µm , .

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Figure 1Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm.TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.


Figure 2Electron micrographs showing different stages of development in WT and α1 - tubulin ( - ) parasites . Key structural elements are highlighted in the following colors : blue : plasma membrane ; yellow : inner membrane complex ; green : microtubules ; light blue : micronemes ; lilac : rhoptries ; brown : rootlet fiber ; cyan : nucleus . Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1 - tubulin ( - ) parasites . The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque ( dark structure in nuclear envelope ) . Panel III corresponds to Fig 1D . Scale bars : 200 nm . α1 - tubulin ( - ) parasites show multiple attachments ( arrowheads ) with the sporoblast membrane at day 17 post infection . Note the highly branched nature of the budding parasite ( same parasite as in panel C ) shown in two views in I and II . Right : slices through the tomogram in grey scale with the outlines used for the 3D model indicated . Two connections with the sporoblast are indicated by 1 and 2 . Scale bars : 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm.


Figure 3Life imaging of wild - type ( WT ) and α1 - tubulin ( - ) oocysts expressing ef1α : GFP ( early and late oocysts ) and csp : mCherry ( late oocysts ) to locate the oocyst cytoplasm . Microtubules were labeled with SiR - tubulin ( green ) and DNA with Hoechst ( blue ) . Oocysts are shown in a chronological order from early ( day 4 ) to late ( day 12 ) development . ( A , B ) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage ( A ) and subsequent DNA replication ( B ) , where some but not all DNA is co - stained by microtubules in the mutant . ( C ) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites . Note the strong SiR - tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant . Sb : sporoblast . ( D ) Oocyst ready to burst with fully formed sporozoites in WT oocysts . Note that α1 - tubulin ( - ) oocysts retained sporozoite nuclei predominantly within the sporoblast ( Sb ) during budding . Scale bars : 5 μm , magnification box in B : 1 µm , . 3D reconstructions from spinning disc confocal microscopy z - stacks across an early oocyst labelled with the DNA dye Hoechst ( left ) . 50 and 19 images with a z - distance of 0 . 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Right : 3D reconstructions from late WT and α1 - tubulin ( - ) oocysts labelled with Hoechst ( blue ) and SiR - tubulin ( green ) . Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites . 10 and 28 images with a z - distance of 1 , 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Sb : sporoblast ; scale bars : 5 µm . TEM cross sections of WT and α1 - tubulin ( - ) oocysts during nuclear division . Spindle microtubules ( arrowheads ) emerging from centriolar plaques ( M ) were seen in WT oocysts . Although individual nuclei ( N ) were visible in α1 ( - ) oocysts , microtubules were absent even in dividing centriolar plaques ( M ) . Scale bars : 0 . 2 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 3Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.


Figure 4Fluorescence images showing the SiR - tubulin stained microtubules , nuclei ( Hoechst ) and cytoplasmic shape ( mCherry ) of the parasite lines . Note the small amount of DNA ( black arrowhead ) and the reduced level of SiR - tubulin fluorescence in the α1cm & ∆ introns - expressing sporozoites ( lower graph ) . See also Fig S6 . Quantitative data was derived from 96 - 120 sporozoites per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . Scale bar : 5 μm . * * * * indicates p < 0 . 0001 , Kruskal - Wallis - test . Note that α1cm & ∆ introns - expressing sporozoites do not express mCherry ; their autofluorescence ( auto - fluo ) indicates an aberrant shape . qRT - PCR analysis of the three parasite lines at day 7 and 12 after infection . Error bars correspond to the standard deviation from two biological replicates . TEM reveals α1cm & ∆ introns - expressing sporozoites with smaller ( A and B ) and larger ( C ) diameters than wild type sporozoites . See also Fig S7 . Scale bar : 0 . 2 µm . α1cm & ∆ introns - expressing sporozoites show a reduced number of subpellicular microtubules ( green arrowheads ) by TEM .

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

Figure 4Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM.


Figure 5Expression of different α1 - α2 chimeric α - tubulins instead of α1 - tubulin leads to sporozoite shape changes as revealed by SEM . See also Fig EV5 . Scale bar : 1 µm . α1 ∆ c - term lacks the last 3 amino acids of α1 - tubulin . α2 + parasites contain an α1 - tubulin with all amino acids that are different between the two α - tubulins changed into those of α2 - tubulin ; see also Fig S4 . α2 + + parasites contain α2 - tubulin ( exons , introns , 3 ' UTR ) and α2 + + + parasites express the exons of α2 - tubulin and introns of α1 - tubulin from the α1 - tubulin promoter . SiR - tubulin staining reveals differences in microtubule lengths and intensity . Between 69 and 156 sporozoites were analysed per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * , and * * * * indicate p < 0 . 001 and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test . qRT - PCR analysis of the parasite lines at day 12 after infection shows different expression level . Note the subtly higher expression of α2 + + compared to α2 + + + . Error bars show standard deviation from the mean of two biological replicates . TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α - tubulin chimeras . Green arrowheads point to microtubules . See also Fig S8 and S9 . Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier . Scale bars : 0 . 2 µm . TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines . Each dot represents the numbers of microtubules counted per section . Red lines indicate median from 23 to 40 analysed sections per line . Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2 + and α1 ∆ c - term parasites compared to wild type sporozoites . Semilog correlation of gene expression as determined by qRT - PCR with microtubule numbers as determine by TEM . Bars show range of values in x and standard deviation in y around the mean ( dots ) of two biological replicates ( x ) and all determined values ( y ) .

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 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 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 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 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

Figure 5Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line.Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites.Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).


Figure 6The microtubule number increases with the increase in the concentration of tubulin dimers . Microtubule number scales with tubulin concentration at low expression . At high expression , microtubule number is limited by the number of nucleation sites . Shown are mean + / - standard deviation from all determined values . The average microtubule length remains close to the maximum length 6 μm . The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data ( blue stars are for ( α2 + , α2 + + , α2 + + + ) strains ) . The error bar shows the standard deviation in the data and simulation results . For the WT expression level ( c0 = 36 . 6 ( m0 + p + n ) ) , the rate of nucleation is faster than the microtubule growth . Then the microtubule number is determined by the number of nucleation sites . The average microtubule length is around 1 μm ( shown by dashed arrow ) when the last nucleus forms . At intermediate tubulin numbers ( c0 = 16 ( m0 + p + n ) ) , corresponding to the α1c - term mutant , the microtubule formation rate is comparable to nucleation rate . The average microtubule length is around 4 μm ( shown by dashed arrow ) when the last nucleus forms .

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 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 O O O O O O O O O O O O O O O O O O O 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 6The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.


Figure 7Quantitative analysis of salivary gland sporozoite lengths from the different lines . n indicates numbers of investigated sporozoites . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * and * * * * indicate p < 0 . 001 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test ; scale bar : 1 µm . Quantification of SiR - tubulin fluorescence from 69 - 127 salivary gland derived sporozoites . Note the decrease in the fluorescence intensity of sporozoites expressing α1 - tubulin without introns as well as the decreased intensity and length of the chimeras . Linear correlation of sporozoite length and microtubule length reveals a R2 = 0 . 94 . * , * * , and * * * * indicate p < 0 . 05 , p < 0 . 01 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test . Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass . Scale bar : 20 µm . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * and * * * * indicate p < 0 . 05 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test . Sporozoites with fewest microtubules ( α2 + + + ) show a motility defect . n indicates numbers of investigated sporozoites ; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites . Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise ( CW ) direction . Numbers on black bars indicate percentage of CW moving sporozoites ; numbers in movie stills indicate time in seconds . CW moving sporozoites move at lower speed than CCW moving ones . Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2 + + infected salivary glands . Green arrowheads point to microtubules . Scale bar : 0 . 2 µm . Error bars show the full range and boxes 50 % of values . Red crosses indicate the mean , red lines the median . Fisher ' s exact test ( two - tailed ) .

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 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 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 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

Figure 7Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).


Figure 1 ( A - C ) Bone marrow derived mouse macrophages ( BMDMs ) from wild type C57BL / 6 mice were treated with 4µ8c ( 100 µM ) or vehicle ( dimethyl sulfoxide ; DMSO ) for 24 hours prior to sample processing for lipidomics analysis : ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP ratio are shown . Data represent mean values ± standard error of mean ( SEM ) of peak areas normalized to internal standards ( n = 3 biological replicates ) . ( D ) BMDMs were treated with 4µ8c ( 100 µM ) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates ; a representative blot is shown ) . ( E ) Wild type C57BL / 6 mice were injected with tunicamycin ( 1 mg / kg ) and 4µ8c ( 10 µg / kg ) for 8 hours prior to sacrifice and collection of thiogylcolate - elicited peritoneal macrophages . Protein lysates were analyzed by western blotting using specific antibodies against , pIRE1S742 , IRE1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin ( n = 3 biological replicates ; a representative blot is shown ) . Western blot quantifications are shown next to the figure . ( F ) IRE1 + / + and IRE1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742 , IRE1 , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates , a representative blot is shown ) .

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Figure 1(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).


Figure 2IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( B ) RNA lysates from the same experiment were analyzed by qRT - PCR for miR - 2137 expression . IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( C ) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742 , IRE1 and β - actin ( n = 3 biological replicates ) . IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( D ) RNA lysates were analyzed by qRT - PCR for sXBP1 expression ( n = 3 biological replicates ) . IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( E - F ) RNA lysates from the same experiment were analyzed by qRT - PCR for ( E ) BLOC1S1 and ( F ) SCARA3 expression ( n = 3 biological replicates ) .

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Figure 2IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).


Figure 3 ( A - B ) BMDMs were treated with PA , steric acid ( SA ) , palmitoleic acid ( PAO ) or oleic acid ( OA ) ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for the expression of ( A ) miR - 2137 and ( B ) sXBP1 ( n = 3 biological replicates ) . ( C , D ) ( C ) C57BL / 6 ( WT ) and ( D ) Apoe - / - mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( WT ; n = 4 - 4 mice per group , Apoe - / - ; n = 5 - 5 mice per group ) . ( E - F ) Apoe - / - mice were fed with chow or WD for 12 weeks and injected with 4µ8c ( 10 mg / kg / day ) or vehicle ( DMSO ) in the final 6 weeks of the diet ( n = 5 - 7 mice per group ) : ( E ) RNA lysates from bone marrow were analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) . ( F ) Total RNA isolated from the aortic root plaques was analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) .

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Figure 3(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).


Figure 4 ( A ) BMDMs were transfected with scrambled ( Scr ) or IRE1 - specific siRNA ( 40 nM ) and treated with palmitate ( PA , 500 µM ) or vehicle for 6 and 9 hours ( hr ) . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( B ) BMDMs were treated with PA ( 500 µM ) and 4µ8c ( 100 µM ) or vehicle for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( C ) BMDM cells were treated with tunicamycin ( Tun ; 5 µg / ml ) and 4µ8c ( 100 µM ) or vehicle ( DMSO ) for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( D ) IRE1 - / - MEFs were transfected with vector ( control ) , WT - IRE1 or K907A - IRE1 ( RNase dead mutant ) plasmids for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( E ) BMDMs were transfected with scrambled or XBP - 1 specific siRNA ( 70 nM ) for 24 hours , followed by treatment with PA ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( F ) XBP1 - / - MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed for miR - 2137 expression by qRT - PCR ( n = 3 biological replicates ) . ( G ) IRE1 cleavage assay performed using synthetic pre - miR - 2137 ( 50 nM ) as substrate with active , recombinant IRE1 ( 100 ng ) at 37 ℃ for 2 hours , followed by sample separation in Urea - PAGE and detection with SYBR gold staining . M indicates micro RNA marker . ( H - I ) BMDMs isolated from WT ( C57BL6 ) and DICER - / - mice were treated with PA ( 500 µM ) for 12 hours . RNA lysates were analyzed by qRT - PCR for ( H ) miR - 2137 and ( I ) sXBP1 expression ( n = 3 biological replicates ) .

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Figure 4(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).


Figure 5 ( B ) HEK293 cells were co - transfected with an INPPL1 3 ` UTR - luciferase plasmid and scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours . Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase ( SEAP ) reporter activity ( n = 3 biological replicates ) . ( C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) and miR - 205 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications are displayed in graph next to the blots ( n = 3 biological replicates ) . ( D ) BMDMs were transfected with negative control antagomir ( 200 nM ) or miR - 2137 antagomir ( 200 nM ) for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications shown in the graph next to blots ( n = 3 biological replicates ) .

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Figure 5(B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).


Figure 6 ( A - C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section . The ratio of lipids from this analysis is shown for ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 . ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP . Data from LC / MS / MS using a Waters Xevo TQ - S MS / MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods . ( n = 3 biological replicates ) BMDMs were transfected with ( D ) scrambled miRNA mimic ( 40 nM ) or miR - 2137 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) . BMDMs were transfected ( E ) with control antagomir ( 200 nM ) or miR - 2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) .

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Figure 6(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).BMDMs were transfected (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


Figure 1Overexpression of USP36 promotes SUMOylation in cells . Whole cell lysates ( WCL ) from H1299 ( A ) , U2OS and HeLa ( B ) cells transfected with control empty vector or Flag - USP36 were assayed by IB .

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Figure 1Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB.


Figure 2H1299 cells transfected with WT USP36 or the indicated mutants and His - SUMO2 were subjected to Ni2 + - NTA agarose beads pull down ( PD ) followed by IB using anti - SUMO2 / 3 antibody .

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Figure 2H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.


Figure 3USP36 interacts with Ubc9 in cells . H1299 cell transfected with the indicated plasmids were subjected to co - IP with anti - Flag antibody followed by IB . USP36 SUMOylates PARP1 in vitro . Recombinant T7 - PARP1 protein ( 0 . 1 μM ) was incubated with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the presence of USP361 - 800 ( 50 nM ) and / or ATP ( 2 . 5 mM ) at 30ºC for 5 hours and then assayed by IB .

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Figure 3USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB.USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.


Figure 4USP36 SUMOylates Nhp2 and Nop58 in vitro . In vitro SUMOylation assays were performed by incubating recombinant Nhp2 ( F , G ) or Nop58 ( H ) ( 0 . 1 μM ) with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the absence or presence of USP361 - 800 ( 50 nM , 1x ; 100 nM , 2x ) and / or ATP ( 2 . 5 mM ) at 37ºC for two hours or the indicated times . The reactions were assayed by IB using anti - Nhp2 and anti - Nop58 antibodies , respectively .

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Figure 4USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.


Figure 5USP36 does not change the levels of endogenous snoRNP proteins . HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB .

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Figure 5USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.


Figure 6USP36 promotes the binding of Nhp2 to snoRNAs . H1299 cells transfected with Flag - Nhp2 and V5 - USP36 individually or together were assayed by RNA - IP with anti - Flag or control mouse IgG , followed by RT - qPCR detection of the indicated box H / ACA snoRNAs ( C ) . Shown is one representative experiment of three independent experiments . Data were presented as mean ± SD , n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 , compared to Nhp2 alone ( Student ' s t - test ) .

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Figure 6USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).