Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Numbers of cFos+ cells in the DMH during SD, RS and sleeping-control (SD-Cont, RS-Cont) detected by cFos immunohistochemistry. The total number of cFos+ cells in the DMH was counted at bregma -1.67 mm to, -1.79 mm and -1.91 mm and summed up (total three sections) each mouse (n=3). The third ventricle (3V) is shown. Values are shown as means ± S.E., *p<0.05, ***p<0.001, and non-significant (ns) by one-way ANOVA with Bonferroni’s post hoc test. b , c , Representative images of DMH sections at bregma -1.67 mm from mice under SD-Cont (left) and SD (right) with cFos. Boxed areas were shown at high magnification in c. d , Images of the ZsGreen signal including the DMH, amygdala (Amg) and tuberal nucleus (TN) at bregma -1.54, -1.79, -1.91 and -2.13 mm of Prdm13 -ZsGreen mice. e , Ratios of cFos + cells within Prdm13 + cells in young mice during SD and SD-Cont detected by RNAscope in situ hybridization (n=7-8). Values are shown as means ± S.E., *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni’s post hoc test. f , g , Representative images of DMH sections from young mice under SD-Cont (left) and SD (right) with Prdm13 (yellow) and cFos (red) visualized by RNAscope. Boxed areas were shown at high magnification in g . Cells were counterstained with DAPI (blue). Scale bars indicate 100 and 10 μm ( f and g , respectively).

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Control, Immunohistochemistry, RNAscope, In Situ Hybridization

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Breeding strategy to generate DMH-specific Prdm13 -knockout (Prdm13-KO) mice. After crossing Prdm13 fl/f ;Rosa26R ZsGreen/ZsGreen mice and Nkx2-1 CreERT2/+ ;Prdm13 fl/fl mice, Prdm13 fl/fl ;Nkx2-1 CreERT2/+ ;Rosa26R ZsGreen/+ mice were used as Prdm13-KO mice and Prdm13 fl/fl ;Nkx2-1 +/+ ;Rosa26R ZsGreen/+ mice were used as control (Cont) mice. b , Expression of Prdm13 in the DMH, tuberal nucleus (TN) and amygdala (Amg) of Prdm13-KO and Cont mice (n=5). Values are shown as means ± S.E., **p<0.01, ***p<0.001 and non-significant (ns) by two-way ANOVA with Bonferroni’s post hoc test. c , d , Number of episodes ( c ) or duration ( d ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) every 3 hours through a day (left) and during the light (L) and dark (D) periods (right) in Prdm13-KO and Cont mice. Shading indicates dark period (n=6). Values are shown as means ± S.E., #p<0.05 by repeated measures ANOVA, listed p-values and *p<0.05 by repeated measures ANOVA with Bonferroni’s post hoc test (left) or unpaired t-test (right). e , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-KO and Cont mice (n=13-14). Values are shown as means ± S.E., *p<0.05, ***p<0.001 by repeated measures ANOVA with Bonferroni’s post hoc test. f , SWA during NREM sleep after SD. Normalized power is relative to the average of the 24-hour baseline day each group (n=6). Values are shown as means ± S.E. g , Total amount of wakefulness, NREM sleep and REM sleep during a 24-hour period (24h total), 12-hour light period (12h light) or 12-hour dark period (12h dark) (n=6). Values are shown as means ± S.E. h , EEG spectra of wakefulness (left), NREM sleep (middle) and REM sleep (right) during the light period (n=5-6). Values are shown as means ± S.E.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Knock-Out, Control, Expressing

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , b , Numbers of episodes ( a ) and duration ( b ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) every 3 hours through a day (left) and during the light (L) and dark (D) periods (right) in old DMH-specific Prdm13 -knockout (Prdm13-KO) and control (Cont) mice (n=5-6). Values are shown as means ± S.E., #p<0.05 and ##p<0.01 by repeated measures ANOVA, *p<0.05 and **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test (left) or unpaired t-test (right). c , EEG spectra of wakefulness (left), NREM sleep (middle) and REM sleep (right) during the light period (n=4-6). Values are shown as means ± S.E. d , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in old Prdm13-KO and Cont mice (n=5-6). Values are shown as means ± S.E., **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test. e , SWA after SD of Prdm13-KO and Cont mice at 20 months of age. Normalized power is relative to the average of the 24-hour baseline day (n=5-6). Values are shown as means ± S.E., **p<0.01 by Bonferroni’s post hoc test. f , Body weight of old Prdm13-KO and Cont mice (n=5-7). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. g , The level of wheel-running activity in old Prdm13-KO and Cont mice for six consecutive days (n=5-7). Values are shown as means ± S.E., #p<0.05 by repeated measures ANOVA. h , Kaplan-Meier curves of Prdm13-KO and Cont mice (n=13-18). Listed p-value was calculated by log-rank test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Knock-Out, Control, Activity Assay

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , DR paradigm in C57BL/6J at 20 months of age. Mice at 20-months-old were fed under 60% diet or AL-diet for 14 to 28 days. b , c , Number of episodes ( b ) and duration ( c ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) during the light (L) and dark (D) periods in AL and DR mice at 20 months of age (n=5). Values are shown as means ± S.E., listed p-value, *p<0.05 and **p<0.01 by unpaired t-test. d , EEG spectra of wakefulness (upper left), NREM sleep (upper right) and REM sleep (lower) during the light period (n=5). Values are shown as means ± S.E. e , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in AL and DR mice at 20 months of age (n=5-6). Values are shown as means ± S.E., **p<0.01 by repeated measures ANOVA with Bonferroni’s post hoc test. f , SWA after SD of AL and DR mice at 20 months of age. Normalized power is relative to the average of the 24-hour baseline day (n=5). Values are shown as means ± S.E., *p<0.05 by Bonferroni’s post hoc test. g , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-KO-AL and Prdm13-KO-DR mice (n=8). Values are shown as means ± S.E. h , Expression of Prdm13 in the hypothalamus of Prdm13 -overexpressing (Prdm13-OE) and control (Cont) mice (n=4-5). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. i , j , Number of episodes ( i ) and duration ( j ) of wakefulness (top), NREM sleep (middle) and REM sleep (bottom) during the light (L) and dark (D) periods in Prdm13-OE and Cont mice (n=5). Values are shown as means ± S.E., *p<0.05 by unpaired t-test. k , Number of sleep attempts during SD from 6am to 8am (6-8), 8am to 9am (8-9), 9am to 10am (9-10), 10am to 11am (10-11) and 11am to 12pm (11-12) in Prdm13-OE and Cont mice (n=4). Values are shown as means ± S.E., *p<0.05 by repeated measures ANOVA with Bonferroni’s post hoc test. l , SWA after SD of Prdm13-OE and Cont mice. Normalized power is relative to the average of the 24-hour baseline day (n=4). Values are shown as means ± S.E.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Western blot of Prdm13 in DMH collected by laser microdissection from DMH-specific Prdm13-KO and Cont mice (n=4 mice/lane). The arrow indicates the band for Prdm13; asterisks (*) indicate non-specific bands. b , Schematic of fractionation protocol from mouse hypothalami (left). Western blot of Prdm13 in hypothalamic fractions of C57BL/6J mice (right). Hypothalami from two C57BL/6J female mice were combined for each lane, and 8% equivalent of each fraction was run on the gel. Cytoplasmic supernatant (S1), RNase-extractable supernatant (S2), DNase-extractable supernatant (S3), and insoluble pellet (P) were run each lane. The arrow indicates the band for Prdm13; asterisks (*) indicate non-specific bands. c , Western blot of Prdm13 in hypothalamic fractions of Prdm13 -PA-Tag (KI) and wild-type (WT) mice. Cytosolic and nuclear fractions were run each lane as indicated. d , Expression of Cck, Grp and Pmch mRNA in the DMH of DMH- Prdm13 -KO (Prdm13-KO) and control (Cont) mice (n=3-5). Values are shown as means ± S.E., listed p-value, **p<0.01 and ***p<0.001 by unpaired t-test. e , Transcriptional activity of Prdm13-202 and Prdm13-mutants for the luciferase reporter vector containing the promoter region of Cck, Grp and Pmch . Schematic representation of Prdm13-202 and Prdm13-mutants are shown above. NIH3T3 cells were co-transfected with 250 ng of luciferase reporter plasmid and plasmid expressing Prdm13-202 (Prdm13), Prdm13-Zif mutant (mutZif) or Prdm13-deltaPR mutant (deltaPR). Obtained luminescence was normalized to total protein concentration (n=3, four individual experiments). Values are shown as means ± S.E., listed p-value, *p<0.05, **p<0.01 and ***p<0.001 by one-way ANOVA with Bonferroni’s post hoc test, # p<0.05 and ## p<0.01 and ### p<0.001 by unpaired t-test. f , Transcriptional activity of hypothalamic Prdm13 (htPrdm13) for the luciferase reporter plasmid containing the promoter region of Cck . NIH3T3 cells were co-transfected with 250 ng of reporter plasmid and 10, 50 or 250 ng of htPrdm13 -expressing plasmid. Obtained luminescence was normalized to total protein concentrations (n=3, three individual experiments). Values are shown as means ± S.E., *p<0.05 by one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: Western Blot, Laser Capture Microdissection, Fractionation, Expressing, Control, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Mutagenesis, Protein Concentration

Journal: bioRxiv

Article Title: Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

doi: 10.1101/2022.09.26.509442

Figure Lengend Snippet: a , Representative images of the DMH with Prdm13 (yellow) and one of the two genes, Cck or Grp (green) visualized by RNAscope. Cells were counterstained with DAPI (blue). White boxes show the DMH, which is divided into medial and lateral areas by dashed lines. White arrows show yellow+green+ cells. Scale bar indicates 100 μm. b , Ratios of Cck + or Grp + cells within Prdm13 + cells in medial, lateral or total (medial and lateral) DMH (n=3-5). Values are shown as means ± S.E., ***p<0.001 by unpaired t-test. c , Distribution of cFos + Prdm13 + cells (n=5). d , Representative images of the DMH from young mice under SD-Cont and SD with Prdm13 (yellow), Cck (green) and cFos (red) visualized by RNAscope. Cells were counterstained with DAPI (blue). White arrows show Prdm13+Cck+cFos+ cells. Scale bar indicates 100 μm. e , f , Ratios of cFos + cells within Prdm13 + Cck -(left) or Prdm13 + Cck + (right) cells in young ( e ) or old ( f ) mice during SD-Cont and SD (n=7-8). Values are shown as means ± S.E., *p<0.05, ***p<0.001 by two-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The 8% equivalent of each fraction by volume was run on a 4-15% TGX gel (Bio-Rad) for Western blotting using affinity-purified polyclonal rabbit anti-mouse Prdm13.

Techniques: RNAscope

Journal: Developmental biology

Article Title: Prdm13 is required for Ebf3+ amacrine cell formation in the retina

doi: 10.1016/j.ydbio.2017.12.003

Figure Lengend Snippet: Prdm13 marks a subset of amacrines in the adult retina. (A–D‴) P30 Prdm13-GFP/+ heterozygous animals co-stained with GFP (green) and amacrine cell markers (grey/red). (A–A‴) A small subset of Prdm13-GFP+ amacrine cells co-express AP2α (grey, arrows) and Bhlhb5 (red, arrowheads). (B–B‴) Nearly half of GFP+ cells co-express calretinin (grey, arrows), while less than 1% of GFP+ cells co-express calbindin (red). No intensely calretinin+ horizontal cells co-express GFP. (C–C‴) A large fraction of GFP+ cells co-express the glycinergic amacrine marker GlyT (grey, arrows), but few GFP+ cells co-express the GABAergic marker GAD65/67 and these were sometimes GlyT+ as well. (D–D‴) The majority of GFP+ cells co-express Ebf3 (grey, arrows), but all Ebf3+ cells in the INL are GFP+. A minority of GFP+ cells co-express Bhlhb5 (red, arrowheads) and cells that co-express Bhlhb5, Ebf3, and GFP are rarely seen. (E) Plot of the percentage of GFP+ cells that co-express a given marker. GFP does not overlap with Brn3, TH, or vGlut3 (not shown). (F) Plot showing the percentage of Ebf3+ amacrines, Pax6+ INL nuclei, and AP2α+ INL cells that co-express Prdm13-GFP. Sample size was 3 mice per condition. Statistical significance determined by unpaired two-sample t tests: * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent standard deviation. Scale bar 50μm. Inset scale bar 10μm. INL, inner nuclear layer.

Article Snippet: Millipore); rabbit anti-ChAT (1:400; AB143, Millipore); sheep anti-Chx10 (1:400; X1179P, Exalpha Biologicals Inc, Shirley, MA, USA); mouse anti-Cre (1:250; MAB3120, Millipore); mouse anti-Ebf3 (1:400; H00253738-M05, Abnova, Taipei, Taiwan); rabbit anti-Gad65/67 (1:400; AB1511, Millipore); mouse anti-GFP (1:1000; ab13970, Abcam Inc, Cambridge, MA, USA); goat anti-GlyT (1:500; AB1770, Millipore); mouse anti-Lhx1 (1:400; 4F2-c, DSHB); goat anti-Otx2 (1:250; AF1979, Bio-Techne Corporation, Minneapolis, MN, USA); rabbit anti-Pax6 (1:500; PRB-278P, Covance, Princeton, NJ, USA); guinea pig anti-Ptf1a ( Hori et al., 2008 ); rabbit anti-Prdm13 (see above) (1:250); rabbit anti-recoverin (1:500; AB5585, Abcam Inc); mouse anti-RFP (1:1000; ab65856, Abcam Inc); goat anti-Sox2 (1:500; sc-17320, Santa Cruz Biotechnology Inc); rabbit anti-tyrosine hydroxylase (TH) (1:100, ab152, Milipore); rabbit anti-vGlut3 (1:100; Cat# 135 203, Synaptic Systems, Göttingen, Germany).

Techniques: Staining, Marker, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of PRDM13 inhibits glioma cells via Rho and GTP enzyme activation protein

doi: 10.3892/ijmm.2018.3679

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative polymerase chain reaction analysis.

Article Snippet: The antibodies used were as follows: PRDM13 (1:1,000; cat. no. ab40542), Flag (1:1,000; cat. no. ab192404), and deleted in liver cancer 1 (DLC1; 1:1,000; cat. no. ab1220) from Abcam (Cambridge, MA, USA), Rho GTPase activating protein 30 (ARHGAP30; 1:500; cat. no. AP4996b; Abgent Biotech Co., Ltd., Wuhan, China), A disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS12; 1:500; cat. no. 24934-1-AP; ProteinTech Group, Inc., Wuhan, China), inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1; 1:500; cat. no. bsm-7899R; BIOSS, Beijing, China), and GAPDH (1:6,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.).

Techniques: Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of PRDM13 inhibits glioma cells via Rho and GTP enzyme activation protein

doi: 10.3892/ijmm.2018.3679

Figure Lengend Snippet: Proliferation and migration-associated genes involved in U87 cells infected with Lenti-PRDM13 and Lenti-Con. (A) Total RNA was extracted and reverse transcription-quantitative polymerase chain reaction analysis was performed to quantify the expression of different proliferation- and migration-associated genes at the mRNA level. The mRNA level was normalized to that of GAPDH. * P<0.05 (B) Protein expression levels of DLC1, ARHGAP30, INCA1 and ADAMTS12 were analyzed by western blot analysis in U87 cells. GAPDH was used as an internal control. (C) Chromatin immuno- precipitation assay in U87 cells exhibited a direct association between PRDM13 and the DLC1 promoter. (D) Diagram summarizing the conclusion that PRDM13 may upregulate DLC1 and ARHGAP30, which suppress U87 cell migration and upregulate INCA1 and ADAMTS12, causing a decrease in U87 cell proliferation. PRDM13, PRDI-BFI and RIZ homology domain containing 13; DLC1, deleted in liver cancer 1; ARHGAP30, Rho GTPase activating protein 30; ADAMTS12, A disintegrin and metalloproteinase with thrombospondin motifs 12; INCA1, inhibitor of cyclin-dependent kinase interacting with cyclin A1; NC, negative control.

Article Snippet: The antibodies used were as follows: PRDM13 (1:1,000; cat. no. ab40542), Flag (1:1,000; cat. no. ab192404), and deleted in liver cancer 1 (DLC1; 1:1,000; cat. no. ab1220) from Abcam (Cambridge, MA, USA), Rho GTPase activating protein 30 (ARHGAP30; 1:500; cat. no. AP4996b; Abgent Biotech Co., Ltd., Wuhan, China), A disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS12; 1:500; cat. no. 24934-1-AP; ProteinTech Group, Inc., Wuhan, China), inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1; 1:500; cat. no. bsm-7899R; BIOSS, Beijing, China), and GAPDH (1:6,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.).

Techniques: Migration, Infection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Chromatin Immunoprecipitation, Negative Control