Figure 1Relative fold repression for let - 7a reporter in RAW 264 . 7 cells during LPS treatment ( mean + / - s . e . m . , n = 3 ) ( B ) . The schematic representation of the constructs used for luciferase assays are shown . Relative levels of pro - inflammatory cytokines TNF - ɑ and IL - 6 mRNA , a target of let - 7a miRNA , have been plotted against time of LPS stimulation , 18S rRNA was used for normalization ( mean + / - s . e . m . , n = 2 ( IL - 6 ) , n = 3 ( TNF - α ) ) ( C ) . Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Ago2 was pulled down using anti - FLAG beads and the levels of Ago - associated let - 7a miRNA were measured by qRT - PCR . Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti - HA antibody and used for normalization of amounts of miRNA detected by qRT - PCR . Levels of cellular let - 7a were measured by Northern blot and U6 RNA was used as loading control . Relative levels of cellular and Ago2 bound let - 7a were plotted ( mean + / - s . e . m . , n = 3 ) ( D ) . Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Phosphorylated Ago2 ( pY - Ago2 ) level was measured using 4G10 antibody specific for phosphorylated tyrosine . Phosphorylated Ago2 ( pY - Ago2 ) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down ( E , upper panel ) . Relative 4G10 intensities were plotted from three independent experiments ( mean + / - s . e . m ) ( E , lower panel ) . Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells . RAW 264 . 7 cells were transfected with FH - Ago2 expression plasmid and treated with LPS ( 1ng / ml ) for different time points . Primary cells ( PEC ) were isolated from BALB / c mice and endogenous Ago2 was immunoprecipitated with anti - Ago2 ( eIF2C2 ) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody . Relative intensities of phospho - Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes ( mean + / - s . e . m . , n = 3 ) ( F , upper panel ) . Similar experiments were done with FH - Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH - Ago1 and FH - Ago3 were measured in RAW 264 . 7 cells ( F , lower panel ) . Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 cells . Knockdown of PP2A , MKP1 , MKP3 or SHP1 / 2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs . β - Actin was used as loading control ( G ) . Effect of knockdown of different phosphatases on miRNA - mediated repression in 24hrs LPS treated RAW 264 . 7 cells . Luciferase based let - 7a miRNA reporter assay was done in LPS - treated cells downregulated for specific phosphatases . Levels at 0 hrs time point were taken as units ( mean + / - s . e . m . , n = 4 ) ( H ) . Effect of knockdown of different phosphatases on miRNA - association of Ago2 in 24hrs LPS treated RAW 264 . 7 cells . Ago2 associated let - 7a was estimated by qRT - PCR in LPS treated cells depleted for specific phosphatases . let - 7a miRNA level was normalized against respective FH - Ago2 bands ( mean + / - s . e . m . , n = 3 ) ( I ) . Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody . HA - Ago2 was detected with anti - HA antibody ( J ) . Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments . Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Cellular miRNA levels ( K , left panel mean + / - s . e . m . , n = 3 ) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT - PCR and plotted ( K , right panel , mean + / - s . e . m . , n = 3 ) . Effect of PP2A knock - down on Ago2 phosphorylation and its miRNA association . Ago2 associated IL - 1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs ( mean + / - s . e . m . , n = 3 ) ( L ) .

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 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 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 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 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 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 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 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 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 1Relative fold repression for let-7a reporter in RAW 264.7 cells during LPS treatment (mean+/- s.e.m., n=3) (B). The schematic representation of the constructs used for luciferase assays are shown.Relative levels of pro-inflammatory cytokines TNF-ɑ and IL-6 mRNA, a target of let-7a miRNA, have been plotted against time of LPS stimulation, 18S rRNA was used for normalization (mean+/- s.e.m., n=2 (IL-6), n=3 (TNF-α)) (C).Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Ago2 was pulled down using anti-FLAG beads and the levels of Ago-associated let-7a miRNA were measured by qRT-PCR. Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti-HA antibody and used for normalization of amounts of miRNA detected by qRT-PCR. Levels of cellular let-7a were measured by Northern blot and U6 RNA was used as loading control. Relative levels of cellular and Ago2 bound let-7a were plotted (mean+/- s.e.m., n=3) (D).Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Phosphorylated Ago2 (pY-Ago2) level was measured using 4G10 antibody specific for phosphorylated tyrosine. Phosphorylated Ago2 (pY-Ago2) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down (E, upper panel). Relative 4G10 intensities were plotted from three independent experiments (mean+/- s.e.m) (E, lower panel).Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Primary cells (PEC) were isolated from BALB/c mice and endogenous Ago2 was immunoprecipitated with anti-Ago2 (eIF2C2) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody. Relative intensities of phospho-Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes (mean+/- s.e.m., n=3) (F, upper panel). Similar experiments were done with FH-Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH-Ago1 and FH-Ago3 were measured in RAW 264.7 cells (F, lower panel).Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Knockdown of PP2A, MKP1, MKP3 or SHP1/2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs. β-Actin was used as loading control (G).Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Luciferase based let-7a miRNA reporter assay was done in LPS-treated cells downregulated for specific phosphatases. Levels at 0 hrs time point were taken as units (mean +/- s.e.m., n=4) (H).Effect of knockdown of different phosphatases on miRNA-association of Ago2 in 24hrs LPS treated RAW 264.7 cells. Ago2 associated let-7a was estimated by qRT-PCR in LPS treated cells depleted for specific phosphatases. let-7a miRNA level was normalized against respective FH-Ago2 bands (mean+/- s.e.m., n=3) (I).Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody. HA-Ago2 was detected with anti-HA antibody (J). Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments.Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Cellular miRNA levels (K, left panel mean +/- s.e.m., n=3) and Ago2 associated miRNAs content in cells treated with siCon or siPP2A were measured using qRT-PCR and plotted (K, right panel, mean+/- s.e.m., n=3).Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Ago2 associated IL-1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs (mean+/- s.e.m., n=3) (L).


Figure 2Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264 . 7 cells after LPS stimulation ( A ) . Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation . The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount ( B ) . Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on miRNA activity . Change in fold repression for a let - 7a reporter upon LPS exposure in control and OA treated cells ( mean + / - s . e . m . , n = 3 ) ( C ) . Schematic representation of the in vitro phosphatase assay ( upper panel ) . PP2A was immunoprecipitated from 24hrs LPS treated RAW 264 . 7 cells and were incubated in vitro with wild type or phosphorylation defective FH - Ago2 mutant ( Ago2Y529F ) isolated from HEK293 cells transfected with respective expression constructs . Phosphorylated Ago2 ( pYAgo2 ) levels were measured by western blot analysis using anti - phospho Tyrosine specific 4G10 antibody . Relative intensities were quantified by densitometric analysis and mentioned below the respective panels . FH - Ago2 was detected by anti - HA antibody ( Lower panel ) . Defective de novo miRNP formation in cells pre - treated with PP2A inhibitor . Ago2 associated ( F ) and total ( G ) miR - 122 levels were measured by qRT - PCR and normalized against immuneprecipitated FH - Ago2 content and U6 RNA respectively . Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted . All samples were treated for 24 hrs with LPS ( mean + / - s . e . m . , n = 4 ) . PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Expression of PTPA after LPS stimulation in RAW 264 . 7 cells as well as in PEC isolated from BALB / c mice . β - actin is used as loading control ( H ) . PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264 . 7 cells ( I ) . Phosphorylated Ago2 level was measured by western blot in HA - immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH - Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody ( I , left panel ) . The PTPA was detected with anti PTPA antibody , β - Actin was used as loading control ( I , right panel ) . Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs . Ago2 - associated miRNA ( J , left panel ) ( mean + / - s . e . m . , n = 3 ) and mRNA levels ( J , right panel ) ( mean + / - s . e . m . , n = 2 ) were also quantified using qRT - PCR . PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264 . 7 cells treated with LPS for 24 hrs and was incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells and upon the assay reaction , the phospho - Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody ( K , right panel ) .

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 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 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 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 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 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

Figure 2Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs. Ago2-associated miRNA (J, left panel) (mean+/- s.e.m., n=3) and mRNA levels (J, right panel) (mean+/- s.e.m., n=2) were also quantified using qRT-PCR.PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).


Figure 3Expression of different cytokines and phosphatases in RAW 264 . 7 cells and PEC infected with Leishmania donovani ( Ld ) parasites . Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264 . 7 ( B , left panel ) and mouse peritoneal macrophage cells PEC ( B , right panel ) . Cytokine mRNA levels like IL - 10 , TNF - α and IL - 6 were quantified after 24 hrs of Ld infection in RAW 264 . 7 ( C , upper panel mean + / - s . e . m . , n = 3 ) and mouse peritoneal macrophage PEC ( mean + / - s . e . m . , n = 3 ) ( C , Lower panel ) . Effect of Ld infection on Ago2 phosphorylation and its miRNA association . RAW 264 . 7 cells were transfected with FH - Ago2 expression construct and infection was done for various time points followed by FH - Ago2 pull down using anti - FLAG beads . Phosphorylated Ago2 levels were checked during the course of Ld infection ( D ) . Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let - 7a level measurement in primary macrophages ( mean + / - s . e . m . , n = 2 ) ( E , left panel ) and in RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) ( E , right panel ) . Schematic representation of in vitro phosphatase assay ( F , upper panel ) . PP2A , MKP1 , MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody ( F , lower panel ) and measured by densitometry . BC ; Bead Control . PP2A MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted ( mean + / - s . e . m . , n = 3 ) ( G ) . BC ; Bead Control . Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells ( H ) . Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Ago2 associated let - 7a levels were also estimated and plotted ( mean + / - s . e . m . , n = 4 ) ( I ) . Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment ( 100 nM ) were detected . Cells were pre - treated with OA ( 100 nM ) for 2 hrs before infection ( J ) . β - Actin was used as loading control . Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells and mouse PEC . Phospho - Ago2 level was measured in OA pre - treated ; Ld infected RAW 264 . 7 cells expressing FH - Ago2 and in PEC post 24 hrs of infection with or without OA pre - treatment . Phosphorylated Ago2 levels were also checked ( K ) . Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots . Alix was used at MVB marker , while Calnexin was used as ER marker ( L ) . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . OptiprepR fractions , positive for MVB ( fraction number 2 , 3 , 4 ) and ER ( fraction number 7 , 8 , 9 ) markers were pooled separately and Ago2 was immunoprecipitated using Anti - FLAG beads . Phosphorylated Ago2 levels were quantified in western blot done with phospho - tyrosine specific 4G10 antibody ( M ) . Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . PP2A percentage intensity plot for each fraction was done for control and infected samples ( N ) .

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 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 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 O O O O 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 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 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 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 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 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

Figure 3Expression of different cytokines and phosphatases in RAW 264.7 cells and PEC infected with Leishmania donovani (Ld) parasites. Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264.7 ( B, left panel) and mouse peritoneal macrophage cells PEC (B, right panel).Cytokine mRNA levels like IL-10, TNF-α and IL-6 were quantified after 24 hrs of Ld infection in RAW 264.7 (C, upper panel mean +/- s.e.m., n=3) and mouse peritoneal macrophage PEC (mean+/- s.e.m., n=3) (C, Lower panel).Effect of Ld infection on Ago2 phosphorylation and its miRNA association. RAW 264.7 cells were transfected with FH-Ago2 expression construct and infection was done for various time points followed by FH-Ago2 pull down using anti-FLAG beads. Phosphorylated Ago2 levels were checked during the course of Ld infection (D).Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let-7a level measurement in primary macrophages (mean+/- s.e.m., n=2) (E, left panel) and in RAW 264.7 cells (mean+/- s.e.m., n=3) (E, right panel) .Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. BC; Bead Control.PP2A MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted (mean+/- s.e.m., n=3) (G). BC; Bead Control.Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H).Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Ago2 associated let-7a levels were also estimated and plotted (mean+/- s.e.m., n=4) (I).Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control.Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots. Alix was used at MVB marker, while Calnexin was used as ER marker (L).RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M).Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. PP2A percentage intensity plot for each fraction was done for control and infected samples (N).


Figure 4Effect of PP2A downregulation or inhibition on pro and anti - inflammatory cytokine levels in Ld infected macrophage . IL - 10 levels were measured after Ld infection in PP2A knocked - down RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) and in PEC ( mean + / - s . e . m . , n = 3 ) . IL - 6 was also measured in RAW cells after OA treatment ( mean + / - s . e . m . , n = 5 ) and also in PEC pre - treated with OA ( mean + / - s . e . m . , n = 3 ) . Values obtained at 0 hrs ( non - infected ) or non - OA treated samples were considered as unit in each case . IL - 6 protein level ( n = 5 ) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock - down RAW 264 . 7 cells . Effect of PP2A knock - down on parasite internalization Internalized parasites were imaged in RAW 264 . 7 cells stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 ( red ) ( B ) . Effect of PP2A knock - down on parasite internalization and cellular IL - 1β level . Number of Ld internalized was measured in control and PP2A knock - down cells and relative numbers of parasites within infected cells were plotted ( C , left panel , ( mean + / - s . e . m . , n = 20 cells ) . IL - 1β mRNA levels were also quantified in those cells by qRT - PCR done for total cellular RNA [ C , right panel , ( mean + / - s . e . m . , n = 3 ) ] . Scale bar 20 µm . 4x Zoomed insets are shown . Effect of PP2A knockdown on IL - 10 level in Ld - infected cells expressing phosphorylation defective mutant of Ago2 . In RAW 264 . 7 cells , depleted for endogeneous Ago2 ( using a specific siRNA against the 3 ' UTR of Ago2 mRNA ) , either the wild type or Y529F mutant of FH - Ago2 ( without having the 3 ' UTR of Ago2 ) was expressed in siCon or siPP2A treated RAW 264 . 7 cells . IL - 10 mRNA level was measured subsequent to Ld infection . Relative IL - 10 levels were plotted . Expression levels in non - infected siCon treated cells in each case were designated as unit ( mean + / - s . e . m . , n = 3 ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . In images obtained , Ago2 was detected with α - HA ( detected at 405nm ) and Leishmania was stained for GP63 ( detected at 564nm ) ( E ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted . Scale bar 10 µm . RNA was isolated from different experimental sets and TNF - α , IL - 1β and IL - 10 mRNA levels were estimated ( mean + / - s . e . m . , n = 3 ) ( F ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . RAW 264 . 7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs . RAW 264 . 7 cells were stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 ( red ) ( G , left panel ) . Number of internalized parasites per 100 macrophages were calculated and relative values were plotted ( G , right panel ) ( mean + / - s . e . m . , n = 20 ) . Scale bar 20 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 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 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 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 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 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

Figure 4Effect of PP2A downregulation or inhibition on pro and anti-inflammatory cytokine levels in Ld infected macrophage. IL-10 levels were measured after Ld infection in PP2A knocked-down RAW 264.7 cells (mean+/- s.e.m., n=3) and in PEC (mean+/- s.e.m., n=3). IL-6 was also measured in RAW cells after OA treatment (mean+/- s.e.m., n=5) and also in PEC pre-treated with OA (mean+/- s.e.m., n=3). Values obtained at 0 hrs (non-infected) or non-OA treated samples were considered as unit in each case. IL-6 protein level (n=5) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock-down RAW 264.7 cells.Effect of PP2A knock-down on parasite internalization Internalized parasites were imaged in RAW 264.7 cells stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 (red) (B).Effect of PP2A knock-down on parasite internalization and cellular IL-1β level. Number of Ld internalized was measured in control and PP2A knock-down cells and relative numbers of parasites within infected cells were plotted (C, left panel, (mean+/- s.e.m., n=20 cells). IL-1β mRNA levels were also quantified in those cells by qRT-PCR done for total cellular RNA [C, right panel, (mean+/- s.e.m., n=3)]. Scale bar 20 µm. 4x Zoomed insets are shown.Effect of PP2A knockdown on IL-10 level in Ld-infected cells expressing phosphorylation defective mutant of Ago2. In RAW 264.7 cells, depleted for endogeneous Ago2 (using a specific siRNA against the 3'UTR of Ago2 mRNA), either the wild type or Y529F mutant of FH-Ago2 (without having the 3'UTR of Ago2) was expressed in siCon or siPP2A treated RAW 264.7 cells. IL-10 mRNA level was measured subsequent to Ld infection. Relative IL-10 levels were plotted. Expression levels in non-infected siCon treated cells in each case were designated as unit (mean+/- s.e.m., n=3).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. In images obtained, Ago2 was detected with α-HA (detected at 405nm) and Leishmania was stained for GP63 (detected at 564nm) (E).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted. Scale bar 10 µm. RNA was isolated from different experimental sets and TNF-α, IL-1β and IL-10 mRNA levels were estimated (mean +/- s.e.m., n=3) (F).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. RAW 264.7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs. RAW 264.7 cells were stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 (red) (G, left panel). Number of internalized parasites per 100 macrophages were calculated and relative values were plotted (G, right panel) (mean+/- s.e.m., n=20). Scale bar 20 µm.


Figure 5LPS induced miRNA export from mammalian macrophage cells . A schematic diagram of the experiments has been described in left panel of ( A ) . Characterization and quantification of exosomes derived from Control and LPS - activated RAW 264 . 7 cells by Nanoparticle Tracking Analysis ( NTA ) done for isolated exosomes . Relative size distributions are shown ( A , right panel ) . Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264 . 7 cells . RNA content was normalized against total exosomal proteins ( B ) . β - Actin as loading control for HuR western blot . Binding of miRNAs with HuR after LPS exposure was measured . Amount of RNA was normalized against HuR content . Anti GFP - antibody was used for immunoprecipitation control ( C ) . Values are mean + / - s . e . m . and , n = 3Effect of GW4869 , the inhibitor of exosome mediated miRNA export , on exosomal miRNA content released by LPS stimulated cells . The level of miRNAs , let - 7a and miR - 155 were measured in exosomes ( EV ) released from LPS activated cell after GW4869 treatment . Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit . Values are mean + / - s . e . m . and , n = 3Effect of GW4869 on cellular cytokine levels in LPS treated cells . The effect of GW4869 on cellular expression of pro - inflammatory cytokines IL - 1β and TNF - α in LPS - activated macrophages were measured and normalized against 18S rRNA . Values in naive RAW 264 . 7 cells without GW4869 and LPS treatment were set as unit . Values are mean + / - s . e . m . and , n = 3Effect of siRNA - mediated downregulation of protein HuR on cellular expression of pro - inflammatory cytokines . In the left panel of F , effect of siRNA treatment on cellular cytokine mRNA levels in LPS - stimulated RAW 264 . 7 cells is shown . Relative levels measured against 18S rRNA are plotted . The effect of HuR depletion on cellular miR - 155 content has been measured and relative values normalized against U6 snRNA are plotted ( right panel , F ) . The amount of let - 7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 ( G ) . Values are mean + / - s . e . m . and , n = 3Effect of HuR expression on inflammatory responses in macrophage cells . Effect of ectopic expression of HuR on exosomal let - 7a content in naive macrophage cells ( H , right panel ) . HuR over expression was checked by western blot ( H , left panel ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n = 3Effect of HuR expression on inflammatory responses in macrophage cells . The effect of HuR expression on production of pro - inflammatory cytokine TNF - α in RAW 264 . 7 cells was measured . mRNA and protein level of TNF - α was quantified . Values obtained with pCINeo expression and without LPS treatment was taken as unit ( I ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n = 3Effect of HA - HuR expression on p38 mediated activation of downstream signalling events . The Levels of p - p38 and p - MSK1 have been monitored by western blotting done with cell extracts prepared from HA - HuR and control plasmid transfected , untreated and LPS treated RAW 264 . 7 cells . β - Actin blot was used as loading control . Downregulation of HuR in Ld - infected macrophages . HuR levels were monitored against time in Ld - infected primary macrophage cells ( PEC ) from BALB / c mice ( K , left upper panel ) as well as in Ld - infected RAW 264 . 7 cells ( K , upper right panel ) . HuR levels were also monitored against time in L . major - infected RAW 264 . 7 cells ( K , lower left panel ) and L . mexicana - infected RAW 264 . 7 cells ( K , lower right panel ) . Downregulation of HuR in Ld - infected macrophages . Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld - infected RAW 264 . 7 cells . In parallel assays levels of pro - inflammatory cytokine TNF - α was measured in Ld - infected cells either with no treatment or pre - treated with MG132 ( L ) . Values are mean + / - s . e . m . and , n = 3 . Downregulation of HuR in Ld - infected macrophages . The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined . Input samples were monitored for HuR downregulation by western blot . Presence of Alix ( marker for early endosomes ) and calnexin ( marker protein for ER ) were used to confirm separation of organelles during density gradient fractionation . Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection ( M upper panel ) . Densitometric analysis of individual fractions was done and the respective values were plotted ( M , lower panel ) .

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 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 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 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 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 O O O O O O O O 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 O O O O O O O O O O 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

Figure 5LPS induced miRNA export from mammalian macrophage cells. A schematic diagram of the experiments has been described in left panel of (A). Characterization and quantification of exosomes derived from Control and LPS-activated RAW 264.7 cells by Nanoparticle Tracking Analysis (NTA) done for isolated exosomes. Relative size distributions are shown (A, right panel).Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264.7 cells. RNA content was normalized against total exosomal proteins (B). β-Actin as loading control for HuR western blot.Binding of miRNAs with HuR after LPS exposure was measured. Amount of RNA was normalized against HuR content. Anti GFP-antibody was used for immunoprecipitation control (C). Values are mean+/- s.e.m. and, n=3Effect of GW4869, the inhibitor of exosome mediated miRNA export, on exosomal miRNA content released by LPS stimulated cells. The level of miRNAs, let-7a and miR-155 were measured in exosomes (EV) released from LPS activated cell after GW4869 treatment. Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit. Values are mean+/- s.e.m. and, n=3Effect of GW4869 on cellular cytokine levels in LPS treated cells. The effect of GW4869 on cellular expression of pro-inflammatory cytokines IL-1β and TNF-α in LPS-activated macrophages were measured and normalized against 18S rRNA. Values in naive RAW 264.7 cells without GW4869 and LPS treatment were set as unit. Values are mean+/- s.e.m. and, n=3Effect of siRNA-mediated downregulation of protein HuR on cellular expression of pro-inflammatory cytokines. In the left panel of F, effect of siRNA treatment on cellular cytokine mRNA levels in LPS-stimulated RAW 264.7 cells is shown. Relative levels measured against 18S rRNA are plotted. The effect of HuR depletion on cellular miR-155 content has been measured and relative values normalized against U6 snRNA are plotted (right panel, F).The amount of let-7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 (G) .Values are mean+/- s.e.m. and, n=3Effect of HuR expression on inflammatory responses in macrophage cells. Effect of ectopic expression of HuR on exosomal let-7a content in naive macrophage cells (H, right panel). HuR over expression was checked by western blot (H, left panel). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3Effect of HuR expression on inflammatory responses in macrophage cells. The effect of HuR expression on production of pro-inflammatory cytokine TNF-α in RAW 264.7 cells was measured. mRNA and protein level of TNF-α was quantified. Values obtained with pCINeo expression and without LPS treatment was taken as unit (I). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3Effect of HA-HuR expression on p38 mediated activation of downstream signalling events. The Levels of p-p38 and p-MSK1 have been monitored by western blotting done with cell extracts prepared from HA-HuR and control plasmid transfected, untreated and LPS treated RAW 264.7 cells. β-Actin blot was used as loading control.Downregulation of HuR in Ld-infected macrophages. HuR levels were monitored against time in Ld-infected primary macrophage cells (PEC) from BALB/c mice (K, left upper panel) as well as in Ld-infected RAW 264.7 cells (K, upper right panel) .HuR levels were also monitored against time in L. major-infected RAW 264.7 cells (K, lower left panel) and L. mexicana-infected RAW 264.7 cells (K, lower right panel).Downregulation of HuR in Ld-infected macrophages. Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld-infected RAW 264.7 cells. In parallel assays levels of pro-inflammatory cytokine TNF-α was measured in Ld-infected cells either with no treatment or pre-treated with MG132 (L). Values are mean+/- s.e.m. and, n=3.Downregulation of HuR in Ld-infected macrophages. The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined. Input samples were monitored for HuR downregulation by western blot. Presence of Alix (marker for early endosomes) and calnexin (marker protein for ER) were used to confirm separation of organelles during density gradient fractionation. Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection (M upper panel). Densitometric analysis of individual fractions was done and the respective values were plotted (M, lower panel).


Figure 6effect of HA - HuR expression on PP2A protein levelseffect of HA - HuR expression on PP2A RNA levels were scored . Expression profiles of HuR , PP2A and miRNA let - 7a are connected to IL - 6 expression in RAW 264 . 7 cells . Data obtained from experiments described in previous figures are summed up to plot the changes in IL - 6 mRNA levels against time of LPS treatment along with changes in PP2A , miRNA let - 7a and HuR ( D ) . Change in expression of IL - 6 is connected to changes in let - 7a concentration . The mathematical equation fitting the curve are shown here . Y represents concentration of IL - 6 and X represents miRNA let - 7a concentration . A1 is the initial IL - 6 levels and A2 is the changed level . LogX0 is the mid - point of value 0 . 34 , a concentration of let - 7a where Il - 6 expression is reduced to halfvariations of IL - 6 expression with the altered level of PP2A ( black ) and HuR ( red ) . For both cases , the data were fitted to an equation , which is shown above . The parameter m denotes the expression level of PP2A / HuR which corresponds to the maximum IL - 6 expression ; w denotes the width of the distributions for both cases . For PP2A and HuR , the values of m were found to be 0 . 6 and 0 . 9 respectively and A is a constant ( F ) .

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

Figure 6effect of HA-HuR expression on PP2A protein levelseffect of HA-HuR expression on PP2A RNA levels were scored.Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D).Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to halfvariations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).


Figure 7Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . HA - HuR was immunostained with anti - HA antibody and detected by secondary antibody tagged with AlexaR564 ( A ) . Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . In the zoomed part of the merged picture are used to show the internalized parasite . Internalized parasites present in HA - HuR positive cells were counted and compared against untransfected cells without HA - HuR expression ( B ) . Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Levels of expression of different cytokines are measured in control or HA - HuR expressing RAW 264 . 7 cells infected with Ld ( C ) . Scale bar 20 µm . Values are mean + / - s . e . m . and , n = 3 . Effect of Ld infection on the HuR expression in mouse liver . BALB / c mice of 6 weeks of age were infected with Ld via cardiac puncture . Animals were sacrificed after subsequent days ; protein was extracted from liver tissue followed by western blot analysis for HuR . β - Actin was used as loading control ( D , left panel ) . Each lane have designated individual animal . Band intensities were determined by ImageJ . In each case , HuR level was normalized by its respective β - Actin bands ( D , right panel mean + / - s . e . m . , n = 5 ) . Animals were injected with GP63 containing liposome ( gp1 - 3 ) or empty liposome ( C1 - 2 ) and after 24 hrs of injection , HuR level was checked against β - Actin in liver tissue . β - Actin was used as loading control ( E ) . Effect of expression of HA - HuR on Ld infection in mice liver . Scheme for animal experiment done with HA - HuR expression in liver is shown . Also the HA - HuR level was detected in the liver tissue by western blot analysis using anti - HA antibody ( F ) . Effect of expression of HA - HuR on Ld infection in mice liver . Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA . Values are mean + / - s . e . m . and , n = 5 ( G ) . Effect of expression of HA - HuR on Ld infection in mice liver . TNF - α ( Values are mean + / - s . e . m . and , n = 5 ) and IL - 10 ( Values are mean + / - s . e . m . and , n = 4 ) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted ( H and I , left panels ) . Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count , TNF - α and IL - 10 mRNA levels ( G mean + / - s . e . m . , ( n = 5 ) , H mean + / - s . e . m . , ( n = 6 ) and I mean + / - s . e . m . , ( n = 4 ) , right panel ) . Effect of HA - HuR plasmid tail - vein injection on mice spleen . Levels of HA - HuR were undetectable in mice spleen after expression plasmid was injected . Sample from one liver tissue of HA - HuR injected group was used as a positive control of HA - HuR expression in moue liver after tail vein injection of HuR encoding plasmid ( J ) . Effect of HA - HuR plasmid tail - vein injection on mice spleen . Simultaneously , TNF - α mRNA levels were checked in spleen total RNA ( K ) .

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 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 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 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 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 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 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

Figure 7Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. HA-HuR was immunostained with anti-HA antibody and detected by secondary antibody tagged with AlexaR564 (A).Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. In the zoomed part of the merged picture are used to show the internalized parasite. Internalized parasites present in HA-HuR positive cells were counted and compared against untransfected cells without HA-HuR expression (B).Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Levels of expression of different cytokines are measured in control or HA-HuR expressing RAW 264.7 cells infected with Ld (C). Scale bar 20 µm. Values are mean+/- s.e.m. and, n=3.Effect of Ld infection on the HuR expression in mouse liver. BALB/c mice of 6 weeks of age were infected with Ld via cardiac puncture. Animals were sacrificed after subsequent days; protein was extracted from liver tissue followed by western blot analysis for HuR. β- Actin was used as loading control (D, left panel). Each lane have designated individual animal. Band intensities were determined by ImageJ. In each case, HuR level was normalized by its respective β- Actin bands (D, right panel mean +/- s.e.m., n=5).Animals were injected with GP63 containing liposome (gp1-3) or empty liposome (C1-2) and after 24 hrs of injection, HuR level was checked against β-Actin in liver tissue. β-Actin was used as loading control (E).Effect of expression of HA-HuR on Ld infection in mice liver. Scheme for animal experiment done with HA-HuR expression in liver is shown. Also the HA-HuR level was detected in the liver tissue by western blot analysis using anti-HA antibody (F).Effect of expression of HA-HuR on Ld infection in mice liver. Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA. Values are mean+/- s.e.m. and, n=5 (G).Effect of expression of HA-HuR on Ld infection in mice liver. TNF-α (Values are mean+/- s.e.m. and, n=5) and IL-10 (Values are mean+/- s.e.m. and, n=4) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted (H and I, left panels). Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count, TNF-α and IL-10 mRNA levels (G mean +/- s.e.m., (n=5), H mean +/- s.e.m., (n=6) and I mean +/- s.e.m., (n=4), right panel).Effect of HA-HuR plasmid tail-vein injection on mice spleen. Levels of HA-HuR were undetectable in mice spleen after expression plasmid was injected. Sample from one liver tissue of HA-HuR injected group was used as a positive control of HA-HuR expression in moue liver after tail vein injection of HuR encoding plasmid (J).Effect of HA-HuR plasmid tail-vein injection on mice spleen. Simultaneously, TNF-α mRNA levels were checked in spleen total RNA (K).


Figure 8Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on cellular cytokine level RAW 264 . 7 cells were infected with Antimony sensitive ( LdS - Ag83 ) and antimony resistant ( LdR - BHU569 ) form of Ld for 24 hours along with uninfected cells kept as control . IL - 10 ( A ) and TNF - α ( B ) were measured at mRNA level by qRT - PCR after infection ( mean + / - s . e . m . and , n = 4 ) . Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on on phospho - ERK level . ( D ) . Densitometry analysis was done for p - ERK level by ImageJ and relative values were plotted ( C ) . β - Actin was used as loading control Values are mean + / - s . e . m . and , n = 3 . Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on phospho - ERK and MDR1 level . p - ERK , ERK and MDR1 level were also measured by western blot ( D ) . Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A . RAW 264 . 7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot ( left panel ) . PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection ( right panel ) . β - Actin was used as loading control . Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell . RAW 264 . 7 cells were transfected with HA - HuR or pCIneo followed by 2 hrs of pretreatment with OA . These cells were then infected with LdR for 24 hrs . Pro - inflammatory cytokines TNF - α ( right panel ; mean + / - s . e . m . , n = 3 ) and IL - 1β ( left panel ; mean + / - s . e . m . and , n = 4 ) were measured at mRNA level and relative values are plotted . Values without OA treatment was set as control . Effect of HuR over expression on LdR infection in mice liver . Pro - inflammatory cytokines TNF - α ( left panel ) and IL - 1β ( right panel ) level were measured at mRNA level in liver ( H ) . Effect of HuR over expression on LdR infection in mice liver . Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld ( I ) . Values are mean + / - s . e . m . and , n = 6 ) .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 8Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on cellular cytokine level RAW 264.7 cells were infected with Antimony sensitive (LdS-Ag83) and antimony resistant (LdR-BHU569) form of Ld for 24 hours along with uninfected cells kept as control. IL-10 (A) and TNF-α (B) were measured at mRNA level by qRT-PCR after infection (mean+/- s.e.m. and, n=4).Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on on phospho-ERK level. (D). Densitometry analysis was done for p-ERK level by ImageJ and relative values were plotted (C). β-Actin was used as loading control Values are mean+/- s.e.m. and, n=3.Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on phospho-ERK and MDR1 level. p-ERK, ERK and MDR1 level were also measured by western blot (D).Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A. RAW 264.7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot (left panel). PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection (right panel). β-Actin was used as loading control.Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.Effect of HuR over expression on LdR infection in mice liver. Pro-inflammatory cytokines TNF-α (left panel) and IL-1β (right panel) level were measured at mRNA level in liver (H).Effect of HuR over expression on LdR infection in mice liver. Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld (I). Values are mean+/- s.e.m. and, n=6).


Figure 2A Actin expression levels shown by western blotting for strains expressing S . cerevisiae ' s actin protein from various coding sequences , with tubulin ( Tub1p ) as a loading control . B Quantification of actin expression levels , showing a decrease when more silent mutations are present . Data are presented as mean + / - SD ( n = 4 for Sc , n = 8 for ScNI ; n = 12 for Sc [ Ca ] , Sc [ Sp ] and Sc [ At ] ; 2 biological replicates with n / 2 technical replicates each ) . * P < 0 . 05 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . F In vivo actin network deviation indexes , defined to evaluate the patch - cable balance compared to S . cerevisiae haploid cells ( value is 0 in S . cerevisiae ' s cells , 1 when cells contain only actin patches and - 1 when cells contain only cables ) . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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 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

Figure 2A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 3E Phalloidin staining of F - actin organization . Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed . Micrographs of Sc and Sc _ NI cells are reproduced from Fig 2E . Scale bar : 3 µm . F In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . I Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm J Quantification of actin patch resistance to CK - 666 treatment . Bar graphs represent the percentage of cells with a given number of visible actin patches after CK - 666 treatment . ( n = 41 for ScNI , 55 for N2 , 31 for Ca , 41 for Op , 55 for Hs ) .

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 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 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

Figure 3E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm.F In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).


Figure 4A ( Top ) Snapshots of actin networks assembled from three different actins sources : Act _ Sc , Act _ N2 and Act _ Ca . ( Bottom left ) Quantification of actin fluorescence on beads . Data are presented as mean + / - SD ( n = 52 for Act _ Sc branched , n = 70 for Act _ N2 branched , n = 81 for Act _ Ca branched , n = 32 for Act _ Sc linear , n = 34 for Act _ N2 linear and n = 47 for Act _ Ca linear ) . * * P < 0 . 01 , * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . ( Bottom right ) In vitro actin network deviation indexes . B ( Left ) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa - 488 - labeled ADF / cofilin ( replacement of unlabeled ADF / cofilin ) . ( Right ) Quantification of ADF / cofilin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 34 for Act _ Sc branched , n = 28 for Act _ N2 branched , n = 43 for Act _ Ca branched , n = 24 for Act _ Sc linear , n = 15 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . C ( Left ) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488 - tropomyosin . ( Right ) Quantification of tropomyosin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 45 for Act _ Sc branched , n = 63 for Act _ N2 branched , n = 57 for Act _ Ca branched , n = 22 for Act _ Sc linear , n = 32 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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 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 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

Figure 4A (Top) Snapshots of actin networks assembled from three different actins sources: Act_Sc, Act_N2 and Act_Ca. (Bottom left) Quantification of actin fluorescence on beads. Data are presented as mean +/- SD (n = 52 for Act_Sc branched, n = 70 for Act_N2 branched, n = 81 for Act_Ca branched, n = 32 for Act_Sc linear, n = 34 for Act_N2 linear and n = 47 for Act_Ca linear). **P<0.01, ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). (Bottom right) In vitro actin network deviation indexes.B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 6C Phalloidin staining depicting F - actin organization . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . D In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . E Polarity Indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . F Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . G In vivo actin network deviation indexes of cells treated with DMSO or CK - 666 . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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 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

Figure 6C Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


Figure 1 ( B ) Coomassie - stained SDS - PAGE of the SEC fractions highlighted in ( A ) with a horizontal dashed line on top . ( C - D ) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP ( C ) or GDP ( D ) . ( G ) SEC profile ( top ) and Coomassie stained SDS gel ( bottom ) of the complex between RabL2 and a minimal binding - region of CEP19 ( residues 107 - 195 ) demonstrating a direct interaction .

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Figure 1(B) Coomassie-stained SDS-PAGE of the SEC fractions highlighted in (A) with a horizontal dashed line on top.(C-D) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP (C) or GDP (D).(G) SEC profile (top) and Coomassie stained SDS gel (bottom) of the complex between RabL2 and a minimal binding-region of CEP19 (residues 107-195) demonstrating a direct interaction.


Figure 2 ( A ) SEC profile of CrRabL2Q83L co - purification with CrIFT81 / 74 / 27 / 25 / 22 ( top ) in the presence of non - hydrolysable GTP homologue , GTPγS . Coomassie stained SDS gel of SEC fraction highlighted by dashed lines ( bottom ) . ( B ) SEC profile for the purification of the HsIFT81 / 74 / 27 / 25 / 22 / RabL2 complex in the presence of GTPγS ( top ) and the corresponding Coomassie stained SDS gel ( bottom ) . ( E - H ) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time . Each experiment was carried out in three technical replicates . Concentrations for experiments in ( E ) were 55µM CrIFT - B1 pentamer / CrRabL2 , 60µM CrIFT - B1 pentamer and 70µM CrIFT - B1 tetramer and 1mM GTP . Concentrations for experiments in ( G ) were 60µM protein and 30µM GTP . The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel ( E ) and every 200s for panel ( G ) . ( F , H ) Quantification of the reaction rates ( arbitrary units of Absorbance ( Abs ) per second ( s ) ) for each experiment shown in panels ( E ) calculated using linear regression of the first 500s and ( H ) using single exponential fit , and agreement with the fit to the curves in ( E , G ) are indicated by the error bars and R2 - value .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC profile of CrRabL2Q83L co-purification with CrIFT81/74/27/25/22 (top) in the presence of non-hydrolysable GTP homologue, GTPγS. Coomassie stained SDS gel of SEC fraction highlighted by dashed lines (bottom). (B) SEC profile for the purification of the HsIFT81/74/27/25/22/RabL2 complex in the presence of GTPγS (top) and the corresponding Coomassie stained SDS gel (bottom).(E-H) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time. Each experiment was carried out in three technical replicates. Concentrations for experiments in (E) were 55µM CrIFT-B1 pentamer/CrRabL2, 60µM CrIFT-B1 pentamer and 70µM CrIFT-B1 tetramer and 1mM GTP. Concentrations for experiments in (G) were 60µM protein and 30µM GTP. The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel (E) and every 200s for panel (G). (F, H) Quantification of the reaction rates (arbitrary units of Absorbance (Abs) per second(s)) for each experiment shown in panels (E) calculated using linear regression of the first 500s and (H) using single exponential fit, and agreement with the fit to the curves in (E, G) are indicated by the error bars and R2-value.


Figure 3 ( A ) SEC profile showing that a minimal CrIFT81460 - 533 / CrIFT74460 - 532 complex co - purifies with CrRabL2Q83L in the presence of GTPγS ( left ) . The right panel displays the Coomassie stained SDS gel of SEC fractions ( horizontal top dashed line ) . ( B ) An N - terminal hexa - histidine tagged CrIFT81460 - 533 / 74460 - 532 complex interacts with untagged CrRabL2Q83L in a GTPγS - dependent manner in pulldown assays . ( E ) Quantification of GTPase reaction rates ( arbitrary units of Absorbance ( Abs ) per second ( s ) ) using single exponential fit ( D ) , error bars and R2 - value indicate the agreement of the fit to the curves in ( D ) .

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 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 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 3(A) SEC profile showing that a minimal CrIFT81460-533/CrIFT74460-532 complex co-purifies with CrRabL2Q83L in the presence of GTPγS (left). The right panel displays the Coomassie stained SDS gel of SEC fractions (horizontal top dashed line).(B) An N-terminal hexa-histidine tagged CrIFT81460-533/74460-532 complex interacts with untagged CrRabL2Q83L in a GTPγS-dependent manner in pulldown assays.(E) Quantification of GTPase reaction rates (arbitrary units of Absorbance (Abs) per second(s)) using single exponential fit (D), error bars and R2-value indicate the agreement of the fit to the curves in (D).


Figure 5 ( C ) SEC profile showing that human RabL2B co - purifies with Chlamydomonas IFT - B1 pentamer ( left ) . Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile ( right ) . ( E ) Quantification of the reaction rates of the GTPase reaction based on single exponential fit of curves in ( D ) error bars and R2 indicate the agreement of the fit .

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Figure 5(C) SEC profile showing that human RabL2B co-purifies with Chlamydomonas IFT-B1 pentamer (left). Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile (right).(E) Quantification of the reaction rates of the GTPase reaction based on single exponential fit of curves in (D) error bars and R2 indicate the agreement of the fit.


Figure 6 ( B ) Affinity pull - down of CrRabL2 with His - tagged WT or mutant CrIFT74T484R / 81 complex in the presence or absence of nucleotides . ( E ) Immunoblot ( IB ) analysis of expression of IFT74 , IFT81 , RabL2B and α - tubulin in control ( sgSafe ) or IFT74 KO RPE cells stably expressing either empty vector , FLAG - IFT74 , or FLAG - IFT74 T438R . Representative images from three experimental replicates are shown . The cells were grown to confluence ( without serum starvation ) , lysed and analyzed by Immunoblot . Molecular weights ( kDa ) estimated from a protein marker are indicated . Red asterisk : endogenous IFT74 ; black asterisks : FLAG tagged IFT74 . ( F ) Quantification of the immunoblots of IFT74 shown in ( E ) . Data are combined from three independent experiments . Error bars represent ± SD . Statistics obtained through one - way analysis of variance ( ANOVA ) with Tukey ' s multiple comparison test . ( G - I ) Box plots showing centrosomal signal intensity of FLAG ( G ) , IFT81 ( H ) , or RabL2 ( I ) in the cells described in ( D ) serum starved for 24 hours . The relative fluorescence signal intensity compared with the average of the control ( IFT74 KO + FLAG - IFT74 WT for ( G ) and sgSafe + empty for ( H ) and ( I ) ) are shown . Data are combined from three experimental replicates . Statistics obtained through nested one - way ANOVA with Šídák ' s multiple comparison test . ( G ) The number of mother centrioles analyzed in each sample per replicate are : 78 , 77 , 78 ( sgSafe + empty ) ; 102 , 95 , 101 ( IFT74 KO + empty ) ; 54 , 77 , 85 ( IFT74 KO + FLAG - IFT74 WT ) ; 49 , 83 , 99 ( IFT74 KO + FLAG - IFT74 T438R ) , ( H ) The number of mother centrioles analyzed in each sample per replicate are : 93 , 137 , 138 ( sgSafe + empty ) ; 109 , 133 , 193 ( IFT74 KO + empty ) ; 62 , 146 , 131 ( IFT74 KO + FLAG - IFT74 WT ) ; 41 , 143 , 133 ( IFT74 KO + FLAG - IFT74 T438R ) , ( I ) The number of mother centrioles analyzed in each sample per replicate are : 64 , 101 , 106 ( sgSafe + empty ) ; 84 , 105 , 129 ( IFT74 KO + empty ) ; 52 , 96 , 121 ( IFT74 KO + FLAG - IFT74 WT ) ; 73 , 92 , 102 ( IFT74 KO + FLAG - IFT74 T438R ) . ( K ) Box plots showing the quantification of number of IFT88 particles per cilium in the experiment described in ( J ) . Data are combined from three experimental replicates : The number of cilia analyzed in each sample per replicate are : 20 , 20 , 18 ( sgSafe + empty ) ; 20 , 20 , 18 ( IFT74 KO + WT ) ; 20 , 20 , 19 ( IFT74 KO + FLAG - IFT74 WT ) . The number of IFT particles in individual cells in each experiment are available from Figures S7A , D , and G . Statistics obtained through nested one - way ANOVA with Tukey ' s multiple comparison test .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 6(B) Affinity pull-down of CrRabL2 with His-tagged WT or mutant CrIFT74T484R/81 complex in the presence or absence of nucleotides.(E) Immunoblot (IB) analysis of expression of IFT74, IFT81, RabL2B and α-tubulin in control (sgSafe) or IFT74 KO RPE cells stably expressing either empty vector, FLAG-IFT74, or FLAG-IFT74 T438R. Representative images from three experimental replicates are shown. The cells were grown to confluence (without serum starvation), lysed and analyzed by Immunoblot. Molecular weights (kDa) estimated from a protein marker are indicated. Red asterisk: endogenous IFT74; black asterisks: FLAG tagged IFT74.(F) Quantification of the immunoblots of IFT74 shown in (E). Data are combined from three independent experiments. Error bars represent ± SD. Statistics obtained through one-way analysis of variance (ANOVA) with Tukey's multiple comparison test.(G-I) Box plots showing centrosomal signal intensity of FLAG (G), IFT81 (H), or RabL2 (I) in the cells described in (D) serum starved for 24 hours. The relative fluorescence signal intensity compared with the average of the control (IFT74 KO + FLAG-IFT74 WT for (G) and sgSafe + empty for (H) and (I)) are shown. Data are combined from three experimental replicates. Statistics obtained through nested one-way ANOVA with Šídák's multiple comparison test. (G) The number of mother centrioles analyzed in each sample per replicate are: 78, 77, 78 (sgSafe + empty); 102, 95, 101 (IFT74 KO + empty); 54, 77, 85 (IFT74 KO + FLAG-IFT74 WT); 49, 83, 99 (IFT74 KO + FLAG-IFT74 T438R), (H) The number of mother centrioles analyzed in each sample per replicate are: 93, 137, 138 (sgSafe + empty); 109, 133, 193 (IFT74 KO + empty); 62, 146, 131 (IFT74 KO + FLAG-IFT74 WT); 41, 143, 133 (IFT74 KO + FLAG-IFT74 T438R), (I) The number of mother centrioles analyzed in each sample per replicate are: 64, 101, 106 (sgSafe + empty); 84, 105, 129 (IFT74 KO + empty); 52, 96, 121 (IFT74 KO + FLAG-IFT74 WT); 73, 92, 102 (IFT74 KO + FLAG-IFT74 T438R).(K) Box plots showing the quantification of number of IFT88 particles per cilium in the experiment described in (J). Data are combined from three experimental replicates: The number of cilia analyzed in each sample per replicate are: 20, 20, 18 (sgSafe + empty); 20, 20, 18 (IFT74 KO + WT); 20, 20, 19 (IFT74 KO + FLAG-IFT74 WT). The number of IFT particles in individual cells in each experiment are available from Figures S7A, D, and G. Statistics obtained through nested one-way ANOVA with Tukey's multiple comparison test.


Figure 1 ( B ) Left , orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM ( in red ) and DNA in blue . Right , dot plot bar representing cell heights in cells from tumor sections , n = 60 cells from 1 tumor . ( C ) On the left , images of low magnification views of healthy tissues labelled with antibodies against pericentrin ( PCNT ) and CDK5RAP2 , shown in red and green , respectively . DNA is in blue . The white dashed squares represent the regions shown in higher magnifications on the right . One centrosome was considered as such when PCNT and CDK5RAP2 signals co - localized ( Inset 1 ) . Lack of co - localization ( Insets 2 and 3 ) was noticed and discarded during quantification . ( D ) Super - resolution microscopy of healthy tissues labelled for the centriole marker CEP135 ( in green ) and PCNT ( in red ) . ( E - F ) Representative images of two different fields of the same tumor ( E ) and of a tumor section to highlight different centrososome phenotypes , labelled as described in ( C ) . The pink dashed line highlights an area of cells without centrosomes . The white dashed squares represent the regions shown in higher magnifications in insets in ( G and I ) and the pink dashed linesurrounds nuclei without centrosomes . ( G ) Insets from ( F ) . ( H ) Examples of tumor sections showing small ( left ) and large ( right ) regions with nuclei without centrosomes . ( I ) Insets from ( F ) showing different types of centrosome amplification events . ( J ) Super - resolution microscopy of tumor tissues labelled as in ( C ) . ( K ) Maximum Z - projection of a tumor section labelled with EpCAM antibodies ( Red ) and DNA in blue . The dashed line shows the absence of EpCAM staining , arrows show a disorganized region .

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 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 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 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 O O O

Figure 1(B) Left, orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM (in red) and DNA in blue. Right, dot plot bar representing cell heights in cells from tumor sections, n=60 cells from 1 tumor.(C) On the left, images of low magnification views of healthy tissues labelled with antibodies against pericentrin (PCNT) and CDK5RAP2, shown in red and green, respectively. DNA is in blue. The white dashed squares represent the regions shown in higher magnifications on the right. One centrosome was considered as such when PCNT and CDK5RAP2 signals co-localized (Inset 1). Lack of co-localization (Insets 2 and 3) was noticed and discarded during quantification.(D) Super-resolution microscopy of healthy tissues labelled for the centriole marker CEP135 (in green) and PCNT (in red).(E-F) Representative images of two different fields of the same tumor (E) and of a tumor section to highlight different centrososome phenotypes, labelled as described in (C). The pink dashed line highlights an area of cells without centrosomes. The white dashed squares represent the regions shown in higher magnifications in insets in (G and I) and the pink dashed linesurrounds nuclei without centrosomes. (G) Insets from (F). (H) Examples of tumor sections showing small (left) and large (right) regions with nuclei without centrosomes.(I) Insets from (F) showing different types of centrosome amplification events.(J) Super-resolution microscopy of tumor tissues labelled as in (C).(K) Maximum Z-projection of a tumor section labelled with EpCAM antibodies (Red) and DNA in blue. The dashed line shows the absence of EpCAM staining, arrows show a disorganized region.