Effect of siRNA - mediated downregulation of HuR on expression of pro - inflammatory cytokines . In left panel of F , effect of siRNA treatment on cytokine mRNA in LPS - stimulated RAW 264 . 7 is shown . Relative measured against 18S rRNA are plotted . effect of HuR depletion on miR - 155 content has been measured relative values normalized against U6 snRNA are plotted ( right panel , F ) .

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Effect 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).


amount of let - 7a bound to Ago2 after LPS stimulation in presence absence of siHuR has been calculated relative values has been normalized to immunoprecipitated Ago2 ( G ) . Values are mean + / - s . e . m . , n =

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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=3


Effect of HuR expression on inflammatory responses in macrophage . Effect of ectopic expression of HuR on exosomal let - 7a content in naive macrophage ( 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 their effect on pro - inflammatory cytokine mRNA in RAW64 . 7 was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . , n =

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Effect 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=3


Effect of HuR expression on inflammatory responses in macrophage . effect of HuR expression on production of pro - inflammatory cytokine TNF - α in RAW 264 . 7 was measured . mRNA level of TNF - α was quantified . Values obtained with pCINeo expression without LPS treatment was taken as unit ( I ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct their effect on pro - inflammatory cytokine mRNA in RAW64 . 7 was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . , 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 . of p - p38 p - MSK1 have been monitored by western blotting done with extracts prepared from HA - HuR control plasmid transfected , untreated LPS treated RAW 264 . 7 . 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 were monitored against time in Ld - infected macrophage ( PEC ) from BALB / c mice ( K , left upper panel ) as well as in Ld - infected RAW 264 . 7 ( K , upper right panel ) . HuR were also monitored against time in L . major - infected RAW 264 . 7 ( K , lower left panel ) L . mexicana - infected RAW 264 . 7 ( 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 in control 6 hrs of Ld - infected RAW 264 . 7 . In parallel assays of pro - inflammatory cytokine TNF - α was measured in Ld - infected either with no treatment or pre - treated with MG132 ( L ) . Values are mean + / - s . e . m . , 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 . isotonic lysates prepared from 6hrs Ld infected were analysed on an OptiprepR density gradient HuR in fractions were determined . Input samples were monitored for HuR downregulation by western blot . Presence of Alix ( marker for early endosomes ) calnexin ( marker 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 of fractions was done 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).


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effect of HA-HuR expression on PP2A protein levels


effect of HA - HuR expression on PP2A were scored .

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


Expression profiles of HuR , PP2A miRNA let - 7a are connected to IL - 6 expression in RAW 264 . 7 . Data obtained from experiments described in previous figures are summed up to plot changes in IL - 6 mRNA against time of LPS treatment along with changes in PP2A , miRNA let - 7a 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 . mathematical equation fitting curve are shown here . Y represents of IL - 6 X represents miRNA let - 7a . A1 is initial IL - 6 A2 is changed level . LogX0 is mid - point of value 0 . 34 , a 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 altered level of PP2A ( black ) HuR ( red ) . For both cases , data were fitted to an equation , which is shown above . parameter m denotes expression level of PP2A / HuR which corresponds to maximum IL - 6 expression ; w denotes width of distributions for both cases . For PP2A HuR , values of m were found to be 0 . 6 0 . 9 respectively 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 . Effect of HuR expression on internalized number cytokine expression in RAW 264 . 7 . HA - HuR was transfected to RAW 264 . 7 infection was given for 24 hrs at a host to 1 : 10 . Ld was stained with CFSE dye internalization was detected by counting CFSE positive structures inside . HA - HuR was immunostained with anti - HA antibody 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 . Effect of HuR expression on internalized number HA - HuR was transfected to RAW 264 . 7 infection was given for 24 hrs at a host to 1 : 10 . Ld was stained with CFSE dye internalization was detected by counting CFSE positive structures inside . In zoomed part of merged picture are used to show internalized . Internalized present in HA - HuR positive were counted compared against untransfected 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 . Effect of HuR expression on cytokine expression in RAW 264 . 7 . HA - HuR was transfected to RAW 264 . 7 infection was given for 24 hrs at a host to 1 : 10 . of expression of different cytokines are measured in control or HA - HuR expressing RAW 264 . 7 infected with Ld ( C ) . Scale bar 20 µm . Values are mean + / - s . e . m . , 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 HuR expression in mouse liver . BALB / c mice of 6 of were infected with Ld via cardiac puncture . were sacrificed after subsequent days ; was extracted from liver tissue followed by western blot for HuR . β - Actin was used as loading control ( D , left panel ) . Each lane have designated . 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).


were injected with GP63 containing liposome ( gp1 - 3 ) or empty liposome ( C1 - 2 ) 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 experiment done with HA - HuR expression in liver is shown . Also HA - HuR level was detected in liver tissue by western blot 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 . load in liver tissues were estimated by measuring Ld in infected tissue using specific primer for Ld minicircle kDNA . Values are mean + / - s . e . m . , 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 . , n = 5 ) IL - 10 ( Values are mean + / - s . e . m . , n = 4 ) mRNA were checked from isolated from liver tissues 18S rRNA normalized Ct values were plotted ( H I , left panels ) . Relative fold change was calculated by ΔΔCt method values plotted for Ld , TNF - α IL - 10 mRNA ( G mean + / - s . e . m . , ( n = 5 ) , H mean + / - s . e . m . , ( n = 6 ) 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 . 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 were checked in spleen total ( 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 sensitive forms of Ld ( LdR LdS ) on cytokine level RAW 264 . 7 were infected with Antimony sensitive ( LdS - Ag83 ) antimony resistant ( LdR - BHU569 ) form of Ld for 24 hours along with uninfected kept as control . IL - 10 ( A ) TNF - α ( B ) were measured at mRNA level by qRT - PCR after infection ( mean + / - s . e . m . , 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 sensitive forms of Ld ( LdR LdS ) on on phospho - ERK level . ( D ) . Densitometry was done for p - ERK level by ImageJ relative values were plotted ( C ) . β - Actin was used as loading control Values are mean + / - s . e . m . , 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 sensitive forms of Ld ( LdR LdS ) on phospho - ERK MDR1 level . p - ERK , ERK 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 antimony resistant form of Ld on HuR PP2A . RAW 264 . 7 were infected with LdS LdR for 12 hrs HuR level was measured by western blot ( left panel ) . PP2A level was also measured by western blot after 4 12 hrs of LdS 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 PP2A inhibitor OA treatment on LdR infection of macrophage . RAW 264 . 7 were transfected with HA - HuR or pCIneo followed by 2 hrs of pretreatment with OA . These were then infected with LdR for 24 hrs . Pro - inflammatory cytokines TNF - α ( right panel ; mean + / - s . e . m . , n = 3 ) IL - 1β ( left panel ; mean + / - s . e . m . , n = 4 ) were measured at mRNA level 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 ) 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 . load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle of Ld ( I ) . Values are mean + / - s . e . m . , 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 ( GSEA ) of differentially expressed genes in colorectal cancer from 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 with or without dysfunction of iron homeostasis in GSE dataset ( n = 590 samples , P = 0 . 016 , Log - rank ( Mantel - Cox ) ) . " Normal " represents with unaltered expression of iron homoeostasis - related genes , including LTF , CP , FTH1 , SLC30A1 TFRC ; " Dysfunctional iron homoeostasis " represents 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 of of IREB2 in colorectal adenocarcinoma its matched adjacent normal tissues ( Left ) , relative level of IREB2 was assessed by ImageJ software ( Right ) ( n = 8 human samples , * P = 0 . 0114 , two - paired Student ' s t - ) .

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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 lysates of colorectal adenocarcinoma its matched adjacent normal tissue , was subsequently analyzed by anti - ubiquitin antibody for 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 of IREB2 - associated . Mock or IREB2 - FLAG was transfected into HEK293T FLAG - tagged were enriched by anti - FLAG M2 beads incubated with lysates from normal mice colon tissues . Five matched peptides corresponding to OTUD1 were shown on 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 of IREB2 . HEK293T were transfected with indicated plasmids subjected to immunoprecipitation with anti - FLAG antibody followed by western blot .

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

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

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

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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 of OTUD1 mRNA in colon tumors matched adjacent normal tissues ( n = 101 human samples , * * * P < 0 . 001 , two - paired Student ' s t - )

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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 OTUD1 in colorectal cancer ( n = 101 human samples , two - Pearson correlation , * * * * 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 of of OTUD1 TFRC in colorectal adenocarcinoma its matched adjacent normal tissues ( n = 4 human samples ) , 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 of OTUD1 in colorectal adenocarcinoma its matched adjacent normal tissue ( 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 ) of OTUD1 in colorectal adenocarcinoma 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 pictures of OTUD1 in normal tissue different colon diseases from tissue microarray CO809a ( 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 was measured in CT26 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 was measured in wild - type ( WT ) or OTUD1 ─ / ─ NCM460 ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - unpaired Student ' s t - )

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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 ) of IREB2 , TFRC 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 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 - unpaired Student ' s t - ) .

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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 - unpaired Student ' s t - ) in wild - type ( WT ) 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 - unpaired Student ' s t - ) in wild - type ( WT ) 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 ( MCHC ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0068 , two - unpaired Student ' s t - ) in wild - type ( WT ) 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.


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

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, 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 - unpaired Student ' s t - ) ( I ) in wild - type ( WT ) 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 ) volume ( mock , n = 7 biological replicates ; OTUD1 , n = 9 biological replicates ; OTUD1C320S , n = 9 biological replicates ; mean ± s . e . m . , * * * P < 0 . 001 , two - unpaired Student ' s t - ) ( B ) of mock , OTUD1 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 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.


volume ( n = 6 biological replicates , mean ± s . e . m . , * * P < 0 . 01 , * * * P = 0 . 0007 , two - unpaired Student ' s t - ) of mock 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 ( n = 5 biological replicates , * * P = 0 . 0021 , Log - rank ( Mantel - Cox ) ) of mock 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 TFRC in murine CT26 tumors were measured by anti - IREB2 antibody anti - TFRC antibody .

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

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


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

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

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

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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 of mock , OTUD1 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 - unpaired Student ' s t - ) .

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

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

O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P < 0 . 0001 , two - unpaired Student ' s t - ) isolated from mock OTUD1 overexpressing LLC tumors . MFI , mean .

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

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 ) OTUD1 ─ / ─ NCM460 . expression of TFRC IREB2 were highlighted with red color . indicates label - free ( LFQ ) of in mass spectrometry .

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

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

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

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

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 stably expressing mock , OTUD1 or OTUD1C320S were treated with Erastin ( 10 μM ) RSL3 ( 5 μM ) , viability was measured by PI ( n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P < 0 . 01 , two - unpaired Student ' s t - ) .

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

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

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

(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 in mock , OTUD1 OTUD1C320S overexpressing CT26 treated with Erastin ( 10 μM ) , AFC ( 50 μM ) or H2O2 ( 100 μM ) were detected by DCFDA . MFI , mean ( n = 2 biological replicates ) .

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

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 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 - unpaired Student ' s t - ) were detected by DCFDA . MFI , mean .

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

(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 ) 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

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

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

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


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

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

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

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

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

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

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 were treated with 5 μM RSL3 , medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - unpaired Student ' s t - ) .

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

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 interstitial fluid ( TIF ) of mock OTUD1 expressing CT26 tumors . Left , example of genes downregulated in OTUD1 overexpressing interstitial fluid ; right , example of genes upregulated in OTUD1 overexpressing interstitial fluid .

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

(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 of of HMGB1 , HSP70 HSP90 in interstitial fluid of mock , OTUD1 OTUD1C320S expressing CT26 tumors .

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

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

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

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

(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 ) .

O O O O O O B-DISEASE I-DISEASE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) pictures of colon tumors ( scale bars represent 1000 μm ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-DISEASE I-DISEASE O O O O O O O O

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


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

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

(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 ) Otud1 ─ / ─ mice treated with AOM / DSS ( n = 3 biological replicates ) . ES , enrichment score ; NES , normalized enrichment score .

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

(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 pictures of CD3 + T in colon tumors ( scale bars represent 400 μm ) .

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

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


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

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

(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 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 - unpaired Student ' s t - ) were detected by DCFDA . MFI , mean .

O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O

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

O O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O B-TISSUE O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 negative control miRNAs in hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected ( aCSF scramble ) at six months of . Sample , n = 9 per group .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O 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. 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 negative control miR - 124 in hippocampus of ant - 132 - scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm .

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

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 miR - 212 in hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to injected with a negative control oligonucleotide ( Ctr ) . Sample , n = 6 per group .

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

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

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

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 ) insoluble ( Formic acid ) Aβ40 Aβ42 in hippocampus of ant - 132 - ( C ) miR - 132 - injected ( D ) at six three months of , respectively . Sample , 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 of total TAU pTAU ( AT8 , AT270 ) upon miR - 132 knockdown ( A ) or overexpression ( B ) at six three months of , respectively . Sample , n = 9 per group ( A ) n = 6 per group ( B ) . Values were normalized to respective control groups presented as mean ± . Student ' s t - 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 indicates mean of scramble control set to 1 . Dashed indicates chosen threshold of 1 . 3 - fold . Sample , n = 9 per group . Values were normalized to scramble - injected group presented as mean ± .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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. 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 of wild - type ( wt ) mutant ( mut ) ITPKB 3 ' UTR in HEK293 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 Aβ42 in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . assays in A B were performed in three independent experiments , each in triplicates .

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

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

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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 Aβ42 in hippocampus of ITPKB siRNA - control - injected at three months of . Sample , n = 6 per group . Values were normalized to control group presented as mean ± .

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

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

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

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

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

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

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

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 of amyloid plaques ( 6E10 ) 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 ) ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD 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 . of miR - 132 , ITPKB hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated values of each signal per were normalized to mean integrated density of each signal across analyzed ( see Materials Methods ) . Sample ( AD ) , n = 3 . Values are presented as mean ± . Two - way Anova was used . Quantifications are summarized in table provided . Arrow directions refer to comparison of each normalized signal to same signal in other group ( in " low miR - 132 " group comparisons are made to " high miR - 132 " group vice versa ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 western blot of ITPKB , phosphorylated ( pERK1 / 2 ) total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full APP ( flAPP ) , APP CTFs sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample , n = 39 for AD n = 15 for ND . Values were normalized to ND presented as mean ± . Student ' s t - was used .

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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 of saponin ( n ≥ 4 ) .

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


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

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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 ) .

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 with 26 Gr mutants for 5 % saponin ( n ≥ 4 ) . 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 - dependent avoidance of saponin in control , Gr28bMi Gr28b Df / Gr28bMi ( n ≥ 4 ) .

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


C Amount of ingestion calculated at indicated time points ( 15 , 30 , 60 , 240 min ) using spectrophotogram for control 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 for control Gr28bMi flies in ( D ) legs or ( E ) proboscis . Flies were initially given 2 % sucrose , then 2 % sucrose in combination with 5 % saponin ( n = 4 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s as a post hoc 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 of sensilla following stimulation with 5 % saponin in control Gr28bMi flies ( we followed Tanimura ' s nomenclature ) ( n ≥ 8 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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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 .

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


C Tip recording of S6 sensilla from control 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 0 . 1 % SDS ( n ≥ 7 ) . error bars represent SEMs .

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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 gene ( hid ) , under Gr33a - GAL4 or Gr66a - GAL4 . heterozygote controls ( UAS - hid / + , Gr33a - GAL4 / + , Gr66a - GAL4 / + ) are shown ( n ≥ 4 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of indicated S - type sensilla genotypes ( n ≥ 7 ) . error bars represent SEMs . asterisks indicate significant differences from that of control detected by a single - factor ANOVA with Scheffe ' s to compare two sets of data ( * P < 0 . 05 , * * P < 0 . 01 ) .

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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 with indicated primers in A for control Gr28bMi flies with tubulin as a control . From left to right , band of tubulin of wild - type , tubulin of Gr28bMi , ladder , Gr28b of wild - type , 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 . asterisks indicate significant differences from that of Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s ( * * P < 0 . 01 ) . C Binary food choice for recovery experiments with each of five alternative Gr28b isoforms using GAL4 / UAS systems . ( n ≥ 4 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD 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 . asterisks indicate significant differences from that of Gr28b mutant detected by a single - factor ANOVA with Scheffe ' s ( * * P < 0 . 01 ) . D Saponin - induced action potential frequencies from indicated transgenic flies on S6 sensilla ( n = 7 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O B-TISSUE I-TISSUE 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 gene ( hid ) , under Gr28b . c - GAL4 ( n = 4 ) .

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

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

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD 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 ( B ) Gr28bMi adult raised on control indicated concentrations of saponin - containing cornmeal diet ( n = 4 ) .

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

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

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - sequencing samples from murine , porcine , bovine oocytes Each graph shows on y - axis reads per million of 19 - 32 nt reads for miRNAs on x - axis miRNAs ordered by name .

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(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.