fluorescently conjugated secondary antibody and dapi  (Lonza)

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: Phenotypic analysis of the Otud7b −/− mouse retina (A) RT-PCR analysis of the Otud7b transcript in mouse tissues at 4 weeks. Otud7b was expressed ubiquitously with strong expression in the retina. β-actin was used as a loading control. (B) Immunofluorescence analysis of the wild-type retina using an anti-Otud7b antibody. Nuclei were stained with DAPI. Otud7b was localized to the photoreceptor inner segment, cell body, and synapse. (C) Immunofluorescence analysis of the wild-type retina using the anti-Otud7b antibody and PNA (a marker for cone outer segments and synapses). Nuclei were stained with DAPI. Otud7b signals were observed in the vicinity of PNA signals in the OPL. (D) RT-PCR analysis of the OTUD7B transcript in the human retina. β-actin was used as a loading control. (E) Immunofluorescence analysis of retinal sections from Otud7b +/+ and Otud7b −/− mice at 1 M using antibodies against Otud7b, Rhodopsin (a marker for rod outer segments), S-opsin (a marker for S-cone outer segments), and M-opsin (a marker for M-cone outer segments). Nuclei were stained with DAPI. No obvious difference was observed comparing the photoreceptor cells of Otud7b +/+ and Otud7b −/− retinas. (F and G) ERG analysis of Otud7b +/+ and Otud7b −/− mice at 1 M. (F) Representative scotopic ERGs elicited by four different stimulus intensities (−4.0 to 1.0 log cd s/m 2 ). (G) Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m 2 ). (H) Immunofluorescence analysis of retinal sections from Otud7b +/+ and Otud7b −/− mice at 6 M using antibodies against Rhodopsin (a marker for rod outer segments), S-opsin (a marker for S-cone outer segments), and M-opsin (a marker for M-cone outer segments). Nuclei were stained with DAPI. No obvious difference was observed between Otud7b +/+ and Otud7b −/− retinas. (I and J) ERG analysis of Otud7b +/+ and Otud7b −/− mice at 6 M. (I) Representative scotopic ERGs elicited by four stimulus intensities (−4.0 to 1.0 log cd s/m 2 ). (J) Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m 2 ). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Staining, Marker

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: Otud7b deficiency exacerbates light-induced retinal damage to cone photoreceptor cells (A) Schematic representation of the exposure of Otud7b +/+ and Otud7b −/− mice to LED light. (B) Immunofluorescence analysis of retinal sections from Otud7b +/+ and Otud7b −/− mice after light exposure using antibodies as follows: Otud7b, Rhodopsin (a marker for rod outer segments), S-opsin (a marker for S-cone outer segments), and M-opsin (a marker for M-cone outer segments). Nuclei were stained with DAPI. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (C–E) Rod outer segment (C), S-cone outer segment (D), and M-cone outer segment (E) lengths were measured. M-cone outer segments were shorter in the Otud7b −/− retina than in the Otud7b +/+ retina. ∗∗p < 0.01, n.s. not significant (unpaired t-test), n = 4 per genotype. (F–H) Scotopic ERG analysis of Otud7b +/+ and Otud7b −/− mice after light exposure. (F) Representative scotopic ERGs elicited by four different stimulus intensities (−4.0 to 1.0 log cd s/m 2 ). Scotopic a-wave (G) and b-wave (H) amplitudes are shown as functions of stimulus intensity. Data are presented as the mean ± SD. ∗p < 0.05 (unpaired t-test). n = 4 per genotype. (I–K) Photopic ERG analysis of Otud7b +/+ and Otud7b −/− mice after light exposure. (I) Representative photopic ERGs elicited by four different stimulus intensities (−0.5 to 1.0 log cd s/m 2 ). Photopic a-wave (J) and b-wave (K) amplitudes are shown as functions of stimulus intensity. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 (unpaired t-test), n = 4 per genotype.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Immunofluorescence, Marker, Staining

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: Otud7b deficiency exacerbates retinal degeneration in Mak −/− mice (A) Immunofluorescence analysis of retinal sections from Otud7b +/+ ; Mak −/− and Otud7b −/− ; Mak −/− at 2 M using antibodies against Rhodopsin (a marker for rod outer segments), S-opsin (a marker for S-cone outer segments), M-opsin (a marker for M-cone outer segments), and Gnat2 (a marker for cone outer segments). Nuclei were stained with DAPI. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (B) Cone outer segment length was measured in Otud7b +/+ ; Mak −/− and Otud7b −/− ; Mak −/− retinas at 2 M Otud7b −/− ; Mak −/− retinas had significantly shorter cone outer segments. Data are presented as the mean ± SD. ∗p < 0.05 (unpaired t-test), n = 3 per genotype. (C) The ONL thickness was measured. The ONL thickness in the Otud7b −/− ; Mak −/− retina was significantly thinner than that in the Otud7b +/+ ; Mak −/− retina. Data are presented as the mean ± SD. ∗∗p < 0.01 (unpaired t-test), n = 6 retinal sections from 3 mice per genotype. (D) The INL+IPL+GCL thickness was measured. Data are presented as the mean ± SD. n.s., not significant (unpaired t-test). n = 6 retinal sections from 3 mice per genotype. (E–G) Scotopic ERG analysis of Otud7b +/+ ; Mak −/− and Otud7b −/− ; Mak −/− mice at 2 M. (E) Representative scotopic ERGs elicited by four different stimulus intensities (−4.0 to 1.0 log cd s/m 2 ). Scotopic a-wave (F) and b-wave (G) amplitudes are shown as functions of stimulus intensity. Data are presented as the mean ± SD (unpaired t-test). n = 3 per genotype. (H–J) Photopic ERG analysis of Otud7b +/+ ; Mak −/− and Otud7b −/− ; Mak −/− mice at 2 M. (H) Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m 2 ). Photopic a-wave (I) and b-wave (J) amplitudes are shown as functions of stimulus intensity. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 (unpaired t-test). n = 3 per genotype.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Immunofluorescence, Marker, Staining

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: Knockdown of Otud7b in neuronal cells increases susceptibility to cell death under stress (A) Inhibition efficacy of shRNA expression constructs for Otud7b knockdown. ShRNA-control or Otud7b-shRNA1 expression plasmids were co-transfected with plasmids expressing an FLAG-tagged Otud7b and a GFP into HEK293T cells. Western blotting was performed using anti-FLAG and anti-GFP antibodies. GFP was used as an internal transfection control. Otud7b-shRNA1 effectively suppressed Otud7b expression. (B and C) ShRNA-control or Otud7b-shRNA1 expression plasmids were co-transfected with a plasmid expressing EGFP into Neuro2A cells. The cells were serum starved for 14 h. (B) Immunofluorescence analysis was performed using an anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. Increased number of cleaved caspase 3 and GFP double-positive cells was observed in the Otud7b-shRNA1 transfected cells. (C) The number of cleaved caspase 3 and GFP double-positive cells per GFP-positive cells was measured. Data are presented as the mean ± SD. ∗∗p < 0.01 (unpaired t-test). n = 3 experiments. (D) Inhibition efficacy of siRNAs for Otud7b knockdown. SiRNA-control, Otud7b-siRNA3, or Otud7b-siRNA4 was transfected into Neuro2A cells. Western blotting was performed using antibodies against Otud7b and α-tubulin. α-tubulin was used as a loading control. Otud7b-siRNA3 and Otud7b-siRNA4 effectively suppressed Otud7b expression. (E and F) Neuro2A cells were transfected with siRNA-control, Otud7b-siRNA3, or Otud7b-siRNA4. The cells were serum starved for 14 h. (E) Immunofluorescence analysis was performed with an anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. Increased number of cleaved caspase 3-positive cells was observed in the cells transfected with Otud7b-siRNA3 and Otud7b-siRNA4. (F) The number of cleaved caspase 3-positive cells was measured. Data are presented as the mean ± SD. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (one-way ANOVA followed by Tukey’s multiple comparisons test). n = 4 experiments.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Knockdown, Inhibition, shRNA, Expressing, Construct, Control, Transfection, Western Blot, Plasmid Preparation, Immunofluorescence, Staining

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: NF-κB is activated by Otud7b deficiency (A) IPA to predict the upstream transcription regulators affecting gene expression changes (fold change >1.5 or < −1.5; FPKM >0.2) in the Otud7b −/− retina after light exposure. The transcription regulators with activation Z scores >2 and p < 0.05 are shown. (B) The IPA networks showing the transcription factor RelA as an upstream regulator. RelA is predicted to be activated in Otud7b −/− retina after light exposure. IPA, ingenuity pathway analysis. (C) Immunofluorescence analysis of retinal sections from Otud7b +/+ and Otud7b −/− mice after light exposure using an anti-RelA antibody. Nuclei were stained with DAPI. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (D) RelA signal intensity in the ONL of Otud7b +/+ and Otud7b −/− retinas after light exposure was measured. RelA signal intensity in the ONL increased in the Otud7b −/− retina. Data are presented as the mean ± SD. ∗p < 0.05 (unpaired t-test). n = 3 experiments. (E) Schematic diagram of the NF-κB response element and minimal promoter. (F) ShRNA-control or Otud7b-shRNA1 expression plasmids were co-transfected into Neuro2A cells with a NanoLuc luciferase reporter construct driven by an NF-κB response element and minimal promoter as well as a Firefly luciferase -expressing construct driven by a minimal promoter. Luciferase activity of the cell lysates was measured 14 h after serum starvation. NanoLuc luciferase activity was normalized to Firefly luciferase activity. Data are presented as the mean ± SD. ∗p < 0.05 (unpaired t-test). n = 3 experiments.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Gene Expression, Activation Assay, Immunofluorescence, Staining, shRNA, Control, Expressing, Transfection, Luciferase, Construct, Activity Assay

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: NF-κB pathway inhibition reduces cell death induced by Otud7b knockdown (A) Neuro2A cells treated with curcumin were transfected with siRNA-control, Otud7b-siRNA3, or Otud7b-siRNA4. The cells were serum starved for 24 h in a medium containing curcumin. Cells were immunostained with an anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. (B) The number of cleaved caspase 3-positive cells was counted. Curcumin treatment suppressed the increase of cleaved caspase 3-positive cells caused by Otud7b knockdown. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test). n = 4 experiments. (C) Immunofluorescence analysis of serum starved Neuro2A cells using an anti-RelA antibody. Curcumin suppressed the nuclear localization of RelA. Nuclei were stained with DAPI. (D) Neuro2A cells treated with BMS-345541 were transfected with siRNA-control or Otud7b-siRNA3. The cells were serum starved for 24 h in the medium containing BMS-345541. Cells were immunostained with an anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. (E) The number of cleaved caspase 3-positive cells was counted. BMS-345541 treatment suppressed the increase of cleaved caspase 3-positive cells caused by Otud7b knockdown. Data are presented as the mean ± SD. ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test). n = 3 experiments. (F) Immunofluorescence analysis of serum starved Neuro2A cells using an anti-RelA antibody. BMS-345541 suppressed the nuclear localization of RelA. Nuclei were stained with DAPI.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Inhibition, Knockdown, Transfection, Control, Staining, Immunofluorescence

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: NF-κB pathway inhibition suppresses light-induced retinal damage in Otud7b −/− mice (A) Schematic representation of LED light exposure to mice treated with curcumin. Curcumin was injected into mice every day for 7 days. (B–D) Immunofluorescence analysis of retinal sections from curcumin-treated Otud7b +/+ and Otud7b −/− mice after light exposure using the following antibodies: Rhodopsin (a marker for rod outer segments, B), S-opsin (a marker for S-cone outer segments, C), and M-opsin (a marker for M-cone outer segments, D). Nuclei were stained with DAPI. (E–G) Rod outer segment (E), S-cone outer segment (F), and M-cone outer segment (G) lengths measured in curcumin-treated Otud7b +/+ and Otud7b −/− mice after light exposure. The M-cone outer segments were longer in curcumin-treated Otud7b −/− retina compared to DMSO-treated Otud7b −/− retina. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test), n = 4 per genotype. (H) Immunofluorescence analysis of retinal sections from curcumin-treated Otud7b +/+ and Otud7b −/− mice after light exposure using an anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. White arrows indicate cleaved caspase 3-positive cells. (I) The number of cleaved caspase 3-positive cells was counted. Curcumin treatment significantly reduced the number of cleaved caspase 3-positive cells in the Otud7b −/− retina. ∗p < 0.05, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test), n = 4 per genotype. (J) Immunofluorescence analysis of retinal sections from DMSO or curcumin-treated Otud7b −/− mice after light exposure using an anti-RelA antibody. Nuclei were stained with DAPI. (K) RelA intensity in the ONL of DMSO or curcumin-treated Otud7b −/− mice after light exposure was measured. RelA signal intensity in the ONL decreased in the curcumin-treated Otud7b −/− retina. Data are presented as the mean ± SD. ∗p < 0.05 (unpaired t-test). n = 4 experiments. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Inhibition, Injection, Immunofluorescence, Marker, Staining

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet: NF-κB pathway inhibition by BMS-345541 suppresses light-induced retinal damage in Otud7b −/− mice (A) Schematic representation of LED light exposure to mice treated with BMS-345541. BMS-345541 was injected into mice every day for 7 days. (B–D) Immunofluorescence analysis of retinal sections from BMS-345541-treated Otud7b +/+ and Otud7b −/− mice after light exposure using the following antibodies: Rhodopsin (a marker for rod outer segments, B), S-opsin (a marker for S-cone outer segments, C), and M-opsin (a marker for M-cone outer segments, D). Nuclei were stained with DAPI. (E–G) Rod outer segment (E), S-cone outer segment (F), and M-cone outer segment (G) lengths measured in BMS-345541-treated Otud7b +/+ and Otud7b −/− mice after light exposure. The M-cone outer segments were longer in BMS-345541-treated Otud7b −/− retina compared to DMSO-treated Otud7b −/− retina. ∗p < 0.05, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test), n = 3 per genotype. (H) Immunofluorescence analysis of retinal sections from BMS-345541-treated Otud7b +/+ and Otud7b −/− mice after light exposure using anti-cleaved caspase 3 antibody. Nuclei were stained with DAPI. White arrows indicate cleaved caspase 3-positive cells. (I) The number of cleaved caspase 3-positive cells was counted. BMS-345541 treatment significantly reduced the number of cleaved caspase 3-positive cells in the Otud7b −/− retina. ∗∗∗∗p < 0.0001, n.s. not significant (two-way ANOVA followed by Tukey’s multiple comparisons test), n = 3 per genotype. (J) Immunofluorescence analysis of retinal sections from DMSO or BMS-345541-treated Otud7b −/− mice after light exposure using an anti-RelA antibody. Nuclei were stained with DAPI. (K) RelA intensity in the ONL of DMSO or BMS-345541-treated Otud7b −/− mice after light exposure was measured. RelA intensity decreased in the BMS-345541-treated Data are presented as the mean ± SD. ∗∗p < 0.01 (unpaired t-test). n = 3 experiments. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Inhibition, Injection, Immunofluorescence, Marker, Staining

Journal: iScience

Article Title: The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases

doi: 10.1016/j.isci.2024.109380

Figure Lengend Snippet:

Article Snippet: After washing with PBS, the samples were incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1,000, Nacalai Tesque).

Techniques: Plasmid Preparation, Recombinant, Imaging, Reporter Assay, Sequencing, shRNA, Software