Figure 2A ) Inspection of specific amphid - neuron - defective mutants revealed that AWA neurons are most likely responsible for pheromone perception . Here , an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant . The color code of edges represents the average C . I . of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities . The region colored green shows the range of wild - type sex pheromone response in the male . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown . B ) AWA - laser - ablated males were not attracted by the sex pheromone in single - worm chemoattraction assays . Both AWA - defective mutant males ( lin - 11 and odr - 7 ) and AWA - laser - ablated males failed to reach the sex pheromone within 30 min . Wild - type C . elegans males responded normally to the sex pheromone and wild - type C . elegans hermaphrodites were not attracted by same - sex pheromone . The single - worm chemoattraction assay was performed as described in Materials and Methods . Sample size of males in AWA - laser - ablation experiment : 12 ; all other experiments : 100 worms .

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Figure 2A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.


Figure 3B ) Mutant males of 4 GPCR signaling cascade components failed to respond to C . remanei sex pheromone , suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown . C ) In males from both AWA - specific - rescue odr - 3 mutant strain and odr - 3 rescued mutant strain , the sex pheromone response was significantly restored in single male worm arrival assays . However , males from the CEM - specific - rescue odr - 3 mutant strain failed to reach the sex pheromone within 30 min . In odr - 3 and him - 5 strain experiments , the sample sizes were 12 and 20 worms , respectively ; in all other experiments , 40 worms were used .

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Figure 3B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.


Figure 4A ) srd - 1 mutant males were not attracted to the sex pheromone but were attracted to the positive - control chemoattractants , 1 : 1000 diacetyl and 10 mg / mL pyrazine . This demonstrated that SRD - 1 is required and specific for sex pheromone perception . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown . B ) Summary of srd - 1 expression pattern . The transcriptional reporter Psrd - 1 : : gfp was detected in both male and hermaphrodite ASI neurons in the head region , and only in male AWA and ADF neurons . C ) SRD - 1 : : GFP fusion construct revealed predominant SRD - 1 localization at the cilia of AWAs in C . elegans males . Scale bar = 5 μm . D ) Examination of srd - 1 transcriptional reporter revealed that srd - 1 is expressed in AWA neurons in males but not hermaphrodites . A transcriptional reporter for odr - 7 indicated the locations of AWAs ( arrowheads ) . Scale bar = 50 μm . F ) AWA - specific rescue of srd - 1 restored sex pheromone perception ability in both males and hermaphrodites . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown . G ) Ectopic expression of srd - 1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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

Figure 4A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons.C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm.D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm.F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


Figure 5A ) The GCaMP5 reporter was used to visualize activation of the SRD - 1 receptor by the sex pheromone in AWA neurons . To facilitate real - time imaging of the process , the heads of the transgenic worms were immobilized on soft - agar pads permeable to sex pheromone . B ) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification . Arrowheads : AWA neurons . Scale bar = 50 μm . C ) Relative fluorescence - intensity change was quantified through calcium imaging . Wild - type males showed receptor excitation by sex pheromones from C . elegans hermaphrodites or C . remanei females . This response was lost in srd - 1 - mutant - strain males but was restored following AWA - specific rescue of srd - 1 . Red triangles : time points of sex pheromone addition . Three males were assayed from each strain .

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Figure 5A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm.C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.


Figure 6B ) Wild - type C . elegans males are less attracted to sex pheromone than wild - type C . remanei males . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown . C ) Expression level of srd - 1 cDNA in srd - 1 transgenic lines . srd - 1 mutant males were rescued using , separately , wild - type C . elegans and wild - type C . remanei srd - 1 cDNAs . Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays . Four independent transgenic lines were selected , and the expression - level data were combined into a single bar . The expression levels were measured using qPCR . Data information : For transgenic - line studies , each bar represents a total of 1200 males from 4 independent transgenic lines ; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines , and the tests were conducted over 2 - 4 separate days . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown . D ) srd - 1 mutant males rescued with Ce - srd - 1 cDNA showed lower responsiveness than those rescued with Cre - srd - 1 cDNA , which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter . The sex pheromone perception defect of srd - 1 mutants was not rescued in males expressing CT - truncated SRD - 1 proteins . The findings show that the CT region is essential for sex pheromone perception . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown . E ) srd - 1 mutant males rescued by using Ce - srd - 1 cDNA together with Cre - srd - 1 CT cDNA were more responsive to sex pheromone than those rescued using Cre - srd - 1 cDNA with Ce - srd - 1 CT cDNA . The results of the CT - swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD - 1 protein . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown .

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

Figure 6B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  C) Expression level of srd-1 cDNA in srd-1 transgenic lines. srd-1 mutant males were rescued using, separately, wild-type C. elegans and wild-type C. remanei srd-1 cDNAs. Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays. Four independent transgenic lines were selected, and the expression-level data were combined into a single bar. The expression levels were measured using qPCR. Data information: For transgenic-line studies, each bar represents a total of 1200 males from 4 independent transgenic lines; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines, and the tests were conducted over 2-4 separate days. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


Figure 1A - C ARR1 / 10 / 12 protein levels were examined by western blot when five - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , and 35S : ARR12 : YFP were treated with or without 200 mM NaCl for the indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - F Quantification analysis of the protein levels of ARR1 / 10 / 12 in ( A - C ) .

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Figure 1A-C ARR1/10/12 protein levels were examined by western blot when five-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP were treated with or without 200 mM NaCl for the indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-F Quantification analysis of the protein levels of ARR1/10/12 in (A-C).


Figure 2C In vivo Coimmunoprecipitation assay of MPK3 / 4 / 6 and ARR1 / 10 / 12 . MPK3 / 4 / 6 - YFP and ARR1 / 10 / 12 - MYC were co - expressed in Arabidopsis protoplast cells . Protein extracts ( Input ) were immunoprecipitated with GFP Trap magnetic agarose beads . Immunoblots were performed with anti - MYC antibody to detect ARR1 / 10 / 12 and with anti - GFP antibody to detect MPK3 / 4 / 6 .

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Figure 2C In vivo Coimmunoprecipitation assay of MPK3/4/6 and ARR1/10/12. MPK3/4/6-YFP and ARR1/10/12-MYC were co-expressed in Arabidopsis protoplast cells. Protein extracts (Input) were immunoprecipitated with GFP Trap magnetic agarose beads. Immunoblots were performed with anti-MYC antibody to detect ARR1/10/12 and with anti-GFP antibody to detect MPK3/4/6.


Figure 3A - C Thiophosphorylation assays of wild type and mutated forms ARR1 / 10 / 12 by MPK3 / 6 in vitro . Potential phosphorylated Ser residues or Thr residues of ARR1 , ARR10 , and ARR12 were mutated to Ala ( ARR1m / ARR10m / ARR12m ) . Recombinant ARR1 / 10 / 12 - His and ARR1m / 10m / 12m - His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD . Phosphorylated ARR1 / 10 / 12 , ARR1m / 10m / 12m , and MPK3 / 6 were visualized by an anti - thiophosphate ester - specific antibody ( α - TPE ) ( upper panel ) . Asterisk in ( B ) indicates non - specific bands . Recombinant MKK5DD , MPK3 , MPK6 , ARR1 / 10 / 12 , and ARR1m / 10m / 12m were detected by comassie brilliant blue ( CBB ) staining as loading controls ( bottom panel ) .

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Figure 3A-C Thiophosphorylation assays of wild type and mutated forms ARR1/10/12 by MPK3/6 in vitro. Potential phosphorylated Ser residues or Thr residues of ARR1, ARR10, and ARR12 were mutated to Ala (ARR1m/ARR10m/ARR12m). Recombinant ARR1/10/12-His and ARR1m/10m/12m-His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD. Phosphorylated ARR1/10/12, ARR1m/10m/12m, and MPK3/6 were visualized by an anti-thiophosphate ester-specific antibody (α-TPE) (upper panel). Asterisk in (B) indicates non-specific bands. Recombinant MKK5DD, MPK3, MPK6, ARR1/10/12, and ARR1m/10m/12m were detected by comassie brilliant blue (CBB) staining as loading controls (bottom panel).


Figure 4A - C Immunoblot analysis with anti - MYC antibody or anti - GFP antibody showing the upshift of ARR1 , ARR10 , and ARR12 in a phos - tag gel . The phosphorylated ARR1 , ARR10 , and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase ( CIAP ) . Seven - day - old seedlings of 35S : ARR1 : MYC ( A ) , 35S : ARR10 : YFP ( B ) , and 35S : ARR12 : YFP ( C ) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Total proteins were extracted and subjected to immunoblot analysis . ARR1 , ARR10 , and ARR12 proteins separated on SDS - PAGE gel without phos - tag were detected as loading controls .

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Figure 4A-C Immunoblot analysis with anti-MYC antibody or anti-GFP antibody showing the upshift of ARR1, ARR10, and ARR12 in a phos-tag gel. The phosphorylated ARR1, ARR10, and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase (CIAP). Seven-day-old seedlings of 35S:ARR1:MYC (A), 35S:ARR10:YFP (B), and 35S:ARR12:YFP (C) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Total proteins were extracted and subjected to immunoblot analysis. ARR1, ARR10, and ARR12 proteins separated on SDS-PAGE gel without phos-tag were detected as loading controls.


Figure 5A - C Immunoblot analysis the effects of MPK3 / 6 on salt stress - induced ARR1 / 10 / 12 protein degradation . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , and 35S : ARR12 : YFP in Col - 0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I Quantification analysis of MPK3 / 6 effect on salt stress - induced ARR1 / 10 / 12 protein degradation in ( A - C ) .

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Figure 5A-C Immunoblot analysis the effects of MPK3/6 on salt stress-induced ARR1/10/12 protein degradation. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP in Col-0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of MPK3/6 effect on salt stress-induced ARR1/10/12 protein degradation in (A-C).


Figure 6A - C Immunoblot analysis stability of ARR1 / 10 / 12 and ARR1T553A / 10S383A / 12S323A proteins under salt stress . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP , 35S : ARR1T553A : MYC , 35S : ARR10S383A : YFP , and 35S : ARR12S323A : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as the internal reference . The relative intensity of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I Quantification analysis of stability of ARR1 / 10 / 12 and ARR1T553A / 10S383A / 12S323A proteins under salt stress in ( A - C ) .

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Figure 6A-C Immunoblot analysis stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, 35S:ARR12:YFP, 35S:ARR1T553A:MYC, 35S:ARR10S383A:YFP, and 35S:ARR12S323A:YFP were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress in (A-C).


Figure 7Four - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR1T553A : MYC , and 35S : ARR1T553D : MYC were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes ( A ) were determined .

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Figure 7Four-day-old seedlings of 35S:ARR1:MYC, 35S:ARR1T553A:MYC, and 35S:ARR1T553D:MYC were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Figure 8Four - day - old seedlings of 35S : ARR10 : YFP , 35S : ARR10S383A : YFP , and 35S : ARR10S383D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes ( A ) were determined .

Figure 8Four-day-old seedlings of 35S:ARR10:YFP, 35S:ARR10S383A:YFP, and 35S:ARR10S383D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Figure 9Four - day - old seedlings of 35S : ARR12 : YFP , 35S : ARR12S323A : YFP , and 35S : ARR12S323D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , plant phenotypes were determined .

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Figure 9Four-day-old seedlings of 35S:ARR12:YFP, 35S:ARR12S323A:YFP, and 35S:ARR12S323D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes were determined.


Figure 10Three - day - old seedlings of Col - 0 , MPK3SR , MPK3SRarr1 / 12 , and arr1 / 12 were transferred to 0 . 5 μM NA - PP1 - containing 1 / 2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1 / 12 and then the plants were transferred to 0 . 2 μM NA - PP1 - containing 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . Pictures were taken and the elongated root length was determined 3 d

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Figure 10Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later


Figure 1 ( B ) Electrophoretic mobility shift assay ( EMSA ) with increasing concentration ( 0 , 15nM , 30nM , 60nM , 1 . 25μM and 2 . 5μM ) of GST - FOXD3 and fixed concentration ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) from Sox15 promoter ( positive control ) , MSR , MERVL - LTR , MERVL - int , L1MdA and Hprt promoter ( negative control ) . The experiment was repeated 3 times . ( D ) ChIP - qPCR enrichment of FOXD3 in mESCs using primers specific for MSR , MERVL - LTR , MERVL - int ( including and excluding the FOXD3 cognate motif ) , L1MdA promoter sequences . Sox15 promoter and Actb promoter primers are used as positive and negative control respectively . Data is represented as percentage of input and the average of 3 biological replicates is plotted . Error bar indicates standard error of the mean ( SEM ) . Asterisks indicate statistically significant differences compared to no antibody control levels ( * : p < 0 . 05 , paired t - test ) .

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Figure 1(B) Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). The experiment was repeated 3 times. (D) ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MSR, MERVL-LTR, MERVL-int (including and excluding the FOXD3 cognate motif), L1MdA promoter sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).


Figure 2 ( B ) Validation of select upregulated repeats in Foxd3 KO cells by RT - qPCR . The data is plotted as average fold change relative to control , after normalization to Gapdh . Error bars indicate SEM ( n = 3 biological replicates ) . Asterisks indicate statistically significant differences when compared to control ( * * : p < 0 . 005 , * : p < 0 . 05 , paired t - test ) . ( C ) Immunofluorescence analysis of the MERVL - gag protein in control and Foxd3 KO cells . Nuclei are labelled with DAPI . The percentage of counted cells ( ~ 150 cells per experiment ) exhibiting immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates . ( E ) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells . Nuclei are labelled with DAPI . The percentage of counted cells ( ~ 150 cells per experiment ) exhibiting the immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates .

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Figure 2(B) Validation of select upregulated repeats in Foxd3 KO cells by RT-qPCR. The data is plotted as average fold change relative to control, after normalization to Gapdh. Error bars indicate SEM (n=3 biological replicates). Asterisks indicate statistically significant differences when compared to control (**: p<0.005, *: p<0.05, paired t-test).(C) Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10 μm, n= 3 biological replicates.(E) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting the immunofluorescence signal is indicated. Scale bar represents 10 μm, n=3 biological replicates.


Figure 3 ( A ) Electrophoretic mobility shift assay ( EMSA ) with increasing concentrations ( 0 , 30nM , 60nM , 1 . 25μM , 2 . 5μM and 5μM ) of recombinant GST - FOXD3 , DNA binding mutants GST - FOXD3 M1 and GST - FOXD3 M2 and fixed concentration ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) representing MERVL and MSR . The experiment was repeated 3 times . ( B ) RT - qPCR analysis of MERVL , MSR and L1MdA in Foxd3 cKO cells , Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4 - OHT . Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 3 biological replicates is plotted , error bars represent standard error mean ( SEM ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - test ) . ( D ) RT - qPCR analysis of , 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells , Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4 - OHT . Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 3 biological replicates is plotted , error bars represent standard error mean ( SEM ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - test ) .

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Figure 3(A) Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST- FOXD3 M2 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. The experiment was repeated 3 times.(B) RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).(D) RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).


Figure 4 ( A ) GFP immunoprecipitation analysis using GFP - SUV39H1 and GFP - SUV39H2 expressing Suv39h dn mESCs . The top panel represents immunoblot using GFP antibody and the bottom panel represents immunoblot using FOXD3 antibody . The experiment was repeated 3 times using biological replicates . ( D ) ChIP - qPCR analysis of endogenous SUV39H1 enrichment over MERVL , MSR and L1MdA in control and Foxd3 KO cells . Actb promoter is used as negative control . Data is represented as average percentage input from 3 biological replicates . Error bar indicates SEM ( * : p < 0 . 05 paired t - test ) . ( E ) ChIP - qPCR analysis revealing H3K9me3 enrichment over MERVL , MSR and L1MdA in control and Foxd3 KO cells . Actb promoter is used as negative control . Data is represented as average percentage input from 3 biological replicates . Error bar indicates SEM ( * * : p < . 005 , * : p < 0 . 05 paired t - test ) . ( G ) Immunofluorescence analysis of control and Foxd3i GFP - SUV39H1 expressing Suv39h dn mESCs stained with DAPI , FOXD3 and H3K9me3 . The percentage of counted cells ( ~ 150 cells per experiment ) exhibiting fluorescence signal is indicated . Scale bar = 2μm . n = 3 biological replicates .

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Figure 4(A) GFP immunoprecipitation analysis using GFP-SUV39H1 and GFP-SUV39H2 expressing Suv39h dn mESCs. The top panel represents immunoblot using GFP antibody and the bottom panel represents immunoblot using FOXD3 antibody. The experiment was repeated 3 times using biological replicates.(D) ChIP-qPCR analysis of endogenous SUV39H1 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (*: p<0.05 paired t-test).(E) ChIP-qPCR analysis revealing H3K9me3 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (**: p<.005, *: p<0.05 paired t-test).(G) Immunofluorescence analysis of control and Foxd3i GFP-SUV39H1 expressing Suv39h dn mESCs stained with DAPI, FOXD3 and H3K9me3. The percentage of counted cells (~150 cells per experiment) exhibiting fluorescence signal is indicated. Scale bar=2μm. n= 3 biological replicates.


Figure 1 ( B ) COUP - TFII expression ( green ) is increased in human kidneys from patients with thrombotic microangiopathy ( TMA ) and diabetic nephropathy ( DN ) ( n = 2 ) . Quantification by confocal micrographs in 400x hpf . * * * * p < 0 . 0001 by one - way ANOVA ; mean ± SD . The majority of these cells were localized in the interstitial region . More than 50 % COUP - TFII ( + ) cells ( green ) are co - localized with expanded αSMA - positive ( red ) areas of fibrosis . Pearson correlation coefficient ( PCC ) = 0 . 56 ± 0 . 1 for patient # 1 and 0 . 65 ± 0 . 13 for patient # 2 . ( C ) COUP - TFII expression ( green ) is increased in lungs from patients with IPF ( n = 2 ) by immunofluorescence . Quantification by confocal micrographs in 400x hpf . * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD . ( E ) COUP - TFII expression increased significantly in organoids treated with TGFβ1 ( 10ng / ml ) , cobalt chloride ( 100 μM ) , IL - 1β ( 10ng / ml ) , but not PDGF ( 50ng / ml ) . Quantification by confocal micrographs in 400x hpf . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD . The higher magnification of the insert from IL - 1β φησwed co - localization of COUP - TFII ( green ) and αSMA ( red ) staining .

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Figure 1(B) COUP-TFII expression (green) is increased in human kidneys from patients with thrombotic microangiopathy (TMA) and diabetic nephropathy (DN) (n=2). Quantification by confocal micrographs in 400x hpf. ****p<0.0001 by one-way ANOVA; mean ± SD. The majority of these cells were localized in the interstitial region. More than 50% COUP-TFII (+) cells (green) are co-localized with expanded αSMA-positive (red) areas of fibrosis. Pearson correlation coefficient (PCC) = 0.56 ± 0.1 for patient#1 and 0.65 ± 0.13 for patient#2.(C) COUP-TFII expression (green) is increased in lungs from patients with IPF (n=2) by immunofluorescence. Quantification by confocal micrographs in 400x hpf. **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.(E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml), cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.


Figure 2 ( C ) Majority COUP - TFII + cells ( green ) were also positive for Foxd1 ( tdTomato - labeled as red ) in the non - injured mice kidney ( n = 2 ) . Quantification by confocal micrographs in 400x hpf .

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Figure 2(C) Majority COUP-TFII+ cells (green) were also positive for Foxd1 (tdTomato-labeled as red) in the non-injured mice kidney (n=2). Quantification by confocal micrographs in 400x hpf.


Figure 3 ( A ) COUP - TFII expression is significantly increased and co - localized with αSMA + cells at day 10 of UUO and day 14 of UIRI in mice ( n = 3 animals per group ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 * * p < 0 . 01 by paired t test ; mean ± SD . ( B ) In a time course of UUO , COUP - TFII expression increased as early as day 2 and preceded the increased expression of αSMA ( n = 2 animals per time point ) . Quantification by confocal micrographs in 200x hpf . * p < 0 . 05 ; * * * p < 0 . 001 by two - way ANOVA test ; mean ± SD . ( E & F ) Gli1 - tdTomato + cells expanded primarily in the outer medullary region . In contrast , COUP - TFII + cells are distributed both in cortex and medulla . A subset of COUP - TFII + cells ( green ) overlap with genetically labeled Gli1 + pericyte / perivascular cells ( red ) in UUO injury model ( n = 3 animals per group ) . CTL : contralateral kidney . Quantification by confocal micrographs in 200x hpf .

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Figure 3(A) COUP-TFII expression is significantly increased and co-localized with αSMA+ cells at day 10 of UUO and day 14 of UIRI in mice (n=3 animals per group). Quantification by confocal micrographs in 200x hpf. *p<0.05 **p<0.01 by paired t test; mean ± SD.(B) In a time course of UUO, COUP-TFII expression increased as early as day 2 and preceded the increased expression of αSMA (n=2 animals per time point). Quantification by confocal micrographs in 200x hpf. *p<0.05; ***p<0.001 by two-way ANOVA test; mean ± SD.(E&F) Gli1-tdTomato+ cells expanded primarily in the outer medullary region. In contrast, COUP-TFII+ cells are distributed both in cortex and medulla. A subset of COUP-TFII+ cells (green) overlap with genetically labeled Gli1+ pericyte/perivascular cells (red) in UUO injury model (n=3 animals per group). CTL: contralateral kidney. Quantification by confocal micrographs in 200x hpf.


Figure 4 ( D ) COUP - TFII + cells decrease significantly in the UUO kidney in TAM - treated homozygous ( F / F ; Cre / + ) mice ( KO group , n = 6 ) compared to wild - type littermates ( WT group , n = 4 ) . Expression of αSMA ( red ) and collagen1 ( yellow ) are also markedly reduced . Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group . Quantification by confocal micrographs in 200x hpf . * * p < 0 . 01 by unpaired t test , mean ± SD .

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Figure 4(D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.


Figure 5 ( D ) COUP - TFII - OE cells , in the absence of TGFβ1 stimulation , displayed an elongated fibroblast shape with increased αSMA expression , similar to WT cells treated with TGFβ1 . Quantification by confocal micrographs in 400x hpf , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD . ( E ) Overexpression of COUP - TFII alone without TGFβ1 induces collagen1 production ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA , mean ± SD .

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Figure 5(D) COUP-TFII-OE cells, in the absence of TGFβ1 stimulation, displayed an elongated fibroblast shape with increased αSMA expression, similar to WT cells treated with TGFβ1. Quantification by confocal micrographs in 400x hpf, *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.(E) Overexpression of COUP-TFII alone without TGFβ1 induces collagen1 production (n=3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.


Figure 6 ( D ) WT , COUP - TFII overexpression ( OE ) and COUP - TFII knockout ( KO ) cells were seeded in Seahorse XF - 24 cell culture microplates . The cells were rendered quiescent in 0 . 5 % FBS DMEM overnight and then treated with or without 10 ng / ml TGFβ1 for 24 hours . All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate , followed by sequential treatments with glucose ( 10mM ) , oligomycin ( 5μg / ml ) and 2 - DG ( 50mM ) . Real - time extracellular acidification rate ( ECAR ) was recorded as the baseline ( before glucose ) , the rate of glycolysis ( after glucose ) , glycolytic capacity ( after oligomycin ) , and glycolytic reserve ( after 2 - DG ) ( n = 10 ) . ( E ) COUP - TFII OE significantly increased , while COUP - TFII KO decreased , in the baseline , rate of glycolysis , glycolytic capacity , and glycolytic reserve when compared to WT cells under TGFβ1 stimulation . Without TGFβ1 treatment , there is no difference on the baseline and glycolysis among WT , OE , and KO cells . COUP - TFII KO significantly decreased glycolytic capacity and reserve compared to WT , even without TGFβ1 stimulation ( n = 10 ) , * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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Figure 6(D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10).(E) COUP-TFII OE significantly increased, while COUP-TFII KO decreased, in the baseline, rate of glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT cells under TGFβ1 stimulation. Without TGFβ1 treatment, there is no difference on the baseline and glycolysis among WT, OE, and KO cells. COUP-TFII KO significantly decreased glycolytic capacity and reserve compared to WT, even without TGFβ1 stimulation (n=10), *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


Figure 7 ( A ) Volcano plot of all detected proteins from proteomics of TGFβ  - treated naïve ( WT ) and COUP - TFII overexpressing ( COUP - TFII - OE ) cells . PDK4 expression is significantly decreased in OE cells ( n = 2 ) . ( E ) PGC1α OE significantly increased expression of Cpt1 and PDK4 , both are involved in key steps of FAO . However , overexpression of PGC1α did not abrogate the TGFβ1 up - regulated genes in glycolysis and αSMA ( n = 6 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by one - way ANOVA , mean ± SD .

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

Figure 7(A) Volcano plot of all detected proteins from proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells. PDK4 expression is significantly decreased in OE cells (n=2).(E) PGC1α OE significantly increased expression of Cpt1 and PDK4, both are involved in key steps of FAO. However, overexpression of PGC1α did not abrogate the TGFβ1 up-regulated genes in glycolysis and αSMA (n=6). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD.


figf1b - d , MEFs were cultured in nutrient - rich medium ( fed ) or Hanks ' buffer ( starved ) for 1 h . Total cell lysates were prepared , and blotted with the indicated antibodies ( b ) . α , β , indicate Atg16L1 isoform α , β ; ∆ CCD indicates Atg16L1 ∆ CCD ; asterisk indicates a non - specific band . Atg16L1 ∆ / ∆ indicates mice expressing ∆ Atg16L1 . The number of endogenous LC3 or GFP - Atg5 dots was counted ( c , d ) . The results shown are mean + s . d . ( n > 20 ) . e , Electron micrograph of starved MEFs . f , Degradation of long - lived proteins in MEFs . Wortmannin is an autophagy inhibitor . The results shown are mean1s . d . Statistical significance ( P value ) was determined by the Student ' s t - test .

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

figf1b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b).α, β, indicate Atg16L1 isoform α, β; ∆CCD indicates Atg16L1∆CCD; asterisk indicates a non-specific band. Atg16L1∆/∆ indicates mice expressing ∆Atg16L1. The number of endogenous LC3 or GFP-Atg5 dots was counted (c, d). The results shown are mean + s.d. (n > 20).e, Electron micrograph of starved MEFs.f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean1s.d. Statistical significance (P value) was determined by the Student's t-test.


figf2a , Cytokine production from macrophages stimulated with LPS ( 100 ng ml - 1 ) for 24 h . Statistical significance was determined by the Student ' s t - test . * P 0 . 01 . b , IL - 1b production from macrophages stimulated with indicated ligands . c , IL - 1b production from LPS - primed macrophages infected with S . typhimurium ( multiplicity of infection , m . o . i . , 1 ) , or stimulated with ATP or MSU for 1 h . d , Expression levels of caspase - 1 and IL - 1b in macrophages . Lys . , cell lysates ; Sup . , culture supernatants . e , LPS - induced production of IL - 1b from macrophages with the indicated phenotype . f , Expression levels of caspase - 1 and IL - 1b in macrophages treated as in e . g , Effect of the ROS scavenger FeTPPS ( 25 mM ) , N - acetyl - L - cysteine ( NAC ; 25mM ) or P2X7 receptor antagonist oxidized ATP ( 250 mM ) on IL - 1b production . h , ROS in LPS - stimulated macrophages were detected by CM - H2DCFDA staining .

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

figf2a, Cytokine production from macrophages stimulated with LPS (100 ng ml-1) for 24 h. Statistical significance was determined by the Student's t-test. *P  0.01.b, IL-1b production from macrophages stimulated with indicated ligands.c, IL-1b production from LPS-primed macrophages infected with S. typhimurium (multiplicity of infection, m.o.i., 1), or stimulated with ATP or MSU for 1 h.d, Expression levels of caspase-1 and IL-1b in macrophages. Lys., cell lysates; Sup., culture supernatants.e, LPS-induced production of IL-1b from macrophages with the indicated phenotype.f, Expression levels of caspase-1 and IL-1b in macrophages treated as in e.g, Effect of the ROS scavenger FeTPPS (25 mM), N-acetyl-L-cysteine (NAC; 25mM) or P2X7 receptor antagonist oxidized ATP (250 mM) on IL-1b production.h, ROS in LPS-stimulated macrophages were detected by CM-H2DCFDA staining.


figf3a , Macrophages were stimulated with LPS ( 100 ng ml - 1 ) for the indicated time period . The number of endogenous LC3 dots within each cell was counted . The results shown are mean + s . d . ( n > 100 ) . b , Expression levels of p62 and LC3 in macrophages . c , IL - 1b and IL - 6 production by wild - type or Atg7 - deficient macrophages stimulated with LPS . Statistical significance was determined by the Student ' s t - test . * P , 0 . 01 . d , Macrophages were pre - treated with or without 10 mM 3MA and then stimulated with the indicated ligands .

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

figf3a, Macrophages were stimulated with LPS (100 ng ml-1) for the indicated time period. The number of endogenous LC3 dots within each cell was counted. The results shown are mean + s.d. (n > 100).b, Expression levels of p62 and LC3 in macrophages.c, IL-1b and IL-6 production by wild-type or Atg7-deficient macrophages stimulated with LPS. Statistical significance was determined by the Student's t-test. *P,0.01.d, Macrophages were pre-treated with or without 10 mM 3MA and then stimulated with the indicated ligands.


figf4a , b , Fetal liver chimaeric mice were given 5 % DSS in drinking water for 7 days . The survival ( a ) and weight loss ( b ) of each mouse genotype were plotted . The results shown are mean ± s . d . Statistical significance was determined by the Students t - test . c , Typical distal colon appearance 6 days after the initiation of DSS administration . The results shown are mean ± s . d . ( n = 3 , each group ) . d , Expression levels of IL - 1b and IL - 18 in serum ( n55 , each group ) . e , f , Atg16L1 - deficient chimaeric mice given 5 % DSS in drinking water were intraperitoneally injected with both anti - IL - 1b and anti - IL - 18 neutralizing antibodies ( squares ; n55 ) or isotype control IgG ( circles ; n55 ) at days 1 , 3 , 5 and 7 . The survival ( e ) and weight loss ( f ) of each mouse genotype were plotted .

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

figf4a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test.c, Typical distal colon appearance 6  days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group).d, Expression levels of IL-1b and IL-18 in serum (n55, each group).e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1b and anti- IL-18 neutralizing antibodies (squares; n55) or isotype control IgG (circles;n55) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.


Figure 1A . HeLa , H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , cell lysates were subjected to Western blot analysis with anti - c - Myc antibody . C . Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA - MIF . Actin was also included as a positive control . D . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with anti - c - Myc and anti - GAPDH antibodies . E . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . F . HeLa cells expressing either control RNA or lncRNA - MIF were treated with or without MG132 ( 20 μM ) for 6 h . Cell lysates were analyzed by Western blot with the indicated antibodies . G . HeLa cells expressing either control shRNA or lncRNA - MIF shRNA were treated with MG132 for 6 h . Cell lysates were analyzed by Western blotting with the indicated antibodies . H . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were treated with cycloheximide ( CHX , 50 μg / ml ) for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the c - Myc protein half - life . Protein band intensity were analyzed by ImageJ . I . Band intensity of c - Myc in Fig 1H was analyzed by ImageJ . J . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 as indicated . Forty - eight hours after infection , cells were treated with CHX for the indicated periods of time . Cell lysates were then analyzed by Western blotting to examine the half - life of c - Myc protein . K . Band intensity of c - Myc in Fig 1J was analyzed by ImageJ .

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

Figure 1A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody.C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control.D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies.E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.


Figure 2A . P493 - 6 cells ( human B cell lymphoma cell line ) carrying a c - Myc tet - off system were treated with Doxycycline ( 1 μg / ml ) for the indicated periods of time . Total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting with the indicated antibodies . B . HeLa , HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c - Myc shRNA . Forty - eight hours after infection , total RNA and cell lysates were analyzed by real - time RT - PCR and Western blotting , respectively . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . C . HeLa , HCT116 and MCF10A cells were transfected with control vector or Flag - c - Myc . Twenty - four hours after transfection , total RNA was extracted from these cells and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Cell lysates were also analyzed by Western blotting . E . HeLa cells were co - transfected with either Flag - c - Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . F . HeLa cells expressing control shRNA or c - Myc shRNA were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . G . P493 - 6 cells were co - transfected with the indicated reporter constructs and Renilla luciferase plasmid . Twenty - four hours after trasfection , cells were treated with doxycycline for 0 hour and 12 hours respectively . Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . H . Lysates from HeLa cells were subjected to ChIP assay . ChIP products were amplified by qPCR reaction with the indicated pairs of primers . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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

Figure 2A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.B. HeLa, HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c-Myc shRNA. Forty-eight hours after infection, total RNA and cell lysates were analyzed by real-time RT-PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).C. HeLa, HCT116 and MCF10A cells were transfected with control vector or Flag-c-Myc. Twenty-four hours after transfection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting.E. HeLa cells were co-transfected with either Flag-c-Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).F. HeLa cells expressing control shRNA or c-Myc shRNA were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).H. Lysates from HeLa cells were subjected to ChIP assay. ChIP products were amplified by qPCR reaction with the indicated pairs of primers. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


Figure 3A . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF or biotin - labeled antisense RNA for biotinpull - down assay , followed by Western blot analysis with the indicated antibodies . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . E . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes against lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . F . Lysates from HeLa cells were incubated with in vitro synthesized biotin - labeled lncRNA - MIF and antisense RNA for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 and miR - 34a levels . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . H . miR - 586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . I . miR - 586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . J . HeLa cells were subjected to fractionation into cytoplasmic ( C ) and nuclear ( N ) extracts . Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . Actin and PARP were used as markers for cytoplasmic and nuclear fractions , respectively . K . HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin - labeled sense or antisense DNA probes of lncRNA - MIF for biotinpull - down assay , followed by real - time RT - PCR analysis to examine miR - 586 levels . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

Figure 3A. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF or biotin-labeled antisense RNA for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).E. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).F. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF and antisense RNA for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 and miR-34a levels. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).H. miR-586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).I. miR-586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).J. HeLa cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). Actin and PARP were used as markers for cytoplasmic and nuclear fractions, respectively.K. HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes of lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


Figure 4B . miR - 586 mimics or negative control mimics ( NC mimics ) were transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . C . miR - 586 inhibitor or negative control inhibitor ( NC inhibitor ) was transfected into HeLa cells together with the indicated pSICHECK2 - based luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . D . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting . E . HeLa cells were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , total RNA was extracted and subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . F . The copy numbers of miR - 586 and lncRNA - MIF transcript per cell in the indicated cells were quantified using a quantitative real - time RT - PCR assay . Data shown are mean ± SD ( n = 3 ) . G . HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . HeLa cells were infected with lentiviruses expressing lncRNA - MIF or control RNA . Forty - eight hours after infection , total RNA was analyzed by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . H . HeLa cells expressing lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting . I . HeLa cells expressing lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor . Twenty - four hours after transfection , cell lysates were analyzed by Western blotting . K . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , lncRNA - MIF harboring nonsense mutation or miR - 586 binding defective lncRNA - MIF . Forty - eight hours after transfection , cell lysates were analyzed by Western blotting .

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

Figure 4B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).C. miR-586 inhibitor or negative control inhibitor (NC inhibitor)was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3).G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR-586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.


Figure 5A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By Forty - eight hours infection , cells were treated with MG132 or DMSO ( vehicle ) for 6 h . Cell lysates were then analyzed by Western blotting with the indicated antibodies . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . Forty - eight hours after infection , total RNA was subjected to analyzing the levels of lncRNA - MIF and lncRNA - MIF - AS by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS . By 48 h infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) . D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . By 48 h infection , cells were treated with MG132 or DMSO for 6 h . Cell lysates were then analyzed by Western blot analysis with the indicated antibodies . E . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

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

Figure 5A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By 48 h infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.E. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


Figure 6A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , acidification of the culture medium was evaluated by visually inspecting the color of the medium . Yellow medium indicates the presence of more lactate . B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies . C . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . D . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . E . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) . F . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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

Figure 6A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


Figure 7A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) . B . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) . C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle . D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle . E . The percentage numbers of cells ( Fig 7C ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) . F . The percentage numbers of cells ( Fig 7D ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

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

Figure 7A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).