Effect 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 =

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Effect 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=3


Effect 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 .

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Effect 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 ) .

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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 .

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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 ) .

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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).


effect of HA - HuR expression on PP2A protein

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effect of HA-HuR expression on PP2A protein levels


effect of HA - HuR expression on PP2A RNA levels were scored .

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effect 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 ) .

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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

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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 half


variations 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 ) .

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variations 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).


Effect 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 ) .

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Effect 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 ) .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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Effect of HA-HuR plasmid tail-vein injection on mice spleen. Simultaneously, TNF-α mRNA levels were checked in spleen total RNA (K).


Effect 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 ) .

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Effect 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 .

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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 ) .

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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 .

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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 .

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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 ) .

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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 ) .

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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).


( A ) Gene set enrichment analysis ( GSEA ) of differentially expressed genes in colorectal cancer from the GEO database ( GDS4382 ) . ES , enrichment score ; NES , normalized enrichment score . n = 3 biological samples .

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(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples.


( B ) Kaplan - Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset ( n = 590 samples , P = 0 . 016 , Log - rank ( Mantel - Cox ) test ) . " Normal " represents the patients with unaltered expression of iron homoeostasis - related genes , including LTF , CP , FTH1 , SLC30A1 and TFRC ; " Dysfunctional iron homoeostasis " represents the patients with altered expression of iron homoeostasis - related genes .

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(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes.


( C ) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues ( Left ) , the relative protein level of IREB2 was assessed by ImageJ software ( Right ) ( n = 8 human samples , * P = 0 . 0114 , two - tailed paired Student ' s t - test ) .

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(C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test).


( D ) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue , and was subsequently analyzed by anti - ubiquitin antibody for the assessment of ubiquitination .

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(D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.


( E ) Mass spectrum analysis of IREB2 - associated proteins . Mock or IREB2 - FLAG was transfected into HEK293T cells and FLAG - tagged proteins were enriched by anti - FLAG M2 beads and incubated with lysates from normal mice colon tissues . Five matched peptides corresponding to OTUD1 were shown on the right panel .

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(E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel.


( F ) In vivo ubiquitination assay of IREB2 . HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti - FLAG antibody followed by western blot analysis .

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(F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.


( G ) Half - life analysis of IREB2 in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . Cells were treated with 100 μg / ml cycloheximide ( CHX ) and collected at the indicated times for western blot analysis ( Up ) , the relative protein level of IREB2 was assessed by ImageJ software ( Down ) ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0049 , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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(G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test).


( H ) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC ( 50 μM ) and Hemin ( 100 μM ) treatment . MFI , mean fluorescence intensity . Control uses isotype - matched control antibody ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0010 , * * * P = 0 . 0008 , two - tailed unpaired Student ' s t - test ) .

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(H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test).


( I ) Flow cytometric analysis of TFRC expression in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells with DFO ( 100 μM ) treatment . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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(I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).


( A ) RT - qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues ( n = 101 human samples , * * * P < 0 . 001 , two - tailed paired Student ' s t - test )

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(A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test)


( B ) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer ( n = 101 human samples , two - tailed Pearson correlation analysis , * * * * P < 0 . 0001 ) .

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(B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001).


( C ) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues ( n = 4 human samples ) , the protein levels relative to GAPDH are marked .

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(C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked.


( D ) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue ( The scale bars represent 1000 μm ) .

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(D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm).


( E ) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05 - 118e .

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(E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e.


( F ) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a ( The scale bars represent 1000 μm ) .

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(F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).


Intracellular iron concentration was measured in CT26 cells stably expressing mock , OTUD1 or OTUD1C320S ( n = 2 biological replicates )

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Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates)


Intracellular iron concentration was measured in wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test )

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Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test)


( C ) The protein levels of IREB2 , TFRC and OTUD1 were assessed by western blot in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice .

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(C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice.


( D ) Intracellular iron concentration was measured in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice with supplementation of ferrous gluconate ( n = 3 biological replicates , mean ± s . e . m . , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) .

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(D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test).


Mean corpuscular volume ( MCV ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0472 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


mean corpuscular hemoglobin ( MCH ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0048 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


mean corpuscular hemoglobin concentration ( MCHC ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0068 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


, red boold cells ( RBCs ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0049 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

, red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


hemoglobin ( HB ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0036 , * * * P = 0 . 0002 , two - tailed unpaired Student ' s t - test ) ( I ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

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hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


( A - B ) Macroscopic evaluation ( A ) and tumor volume ( mock , n = 7 biological replicates ; OTUD1 , n = 9 biological replicates ; OTUD1C320S , n = 9 biological replicates ; mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( B ) of mock , OTUD1 and OTUD1C320S expressing CT26 tumors in BALB / c mice .

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(A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice.


Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


tumor volume ( n = 6 biological replicates , mean ± s . e . m . , * * P < 0 . 01 , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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tumor volume (n=6 biological replicates, mean ± s.e.m., **P < 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


survival analysis ( n = 5 biological replicates , * * P = 0 . 0021 , Log - rank ( Mantel - Cox ) test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice .

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survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.


( F ) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti - IREB2 antibody and anti - TFRC antibody .

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(F) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti-IREB2 antibody and anti-TFRC antibody.


( G ) Flow cytometric analysis of TFRC expression in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors . MFI , mean fluorescence intensity ( mean ± s . e . m . , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) .

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(G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P < 0.01, two-tailed unpaired Student's t-test).


( H ) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors . MFI , mean fluorescence intensity . ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0011 , two - tailed unpaired Student ' s t - test ) .

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(H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test).


( I ) Iron concentration of mock , OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry ( n = 3 biological replicates , mean ± s . e . m . , * P = 0 . 0237 , * * P = 0 . 0098 , two - tailed unpaired Student ' s t - test ) .

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(I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test).


( J ) Analysis of tumor infiltrating CD8 + T cells or CD4 + T cells in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors in BALB / c mice ( mean ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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(J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test).


Flow cytometric analysis of CD8 + T cells , CD4 + T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P = 0 . 0003 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors .

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Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors.


PD - 1 expression in T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors . MFI , mean fluorescence intensity .

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PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P < 0.0001, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity.


A ) Quantitative proteomics study of wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . The expression of TFRC and IREB2 were highlighted with red color . Intensity indicates label - free quantification ( LFQ ) intensity of proteins in mass spectrometry .

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A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry.


( B ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) .

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(B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test).


( C ) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer - 1 ( 10 μM ) , Z - VAD ( 25 μM ) and nec - 1 ( 20 μM ) . Cell viability was measured by PI staining n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0119 , * * P = 0 . 0015 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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(C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test).


( D ) CT26 cells stably expressing mock , OTUD1 or OTUD1C320S were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) , and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) .

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(D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test).


( E ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0082 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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(E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test).


F ) Intracellular ROS levels in mock , OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin ( 10 μM ) , AFC ( 50 μM ) or H2O2 ( 100 μM ) were detected by DCFDA staining . MFI , mean fluorescence intensity ( n = 2 biological replicates ) .

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F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates).


( G ) Intracellular ROS levels in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) or OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors ( mean ± s . e . m . , * P = 0 . 0189 , * * P = 0 . 0086 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity .

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(G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.


( I ) Tumor volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological replicates

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(I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates


( I ) Tumor volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological

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Flow cytometric analysis of DCFDA ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE . MFI , mean fluorescence intensity .

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Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity.


Flow cytometric analysis of the percentage of tumor infiltrating CD8 + T cells ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0433 , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE .

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Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE.


CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).


wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).


( C ) Volcano plots of genes differentially expressed in tumor interstitial fluid ( TIF ) of mock and OTUD1 expressing CT26 tumors . Left , example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid ; right , example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid .

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(C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid.


( D ) Immunoblot analysis of protein levels of HMGB1 , HSP70 and HSP90 in tumor interstitial fluid of mock , OTUD1 and OTUD1C320S expressing CT26 tumors .

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(D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors.


( E ) ATP level in tumor interstitial fluid ( TIF ) of mock or OTUD1 overexpressing CT26 cells ( n = 5 mice , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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(E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test).


( F and G ) BALB / c mice were immunized with 1 × 106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2 × 106 wild - type CT26 cells in the right flank 10 days later . Macroscopic evaluation ( F ) and tumor volume ( n = 7 mice , mean ± s . e . m . , * * P = 0 . 0053 , two - tailed unpaired Student ' s t - test ) ( G ) of tumors in the right flank were shown .

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(F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown.


( A ) Representative pictures of colon tumors in AOM / DSS - induced Colitis - Associated Cancer ( CAC ) model ( n = 6 biological replicates ) .

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(A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates).


( B and C ) The tumor load ( B ) or tumor number ( C ) in the whole colon was measured ( n = 12 biological replicates , mean ± s . e . m . , * P = 0 . 0431 , * * P = 0 . 003 , two - tailed unpaired Student ' s t - test ) .

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(B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test).


( D ) Representative H & E ( Hematoxylin - eosin ) staining pictures of colon tumors ( The scale bars represent 1000 μm ) .

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(D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm).


( E ) Flow cytometric analysis of TFRC expression in intestinal epithelial cells ( IECs ) from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0062 , two - tailed unpaired Student ' s t - test ) .

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(E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test).


( F ) GSEA of genes expressed in colon tissues from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 3 biological replicates ) . ES , enrichment score ; NES , normalized enrichment score .

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(F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score.


( G ) Representative immunohistochemistry staining pictures of CD3 + T cells in colon tumors ( The scale bars represent 400 μm ) .

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(G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ).


( H ) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells ( LPMC ) isolated from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0025 , * * * P = 0 . 0005 , two - tailed unpaired Student ' s t - test ) .

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(H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test).


( I ) Intracellular ROS levels in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0072 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity .

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(I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.


( J ) ATP level in tumor interstitial fluid ( TIF ) of mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0292 , two - tailed unpaired Student ' s t - test ) .

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(J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test).


B . Semi - quantitative PCR of miR - 132 and negative control miRNAs in the hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected animals ( aCSF and scramble ) at six months of age . Sample size , n = 9 per group .

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B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group.


C . FISH of miR - 132 and negative control miR - 124 in the hippocampus of ant - 132 - and scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm .

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C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm.


D . Semi - quantitative PCR of miR - 132 and miR - 212 in the hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to animals injected with a negative control oligonucleotide ( Ctr ) . Sample size , n = 6 per group .

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D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group.


A . Amyloid staining ( 6E10 ) combined with miR - 132FISH in ant - 132 - and scramble - injected mice at six months of age . Scale bar , 500 μm . B . Quantification of amyloid burden in hippocampus and cortex of ant - 132 - injected and control animals ( see Materials and Methods ) . Sample size , n = 4 per group .

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A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group.


C , D . ELISA of soluble ( TBS ) and insoluble ( Formic acid ) Aβ40 and Aβ42 levels in the hippocampus of ant - 132 - ( C ) and miR - 132 - injected ( D ) animals at six and three months of age , respectively . Sample size , n = 6 per group .

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C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.


Western blot analysis of total TAU and pTAU ( AT8 , AT270 ) levels upon miR - 132 knockdown ( A ) or overexpression ( B ) at six and three months of age , respectively . Sample size , n = 9 per group ( A ) and n = 6 per group ( B ) . Values were normalized to the respective control groups and presented as mean ± SEM . Student ' s t - test was used .

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Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.


B . Positive hits were validated by semi - quantitative qPCR in ant - 132 - injected mousehippocampus . Black line indicates mean of scramble control set to 1 . Dashed line indicates chosen threshold of 1 . 3 - fold . Sample size , n = 9 per group . Values were normalized to scramble - injected group and presented as mean ± SEM .

B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.


A . Luciferasereporter assay of wild - type ( wt ) and mutant ( mut ) ITPKB 3 ' UTR in HEK293 cells co - transfected with a synthetic miR - 132 ( miR - 132 ) or a negative control ( Neg Ctr ) oligonucleotide .

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A. Luciferasereporter assay of wild-type (wt) and mutant (mut) ITPKB 3'UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide.


B . ELISA of Aβ40 and Aβ42 levels in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . The assays in A and B were performed in three independent experiments , each in triplicates .

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B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswecells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates.


C . Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB . Sample size , n = 6 per group . Values were normalized to control - injected group ( Ctr ) and presented as mean ± SEM .

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C. Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM.


D . ELISA of insoluble ( Formic acid - soluble ) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA - and control - injected animals at three months of age . Sample size , n = 6 per group . Values were normalized to control group and presented as mean ± SEM .

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D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.


E , F . Western blot analysis of ITPKB levels upon miR - 132 down - ( E ) or up - regulation ( F ) at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to control groups and presented as mean ± SEM .

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E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM.


Western blot analysis of CTFβ ( A ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of CTFβ (A) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot analysis of sAPPβ - swe ( B ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of sAPPβ-swe (B) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot analysis of phosphorylated ERK1 / 2 ( pERK1 / 2 ) ( C ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


A . Double immunofluorescence staining of amyloid plaques ( 6E10 ) and ITPKB in AD prefrontal cortex . Scale bar , 50 μm . Arrowheads indicate ITPKB immunopositivity .

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A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity.


B . miR - 132FISH coupled with double immunofluorescence against hyperphosphorylated TAU ( AT8 ) - containing neurofibrillary tangles ( NFTs ) and ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

B. miR-132FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm.


C . Quantification of miR - 132 , ITPKB and hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed ( see Materials and Methods ) . Sample size ( AD patients ) , n = 3 . Values are presented as mean ± SEM . Two - way Anova was used . Quantifications are summarized in the table provided . Arrow directions refer to the comparison of each normalized signal to the same signal in the other group ( in the " low miR - 132 " group comparisons are made to the " high miR - 132 " group and vice versa ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the "low miR-132" group comparisons are made to the "high miR-132" group and vice versa).


Semi - quantitative PCR of miR - 132 and western blot analysis of ITPKB , phosphorylated ( pERK1 / 2 ) and total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) and total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full length APP ( flAPP ) , APP CTFs and sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample size , n = 39 for AD and n = 15 for ND . Values were normalized to ND and presented as mean ± SEM . The Student ' s t - test was used .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student's t-test was used.


A Dose - response binary food choice assay of saponin ( n ≥ 4 ) .

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A Dose-response binary food choice assay of saponin (n ≥ 4).


B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla ( n ≥ 8 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8).


C Sample traces of saponin - induced action potentials in ( B ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O

C Sample traces of saponin-induced action potentials in (B).


D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla ( n ≥ 8 ) . E Sample traces from ( D ) . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * * P < 0 . 01 ) . The error bars represent SEMs .

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D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.


A Binary food choice assay with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . The error bars represent SEMs .

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A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4). The error bars represent SEMs.


B Concentration - dependent avoidance of saponin in control , Gr28bMi and Gr28b Df / Gr28bMi ( n ≥ 4 ) .

O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O

B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).


C Amount of ingestion calculated at the indicated time points ( 15 , 30 , 60 , and 240 min ) using spectrophotogram analysis for control and 5 % saponin - containing food ( n = 8 ) .

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C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).


D , E PER assay for control and Gr28bMi flies in the ( D ) legs or ( E ) proboscis . Flies were initially given 2 % sucrose , then 2 % sucrose in combination with 5 % saponin ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis as a post hoc test to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01).


A Mapping analysis of all the sensilla following stimulation with 5 % saponin in control and Gr28bMi flies ( we followed Tanimura ' s nomenclature ) ( n ≥ 8 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


B Sample traces of saponin - induced action potentials on S6 sensilla .

B Sample traces of saponin-induced action potentials on S6 sensilla.


C Tip recording analysis of S6 sensilla from control and Gr28bMi flies . Stimuli used are 0 . 2 % DEET , 1 . 0 mM coumarin , 0 . 5 mM quinine , 10 mM umbelliferone , 10 mM caffeine , 0 . 3 mM strychnine , 0 . 3 mM lobeline , 0 . 1 mM chloroquine , 0 . 2 mM denatonium , 0 . 5 mM papaverine , 0 . 1 mM berberine and 0 . 1 % SDS ( n ≥ 7 ) . The error bars represent SEMs .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7). The error bars represent SEMs.


D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr33a - GAL4 or Gr66a - GAL4 . All heterozygote controls ( UAS - hid / + , Gr33a - GAL4 / + , and Gr66a - GAL4 / + ) are shown ( n ≥ 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr33a-GAL4 or Gr66a-GAL4. All heterozygote controls (UAS-hid/+, Gr33a-GAL4/+, and Gr66a-GAL4/+) are shown (n ≥ 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


E Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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

E Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).


B RT - PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control . From left to right , the band of tubulin of wild - type , tubulin of Gr28bMi , DNA ladder , Gr28b of wild - type , and Gr28b of Gr28bMi .

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B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi.


Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4 / UAS systems . ( n ≥ 4 ) .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O

Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4).


D Each isoform from Gr28b . a to Gr28b . e was driven by Gr28b . a - GAL4 or Gr28b . c - GAL4 in a Gr28bMi background . The asterisks indicate significant differences from that of the Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s analysis ( * * P < 0 . 01 ) . D Saponin - induced action potential frequencies from the indicated transgenic flies on S6 sensilla ( n = 7 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).


A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4).


B Tip recording analysis of the indicated S - type sensilla and genotypes ( n ≥ 7 ) . The error bars represent SEMs . The asterisks indicate significant differences from that of the control detected by a single - factor ANOVA with Scheffe ' s analysis to compare two sets of data ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01).


C Expression of the Gr28b . c - GAL4 reporter in subsets of Gr66a - positive GRNs . First panel shows that all bitter responsive GRNs are labeled by Gr66a - I - GFP ( anti - GFP , green ) . Second panel shows the expression of the Gr28b . c - GAL4 , crossed with UAS - DsRed ( anti - DsRed , red ) . Third panel shows merged image of first and second panels . Forth panel shows a schematic diagram of sensilla arrangement . Red - marked sensilla indicate the expression of Gr28b . c - GAL4 reporter in second panel . Scale bars represent 40 μm .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.


A , B Survival rate of ( A ) control and ( B ) Gr28bMi adult raised on control and the indicated concentrations of saponin - containing cornmeal diet ( n = 4 ) .

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A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4).


C , D Survival rate based on binary food choice . control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM ( C ) or 200 mM fructose ( D ) mixed with 5 % or 7 . 5 % saponin .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O

C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin.


E , F Cumulative pupation rate of ( E ) control and ( F ) Gr28bMi larvae raised on the indicated saponin - containing food as well as control food ( n = 4 ) . The error bars represent SEMs . The asterisks indicate significant differences from normal diet fed which were detected by a single - factor ANOVA with Scheffe ' s analysis ( * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).


( A ) miRNA abundance in RNA - sequencing samples from murine , porcine , and bovine oocytes Each graph shows on the y - axis reads per million of 19 - 32 nt reads for miRNAs and on the x - axis miRNAs ordered by name .

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

(A) miRNA abundance in RNA-sequencing samples from murine, porcine, and bovine oocytes Each graph shows on the y-axis reads per million of 19-32 nt reads for miRNAs and on the x-axis miRNAs ordered by name.


( C ) qPCR quantification of ssc - miR - 205 and bta - miR - 10b miRNAs in murine ( M . m . ) , porcine ( S . s . ) , and bovine ( B . t . ) oocytes , respectively . Shown are a mean values from three independent experiments . Error bars = SD . Detailed information concerning miRNA quantification is provided in the Methods section .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) qPCR quantification of ssc-miR-205 and bta-miR-10b miRNAs in murine (M.m.), porcine (S.s.), and bovine (B.t.) oocytes, respectively. Shown are a mean values from three independent experiments. Error bars = SD. Detailed information concerning miRNA quantification is provided in the Methods section.


( A ) bta - miR - 10b and ssc - miR - 205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary ( 1x perfect ) miRNA binding site or four partially complementary ( bulged ) miRNA binding sites . Error bars = SD . Each data point represents a value obtained from five microinjected oocytes . Data were collected in two microinjection sessions , each microinjection session used a new batch of in vitro transcribed reporter mRNAs . Data information : asterisks indicate statistical significance ( p - value ) of one - tailed t - test , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) bta-miR-10b and ssc-miR-205 miRNAs efficiently repress microinjected NanoLuc luciferase reporters carrying a perfectly complementary (1x perfect) miRNA binding site or four partially complementary (bulged) miRNA binding sites. Error bars = SD. Each data point represents a value obtained from five microinjected oocytes. Data were collected in two microinjection sessions, each microinjection session used a new batch of in vitro transcribed reporter mRNAs. Data information: asterisks indicate statistical significance (p-value) of one-tailed t-test, (*p < 0.05, **p < 0.01, ***p < 0.001).