samd7 fusion protein Search Results


samd7 fusion protein  (GE Healthcare)
96
GE Healthcare samd7 fusion protein
<t>Samd7</t> expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.
Samd7 Fusion Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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glutathione sepharose 4b  (GE Healthcare)
99
GE Healthcare glutathione sepharose 4b
<t>Samd7</t> expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.
Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/glutathione sepharose 4b/product/GE Healthcare
Average 99 stars, based on 1 article reviews
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monoclonal smad2  (Proteintech)
96
Proteintech monoclonal smad2
Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of <t>smad2/3</t> and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.
Monoclonal Smad2, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/monoclonal smad2/product/Proteintech
Average 96 stars, based on 1 article reviews
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anti gapdh polyclonal antibodies  (Proteintech)
96
Proteintech anti gapdh polyclonal antibodies
Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of <t>smad2/3</t> and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.
Anti Gapdh Polyclonal Antibodies, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti gapdh polyclonal antibodies/product/Proteintech
Average 96 stars, based on 1 article reviews
anti gapdh polyclonal antibodies - by Bioz Stars, 2026-06
96/100 stars
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Image Search Results


Samd7 expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Marker, Staining

Samd7 expression and generation of the Samd7−/− allele. (A) Northern blot analysis of mouse Samd7 in developing and adult retinas. Northern blot analysis of Samd7 transcripts was performed using mRNAs purified from retinas of mice between P1 and 4 wk of age. (Upper) A major band of ≈2.4 kb Samd7 mRNA was detected. (Lower) Ethidium bromide staining of RNAs. (B) Immunostaining of P6 WT mouse retinal sections using antibodies against Samd7 (red), Thrb2 (a cone precursor marker, green), and S-opsin (an S-cone marker, blue). Dotted lines indicate Thrb2-positive cone photoreceptor nuclei. The Samd7 signal is obviously weaker both in S-opsin–positive S-cones (arrows) and in S-opsin–negative M-cones (arrowheads) than in rods. (C) Diagram of the targeting vector and the Samd7−/− allele. Removal of exons 4–6 is predicted to result in a translational frame shift and a complete loss of Samd7 function. (D) RT-PCR analysis of the Samd7 transcript in the Samd7−/− retina. An approximately 500-bp fragment of Samd7 was amplified from cDNA prepared from the WT retina. No Samd7 transcript was detected in the Samd7−/− retina. β-Actin was used as a loading control. (E) Western blot analysis of the Samd7 protein in the Samd7−/− retina. An approximately 56-kDa Samd7 band was detected in the WT retina. No Samd7 band was detected in the Samd7−/− retina. β-Actin was used as a loading control.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and generation of the Samd7−/− allele. (A) Northern blot analysis of mouse Samd7 in developing and adult retinas. Northern blot analysis of Samd7 transcripts was performed using mRNAs purified from retinas of mice between P1 and 4 wk of age. (Upper) A major band of ≈2.4 kb Samd7 mRNA was detected. (Lower) Ethidium bromide staining of RNAs. (B) Immunostaining of P6 WT mouse retinal sections using antibodies against Samd7 (red), Thrb2 (a cone precursor marker, green), and S-opsin (an S-cone marker, blue). Dotted lines indicate Thrb2-positive cone photoreceptor nuclei. The Samd7 signal is obviously weaker both in S-opsin–positive S-cones (arrows) and in S-opsin–negative M-cones (arrowheads) than in rods. (C) Diagram of the targeting vector and the Samd7−/− allele. Removal of exons 4–6 is predicted to result in a translational frame shift and a complete loss of Samd7 function. (D) RT-PCR analysis of the Samd7 transcript in the Samd7−/− retina. An approximately 500-bp fragment of Samd7 was amplified from cDNA prepared from the WT retina. No Samd7 transcript was detected in the Samd7−/− retina. β-Actin was used as a loading control. (E) Western blot analysis of the Samd7 protein in the Samd7−/− retina. An approximately 56-kDa Samd7 band was detected in the WT retina. No Samd7 band was detected in the Samd7−/− retina. β-Actin was used as a loading control.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Northern Blot, Purification, Staining, Immunostaining, Marker, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

Immunohistochemical analysis of the Samd7−/− retina. (A) Retinal sections from 2-mo-old WT and Samd7−/− mice were immunostained with the anti–M-opsin antibody (a marker for cone outer segments, green) and PNA (a cone photoreceptor outer and inner segment marker, red). No obvious difference was observed between WT and Samd7−/− retinas. (B) The distribution of S-opsin (red) and rhodopsin (green) signals was normal in the Samd7+/− retina at 2 mo. (C) At P9, ectopic S-opsin signals in premature rod photoreceptor outer segments were observed in the Samd7−/− retina. (D) Retinal sections from dorsal and ventral regions of 2-mo-old WT control and Samd7−/− mice were immunostained using anti–S-opsin (red) and anti-rhodopsin (green) antibodies with DAPI (blue). There were fewer S-opsin–positive cones in the dorsal region than in the ventral region of the control retina. Ectopic S-opsin expression in rod outer segments was similar in the dorsal and ventral regions of the Samd7−/− retina. (E) Flat-mount immunostaining of dorsal and ventral regions of WT control and Samd7−/− retinas. M-opsin (green) and Rhodamine-PNA (red) were used for visualizing cone photoreceptor cells. The numbers of M-opsin and PNA double-positive cells decreased in the ventral region in both WT control and Samd7−/− retinas. (F) Retinal sections from WT and Samd7−/− mice were immunostained with anti-Pikachurin (a marker of photoreceptor synaptic clefts, red) and anti-Ctbp2 (a marker of synaptic ribbons, green) antibodies. No obvious difference was observed between WT and Samd7−/− retinas at 2 mo. (G) Immunofluorescent examination of WT and Samd7−/− retinal sections from 2-mo-old mice using anti-Calbindin (a marker for amacrine and horizontal cells, red), anti-S100b (a maker for Müller glia, green), anti-Chx10 (a marker for bipolar cells, red), and anti-Pax6 (a marker for amacrine and ganglion cells, green) antibodies showed no obvious difference between WT and Samd7−/− retinas. (H) Retinal sections from WT and Samd7−/− mice at 12M were stained with toluidine blue. The thickness of the ONL of WT and Samd7−/− retinas was measured. Average layer thickness in the WT retina was set to 100%. No significant change in ONL thickness was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; n.s., not significant; ONL, outer nuclear layer; OS, outer segments.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Immunohistochemical analysis of the Samd7−/− retina. (A) Retinal sections from 2-mo-old WT and Samd7−/− mice were immunostained with the anti–M-opsin antibody (a marker for cone outer segments, green) and PNA (a cone photoreceptor outer and inner segment marker, red). No obvious difference was observed between WT and Samd7−/− retinas. (B) The distribution of S-opsin (red) and rhodopsin (green) signals was normal in the Samd7+/− retina at 2 mo. (C) At P9, ectopic S-opsin signals in premature rod photoreceptor outer segments were observed in the Samd7−/− retina. (D) Retinal sections from dorsal and ventral regions of 2-mo-old WT control and Samd7−/− mice were immunostained using anti–S-opsin (red) and anti-rhodopsin (green) antibodies with DAPI (blue). There were fewer S-opsin–positive cones in the dorsal region than in the ventral region of the control retina. Ectopic S-opsin expression in rod outer segments was similar in the dorsal and ventral regions of the Samd7−/− retina. (E) Flat-mount immunostaining of dorsal and ventral regions of WT control and Samd7−/− retinas. M-opsin (green) and Rhodamine-PNA (red) were used for visualizing cone photoreceptor cells. The numbers of M-opsin and PNA double-positive cells decreased in the ventral region in both WT control and Samd7−/− retinas. (F) Retinal sections from WT and Samd7−/− mice were immunostained with anti-Pikachurin (a marker of photoreceptor synaptic clefts, red) and anti-Ctbp2 (a marker of synaptic ribbons, green) antibodies. No obvious difference was observed between WT and Samd7−/− retinas at 2 mo. (G) Immunofluorescent examination of WT and Samd7−/− retinal sections from 2-mo-old mice using anti-Calbindin (a marker for amacrine and horizontal cells, red), anti-S100b (a maker for Müller glia, green), anti-Chx10 (a marker for bipolar cells, red), and anti-Pax6 (a marker for amacrine and ganglion cells, green) antibodies showed no obvious difference between WT and Samd7−/− retinas. (H) Retinal sections from WT and Samd7−/− mice at 12M were stained with toluidine blue. The thickness of the ONL of WT and Samd7−/− retinas was measured. Average layer thickness in the WT retina was set to 100%. No significant change in ONL thickness was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; n.s., not significant; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Immunohistochemical staining, Marker, Expressing, Immunostaining, Staining

ERG analysis of Samd7−/− mice. ERGs were recorded from WT (n = 5) and Samd7−/− (n = 5) mice at 12 wk. (A and B) Representative scotopic (A) and photopic (B) ERGs in WT and Samd7−/− mice elicited by white light. (C) The amplitude of the scotopic ERG a-wave as a function of the stimulus intensity (1.0 log cd⋅s⋅m−2) was significantly reduced in Samd7−/− mice. (D) The amplitude of the scotopic ERG b-wave as a function of the stimulus intensity (−1.4 log cd⋅s⋅m−2) decreased significantly in Samd7−/− mice. Error bars show the SD. **P < 0.03.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. ERGs were recorded from WT (n = 5) and Samd7−/− (n = 5) mice at 12 wk. (A and B) Representative scotopic (A) and photopic (B) ERGs in WT and Samd7−/− mice elicited by white light. (C) The amplitude of the scotopic ERG a-wave as a function of the stimulus intensity (1.0 log cd⋅s⋅m−2) was significantly reduced in Samd7−/− mice. (D) The amplitude of the scotopic ERG b-wave as a function of the stimulus intensity (−1.4 log cd⋅s⋅m−2) decreased significantly in Samd7−/− mice. Error bars show the SD. **P < 0.03.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

ERG analysis of Samd7−/− mice. (A) The a-wave amplitudes of scotopic ERGs from WT (n = 5) and Samd7−/− (n = 5) mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2) at 12 wk. The amplitude of the a-wave at +1.0 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (B) The b-wave amplitudes of scotopic ERG from WT and Samd7−/− mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2). The amplitudes of the b-wave at −2.6 and −1.4 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (C) The b-wave amplitudes of photopic ERG from WT and Samd7−/− mice elicited by four different white light stimuli (−0.8, −0.2, 0.4, 1.0 log cd⋅s⋅m−2). No obvious change in amplitude was observed between WT and Samd7−/− mice. Error bars show the SD.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. (A) The a-wave amplitudes of scotopic ERGs from WT (n = 5) and Samd7−/− (n = 5) mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2) at 12 wk. The amplitude of the a-wave at +1.0 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (B) The b-wave amplitudes of scotopic ERG from WT and Samd7−/− mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2). The amplitudes of the b-wave at −2.6 and −1.4 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (C) The b-wave amplitudes of photopic ERG from WT and Samd7−/− mice elicited by four different white light stimuli (−0.8, −0.2, 0.4, 1.0 log cd⋅s⋅m−2). No obvious change in amplitude was observed between WT and Samd7−/− mice. Error bars show the SD.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

Global change of retinal expression profile in the Samd7−/− retina. (A) Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and cone-enriched (red circle) and rod-enriched (yellow circle) genes. Microarray analysis was performed using mRNAs from the WT and Samd7−/− retinas at P12. One hundred sixty-three genes were up-regulated (signal log ratio greater than +1.0, blue circle), and 251 genes were down-regulated (signal log ratio less than −0.5, green circle) in the Samd7−/− retina compared with those in the WT retina. (B) Lists of the 33 genes that overlap between cone-enriched genes and up-regulated genes in the Samd7−/− retina (Left) and the 23 genes that overlap between rod-enriched genes and down-regulated genes in the Samd7−/− retina (Right). Cone- and rod-enriched genes (more than fourfold FPKM value) were identified using RNA-seq data from a previous study (22). (C and D) The expression levels of the selected genes were measured by qRT-PCR using mRNAs of WT and Samd7−/− retinas at P12. Up-regulation of nonrod genes (C) and down-regulation of rod genes (D) in the Samd7−/− retina by microarray analysis were confirmed. (E) Nonrod genes are ectopically expressed in the ONL of the Samd7−/− retina. In situ hybridization analysis of WT and Samd7−/− retinal sections at P12 was performed using probes for the up-regulated genes in the Samd7−/− retina: Cacna1h (a bipolar cell gene), Gngt2, Cnga3, and S-opsin (cone-specific genes). The vertical brackets indicate the extent of the ONL. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Error bars show the SD (n = 4). **P < 0.03. n.s., not significant.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Global change of retinal expression profile in the Samd7−/− retina. (A) Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and cone-enriched (red circle) and rod-enriched (yellow circle) genes. Microarray analysis was performed using mRNAs from the WT and Samd7−/− retinas at P12. One hundred sixty-three genes were up-regulated (signal log ratio greater than +1.0, blue circle), and 251 genes were down-regulated (signal log ratio less than −0.5, green circle) in the Samd7−/− retina compared with those in the WT retina. (B) Lists of the 33 genes that overlap between cone-enriched genes and up-regulated genes in the Samd7−/− retina (Left) and the 23 genes that overlap between rod-enriched genes and down-regulated genes in the Samd7−/− retina (Right). Cone- and rod-enriched genes (more than fourfold FPKM value) were identified using RNA-seq data from a previous study (22). (C and D) The expression levels of the selected genes were measured by qRT-PCR using mRNAs of WT and Samd7−/− retinas at P12. Up-regulation of nonrod genes (C) and down-regulation of rod genes (D) in the Samd7−/− retina by microarray analysis were confirmed. (E) Nonrod genes are ectopically expressed in the ONL of the Samd7−/− retina. In situ hybridization analysis of WT and Samd7−/− retinal sections at P12 was performed using probes for the up-regulated genes in the Samd7−/− retina: Cacna1h (a bipolar cell gene), Gngt2, Cnga3, and S-opsin (cone-specific genes). The vertical brackets indicate the extent of the ONL. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Error bars show the SD (n = 4). **P < 0.03. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Microarray, RNA Sequencing Assay, Quantitative RT-PCR, In Situ Hybridization

Samd7 interacts with Phc proteins in PRC1 and a rod TF Nr2e3. (A) Yeast two-hybrid assay using full-length Samd7 as the bait. Proteins containing SAM domains with high similarity to Samd7 (Samd7/11, Phc1/2/3, and L3mbtl3) were identified as Samd7 interactor proteins. Growth on selective plates lacking histidine (−His) and adenine indicates a physical interaction between the bait and prey constructs. (B) Immunoprecipitation analysis of Samd7 and Phc2. A plasmid expressing FLAG-Samd7 or FLAG-Phc2 was transfected with a plasmid expressing GFP-Samd7 into HEK293 cells. FLAG-tagged proteins were immunoprecipitated using an anti-FLAG antibody. GFP-Samd7 was coimmunoprecipitated with FLAG-Samd7 or FLAG-Phc2. (C) Immunoprecipitation was performed using mouse retinal lysate from 2-mo-old mice using an anti-Phc2 antibody. Immunoprecipitated Samd7 with Phc2 was detected by Western blot analysis using the anti-Samd7 antibody. (D) A 3D structural model of the EH and ML surfaces of the Samd7 SAM domain. The putative 3D structure of the Samd7 SAM domain was obtained by MODELLAR (https://salilab.org/modeller/) using the Phc3 SAM domain as a template [Protein Data Bank (PDB) ID code 4PZN]. Two molecules of the Samd7 SAM domain (green and blue) are shown. Amino acids essential for interaction on the EH surface (L372) and the ML surface (L358/H363) are indicated in red. (E) Immunoprecipitation of FLAG-Samd7-WT with GFP-Samd7-WT or GFP-Samd7-LR (with the L372R mutation on EH surface of the SAM domain). Reduced interaction between FLAG-Samd7-WT or FLAG-Samd7-LR and GFP-Samd7-LR was observed compared with that between FLAG-Samd7-WT and GFP-Samd7-WT. (F) FSEC analysis of the Samd7 protein. Plasmids expressing GFP-fused WT Samd7 (GFP-Samd7-WT) and Samd7 with a mutation on the EH surface of the SAM domain (GFP-Samd7-L372R) were transfected into HEK293 cells, and the fluorescent signals in lysates were analyzed by FSEC. In GFP-Samd7-WT lysates, a putative monomer peak between the 44- and 158-kDa size markers and a broad peak likely corresponding to polymers larger than 669 kDa (arrowhead) were observed. Putative monomer and oligomer peaks but no obvious peaks at a higher molecular size were detected in GFP-Samd7-L372R lysates. (G) Yeast two-hybrid assay using Samd7 with mutations in the SAM domain ML (L358R/H363R, HR) and/or EH (L372R, LR) surfaces as baits. The interaction of Samd7 constructs with SAM domain proteins (Samd7/11, Phc1/2/3, L3mbtl3) was analyzed. Samd7 with mutations both on the ML and EH surfaces (L372R/L358R/H363R, LRHR) showed no interaction with any of the SAM domain proteins tested (Samd7/11, Phc1/2/3, L3mbtl3). (H) Immunoprecipitation analysis of Samd7 with rod photoreceptor transcription factors. A plasmid expressing FLAG-Samd7 was transfected with a plasmid expressing HA-Nr2e3 or HA-Nrl into HEK293 cells. FLAG-tagged Samd7 was immunoprecipitated using an anti-FLAG antibody. The interaction of Samd7 with Nr2e3 was observed, whereas Samd7 showed no substantial interaction with Nrl.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 interacts with Phc proteins in PRC1 and a rod TF Nr2e3. (A) Yeast two-hybrid assay using full-length Samd7 as the bait. Proteins containing SAM domains with high similarity to Samd7 (Samd7/11, Phc1/2/3, and L3mbtl3) were identified as Samd7 interactor proteins. Growth on selective plates lacking histidine (−His) and adenine indicates a physical interaction between the bait and prey constructs. (B) Immunoprecipitation analysis of Samd7 and Phc2. A plasmid expressing FLAG-Samd7 or FLAG-Phc2 was transfected with a plasmid expressing GFP-Samd7 into HEK293 cells. FLAG-tagged proteins were immunoprecipitated using an anti-FLAG antibody. GFP-Samd7 was coimmunoprecipitated with FLAG-Samd7 or FLAG-Phc2. (C) Immunoprecipitation was performed using mouse retinal lysate from 2-mo-old mice using an anti-Phc2 antibody. Immunoprecipitated Samd7 with Phc2 was detected by Western blot analysis using the anti-Samd7 antibody. (D) A 3D structural model of the EH and ML surfaces of the Samd7 SAM domain. The putative 3D structure of the Samd7 SAM domain was obtained by MODELLAR (https://salilab.org/modeller/) using the Phc3 SAM domain as a template [Protein Data Bank (PDB) ID code 4PZN]. Two molecules of the Samd7 SAM domain (green and blue) are shown. Amino acids essential for interaction on the EH surface (L372) and the ML surface (L358/H363) are indicated in red. (E) Immunoprecipitation of FLAG-Samd7-WT with GFP-Samd7-WT or GFP-Samd7-LR (with the L372R mutation on EH surface of the SAM domain). Reduced interaction between FLAG-Samd7-WT or FLAG-Samd7-LR and GFP-Samd7-LR was observed compared with that between FLAG-Samd7-WT and GFP-Samd7-WT. (F) FSEC analysis of the Samd7 protein. Plasmids expressing GFP-fused WT Samd7 (GFP-Samd7-WT) and Samd7 with a mutation on the EH surface of the SAM domain (GFP-Samd7-L372R) were transfected into HEK293 cells, and the fluorescent signals in lysates were analyzed by FSEC. In GFP-Samd7-WT lysates, a putative monomer peak between the 44- and 158-kDa size markers and a broad peak likely corresponding to polymers larger than 669 kDa (arrowhead) were observed. Putative monomer and oligomer peaks but no obvious peaks at a higher molecular size were detected in GFP-Samd7-L372R lysates. (G) Yeast two-hybrid assay using Samd7 with mutations in the SAM domain ML (L358R/H363R, HR) and/or EH (L372R, LR) surfaces as baits. The interaction of Samd7 constructs with SAM domain proteins (Samd7/11, Phc1/2/3, L3mbtl3) was analyzed. Samd7 with mutations both on the ML and EH surfaces (L372R/L358R/H363R, LRHR) showed no interaction with any of the SAM domain proteins tested (Samd7/11, Phc1/2/3, L3mbtl3). (H) Immunoprecipitation analysis of Samd7 with rod photoreceptor transcription factors. A plasmid expressing FLAG-Samd7 was transfected with a plasmid expressing HA-Nr2e3 or HA-Nrl into HEK293 cells. FLAG-tagged Samd7 was immunoprecipitated using an anti-FLAG antibody. The interaction of Samd7 with Nr2e3 was observed, whereas Samd7 showed no substantial interaction with Nrl.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Y2H Assay, Construct, Immunoprecipitation, Plasmid Preparation, Expressing, Transfection, Western Blot, Mutagenesis

Partial colocalization of Samd7 and Phc2 in rod photoreceptor nuclei. Retinal sections at P6, P12, and 1 mo (A and B) and at 2 mo (A and C) were immunostained with the anti-Samd7 (red) and anti-Phc2 (green) antibodies. Small points of Samd7 and Phc2 partially colocalized (arrowheads in A) surrounding heterochromatin regions of rod photoreceptor nuclei stained with DAPI (blue). Subnuclear localization of Phc2 was unchanged in the photoreceptor nuclei in the Samd7−/− retina compared with that in the control retina (C).

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Partial colocalization of Samd7 and Phc2 in rod photoreceptor nuclei. Retinal sections at P6, P12, and 1 mo (A and B) and at 2 mo (A and C) were immunostained with the anti-Samd7 (red) and anti-Phc2 (green) antibodies. Small points of Samd7 and Phc2 partially colocalized (arrowheads in A) surrounding heterochromatin regions of rod photoreceptor nuclei stained with DAPI (blue). Subnuclear localization of Phc2 was unchanged in the photoreceptor nuclei in the Samd7−/− retina compared with that in the control retina (C).

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Staining

Analysis of Samd7 function in transcriptional regulation. (A) Samd7 did not affect transcriptional activation of the Rhodopsin promoter by Crx and Nrl. A luciferase reporter construct driven by the Rhodopsin promoter was cotransfected with Crx and Nrl expression plasmids into HEK293 cells, and luciferase activities of cell lysates were measured at 48 h after transfection. Cotransfection of the Samd7 expression plasmid did not affect the transactivation of the Rhodopsin promoter by Crx and Nrl. (B) Localization of Samd7 in Polycomb bodies. Phc2-dependent colocalization of Samd7 with Ring1B in Polycomb bodies in HEK293 cells. A plasmid expressing GFP-Samd7-WT was transfected with a plasmid expressing FLAG-Phc2 into HEK293 cells. Cells were stained with anti-FLAG and anti-Ring1B antibodies. Puncta of GFP-Samd7 (green) colocalized with the Ring1B signals (blue, a marker for Polycomb bodies) were observed in Polycomb bodies (arrowheads) in a Phc2-dependent manner (red). (C) Localization of Samd7 in Polycomb bodies. Subcellular localization of various Samd7-mutant proteins in U2OS cells was analyzed. U2OS cells were transfected with FLAG-tagged Samd7-mutant protein expression plasmid and were immunostained with anti-FLAG and anti-Ring1B antibodies with DAPI (blue). Puncta of the FLAG signal (green) colocalized with Ring1B signals (red) in Polycomb bodies of cells transfected with Samd7-WT or Samd7-del2 expression plasmids (arrowheads). In contrast, FLAG signals did not colocalize with the Ring1B signal in Polycomb bodies of cells transfected with the Samd7-del1, -del3, or -L372R expression plasmid. In cells with a high-level expression of Samd7-WT, -del1, or -del2 proteins, Polycomb bodies are not formed, likely due to Ring1B depletion. Dotted lines indicate cell nuclei. (D) Schematic diagrams showing the structure of Samd7-mutant proteins. Two highly conserved homology domains (HD1 and HD2) in the Samd7 protein, based on amino acid residue homology among species from mouse to zebrafish, were identified. Samd7-del1, -del2, and -del3 constructs lack the HD1, HD2, and SAM domain, respectively. The Samd7-L372R construct has a point mutation in the SAM domain disrupting its oligomerization activity. (E) Indirect physical interaction between Samd7 and Ring1B through Phc2. The immunoprecipitation assay was performed using HEK293 cells transfected with FLAG-Samd7, Myc-Ring1B, and HA-Phc2 expression plasmids. Myc-Ring1B was coimmunoprecipitated with FLAG-Samd7 in a Phc2-dependent manner. (F) Partial colocalization of Samd7 with Suz12, H2A119ub, and H3K27me3 but not with H3K4me3 marks in U2OS cells. The plasmid expressing FLAG-Samd7-WT was transfected into U2OS cells and was immunostained with anti-Suz12 (a component of PRC2), anti-H2A119ub, anti-H3K27me3, and anti-H3K4me3 antibodies. Samd7 colocalized with Suz12, H3K27me3, and H2AK119ub in some of Polycomb bodies (arrowheads). In contrast, the active histone mark H3K4me3 did not colocalize with Samd7-positive puncta in cell nuclei.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Analysis of Samd7 function in transcriptional regulation. (A) Samd7 did not affect transcriptional activation of the Rhodopsin promoter by Crx and Nrl. A luciferase reporter construct driven by the Rhodopsin promoter was cotransfected with Crx and Nrl expression plasmids into HEK293 cells, and luciferase activities of cell lysates were measured at 48 h after transfection. Cotransfection of the Samd7 expression plasmid did not affect the transactivation of the Rhodopsin promoter by Crx and Nrl. (B) Localization of Samd7 in Polycomb bodies. Phc2-dependent colocalization of Samd7 with Ring1B in Polycomb bodies in HEK293 cells. A plasmid expressing GFP-Samd7-WT was transfected with a plasmid expressing FLAG-Phc2 into HEK293 cells. Cells were stained with anti-FLAG and anti-Ring1B antibodies. Puncta of GFP-Samd7 (green) colocalized with the Ring1B signals (blue, a marker for Polycomb bodies) were observed in Polycomb bodies (arrowheads) in a Phc2-dependent manner (red). (C) Localization of Samd7 in Polycomb bodies. Subcellular localization of various Samd7-mutant proteins in U2OS cells was analyzed. U2OS cells were transfected with FLAG-tagged Samd7-mutant protein expression plasmid and were immunostained with anti-FLAG and anti-Ring1B antibodies with DAPI (blue). Puncta of the FLAG signal (green) colocalized with Ring1B signals (red) in Polycomb bodies of cells transfected with Samd7-WT or Samd7-del2 expression plasmids (arrowheads). In contrast, FLAG signals did not colocalize with the Ring1B signal in Polycomb bodies of cells transfected with the Samd7-del1, -del3, or -L372R expression plasmid. In cells with a high-level expression of Samd7-WT, -del1, or -del2 proteins, Polycomb bodies are not formed, likely due to Ring1B depletion. Dotted lines indicate cell nuclei. (D) Schematic diagrams showing the structure of Samd7-mutant proteins. Two highly conserved homology domains (HD1 and HD2) in the Samd7 protein, based on amino acid residue homology among species from mouse to zebrafish, were identified. Samd7-del1, -del2, and -del3 constructs lack the HD1, HD2, and SAM domain, respectively. The Samd7-L372R construct has a point mutation in the SAM domain disrupting its oligomerization activity. (E) Indirect physical interaction between Samd7 and Ring1B through Phc2. The immunoprecipitation assay was performed using HEK293 cells transfected with FLAG-Samd7, Myc-Ring1B, and HA-Phc2 expression plasmids. Myc-Ring1B was coimmunoprecipitated with FLAG-Samd7 in a Phc2-dependent manner. (F) Partial colocalization of Samd7 with Suz12, H2A119ub, and H3K27me3 but not with H3K4me3 marks in U2OS cells. The plasmid expressing FLAG-Samd7-WT was transfected into U2OS cells and was immunostained with anti-Suz12 (a component of PRC2), anti-H2A119ub, anti-H3K27me3, and anti-H3K4me3 antibodies. Samd7 colocalized with Suz12, H3K27me3, and H2AK119ub in some of Polycomb bodies (arrowheads). In contrast, the active histone mark H3K4me3 did not colocalize with Samd7-positive puncta in cell nuclei.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Activation Assay, Luciferase, Construct, Expressing, Transfection, Cotransfection, Plasmid Preparation, Staining, Marker, Mutagenesis, Activity Assay, Immunoprecipitation

Samd7 regulates H3K27me3 in the retina. (A and B) ChIP-seq profiles of Cnga3 (A) and Cacna1h (B) loci for H3K27me3 in developing rod photoreceptor cells at three different stages (P2, P10, and P28). H3K27me3 levels at Cnga3 and Cacna1h genes, which were up-regulated in the Samd7−/− retina, increased along with rod photoreceptor development. The ChIP-seq profiles were analyzed using previous ChIP-seq data (21). (C) A Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in the developing rods (red circle). Thirty-one genes overlapped between the up-regulated genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in developing rods. (D–F) ChIP-qPCR analysis of H3K27me3 (D) and H2AK119ub (E) on the selected up-regulated genes in Samd7−/− retinas. H3K27me3 levels of the selected genes in WT and Samd7−/− retinas were analyzed by ChIP-qPCR. The H3K27me3 levels of the selected up-regulated genes (Cacna1h, Cnga3, Gngt2, En2, S-opsin, and Rxrg) in WT and Samd7−/− retinas are indicated. No significant change in H3K27me3 levels was observed for the Hoxc13 and Gnat2 promoters. The H2AK119ub levels of the selected up-regulated genes in the Samd7−/− retina (Cacna1h, Cnga3, En2, and S-opsin) significantly decreased in the Samd7−/− retina. (F) ChIP with IgG was performed as a negative control. Error bars show the SD (n = 3). **P < 0.03, *P < 0.05. n.s., not significant.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 regulates H3K27me3 in the retina. (A and B) ChIP-seq profiles of Cnga3 (A) and Cacna1h (B) loci for H3K27me3 in developing rod photoreceptor cells at three different stages (P2, P10, and P28). H3K27me3 levels at Cnga3 and Cacna1h genes, which were up-regulated in the Samd7−/− retina, increased along with rod photoreceptor development. The ChIP-seq profiles were analyzed using previous ChIP-seq data (21). (C) A Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in the developing rods (red circle). Thirty-one genes overlapped between the up-regulated genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in developing rods. (D–F) ChIP-qPCR analysis of H3K27me3 (D) and H2AK119ub (E) on the selected up-regulated genes in Samd7−/− retinas. H3K27me3 levels of the selected genes in WT and Samd7−/− retinas were analyzed by ChIP-qPCR. The H3K27me3 levels of the selected up-regulated genes (Cacna1h, Cnga3, Gngt2, En2, S-opsin, and Rxrg) in WT and Samd7−/− retinas are indicated. No significant change in H3K27me3 levels was observed for the Hoxc13 and Gnat2 promoters. The H2AK119ub levels of the selected up-regulated genes in the Samd7−/− retina (Cacna1h, Cnga3, En2, and S-opsin) significantly decreased in the Samd7−/− retina. (F) ChIP with IgG was performed as a negative control. Error bars show the SD (n = 3). **P < 0.03, *P < 0.05. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: ChIP-sequencing, Negative Control

A proposed model of the Samd7 function in rod photoreceptor cells. (Left) In WT rod photoreceptor cells the Samd7-PRC1 complex induces chromatin condensation together with PRC2 and increases H3K27me3 marks on the S-opsin promoter. Increased H3K27me3 represses S-opsin expression in rod photoreceptor cells. (Right) In the Samd7−/− retina, Samd7 deletion causes a reduction of H3K27me3 and H2AK119ub on the S-opsin promoter, resulting in the ectopic expression of S-opsin in rod photoreceptor cells.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: A proposed model of the Samd7 function in rod photoreceptor cells. (Left) In WT rod photoreceptor cells the Samd7-PRC1 complex induces chromatin condensation together with PRC2 and increases H3K27me3 marks on the S-opsin promoter. Increased H3K27me3 represses S-opsin expression in rod photoreceptor cells. (Right) In the Samd7−/− retina, Samd7 deletion causes a reduction of H3K27me3 and H2AK119ub on the S-opsin promoter, resulting in the ectopic expression of S-opsin in rod photoreceptor cells.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing

Primer sequences

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Primer sequences

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: In Situ

Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of smad2/3 and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Journal: International Journal of Biological Sciences

Article Title: Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

doi: 10.7150/ijbs.69373

Figure Lengend Snippet: Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of smad2/3 and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: The following antibodies, monoclonal anti-GAPDH (Catalogue #5174, 1:1000), monoclonal anti-E-cadherin (Catalogue #3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal smad2/3 (Catalogue#8685T, 1:1000) were purchased from Cell signaling Technology; monoclonal anti-EGR1 (Catalogue#55160, 1:1000) was purchased from abcam; monoclonal anti-samd7 (Catalogue#66478-1-lg, 1:1000) was purchased from proteintech; monoclonal smad2/3-phosphorylation (Catalogue#abs130992, 1:1000) were purchased from Absin; anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell signaling Technology.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Binding Assay, Inhibition, Transfection, Migration

CircNDRG1 regulated smad7 expression and inhibited metastasis by targeting miR-520h. (A and B) Transwell and wound-healing assay demonstrated nitroxoline could inhibit the migration of bladder cancer cells, circNDRG1-si further promoted the migration, which was significantly rescued by preventing the expression of miR-520h. (C) Western-blotting assay showed that alteration of the expression levels of smad7, smad2/3 and the EMT-associated markers E-cadherin and N-cadherin, caused by nitroxoline plus circNDRG1-si treatment, were rescued by transfection with the miR-520h inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Journal: International Journal of Biological Sciences

Article Title: Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

doi: 10.7150/ijbs.69373

Figure Lengend Snippet: CircNDRG1 regulated smad7 expression and inhibited metastasis by targeting miR-520h. (A and B) Transwell and wound-healing assay demonstrated nitroxoline could inhibit the migration of bladder cancer cells, circNDRG1-si further promoted the migration, which was significantly rescued by preventing the expression of miR-520h. (C) Western-blotting assay showed that alteration of the expression levels of smad7, smad2/3 and the EMT-associated markers E-cadherin and N-cadherin, caused by nitroxoline plus circNDRG1-si treatment, were rescued by transfection with the miR-520h inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: The following antibodies, monoclonal anti-GAPDH (Catalogue #5174, 1:1000), monoclonal anti-E-cadherin (Catalogue #3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal smad2/3 (Catalogue#8685T, 1:1000) were purchased from Cell signaling Technology; monoclonal anti-EGR1 (Catalogue#55160, 1:1000) was purchased from abcam; monoclonal anti-samd7 (Catalogue#66478-1-lg, 1:1000) was purchased from proteintech; monoclonal smad2/3-phosphorylation (Catalogue#abs130992, 1:1000) were purchased from Absin; anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell signaling Technology.

Techniques: Expressing, Wound Healing Assay, Migration, Western Blot, Transfection