Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 1. Curcumin activates Nrf2 signaling. (A) Mouse cortical cells were treated with either DMSO or 10 μM curcumin for 12 h. The mRNA levels of Nrf2-response genes were analyzed by qRT-PCR as described in the Methods. The bar graph shows the relative mRNA level of genes in the curcumin-treated group compared to the DMSO group. (B) The protein levels of Nrf2-response genes in the cells were analyzed by immunoblotting using anti-Nrf2, HO-1, NDP52, GST-mu1, NQO1, p62, and actin antibodies, respectively. Full blots are provided in Supplementary Fig. S11. (C) Mouse cortical cells were treated with either DMSO (Veh) or 10 μM curcumin (CCM) for 6 h. The cells were fixed with 4% paraformaldehyde and immunostained using anti-Nrf2 antibody. Fluorescence signals were observed using a confocal laser scanning microscope. (D) Mouse cortical cells were transiently transfected with the ARE-Luc reporter and TK-Renilla plasmids. After treatment with either DMSO (Veh) or 10 μM curcumin (CCM) for 12 h, the cells were assayed for the luciferase activity. Data shown are mean ± S.E. of three independent experiments and were analyzed using the Student’s t-test. (*p < 0.05, ***p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Quantitative RT-PCR, Western Blot, Fluorescence, Laser-Scanning Microscopy, Transfection, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 3. Curcumin-mediated Nrf2 activation is dependent on p62. (A) Mouse cortical cells were treated with DMSO (0 h) or 10 μM curcumin (CCM) for the indicated times. The levels of phosphorylated p62 (S351), p62, Keap1, and actin proteins were analyzed by immunoblotting using anti-phospho p62 (S349), p62, Keap1, and actin antibodies, respectively. (B) MEFs were treated with DMSO (Veh) or 10 μM curcumin (CCM) for 12 h. The protein levels of Nrf2, NQO1, p62, and actin were analyzed by immunoblotting using anti-Nrf2, NQO1, p62, and actin antibodies, respectively. Full blots are provided in Supplementary Fig. S11.

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 4. Curcumin-mediated Nrf2 activation is dependent on the phosphorylation of p62 on S351. (A) HEK293 cells were transiently transfected with the Myc-Nrf2 expression plasmid, and then treated with DMSO (−) or 10 μM curcumin (+) for 12 h. The cell lysates were used for Nrf2 immunoprecipitation using an anti- Myc antibody. The protein level of Keap1 co-immunoprecipitated with Nrf2 was examined by immunoblotting using an anti-Keap1 antibody. Full blots are provided in Supplementary Fig. S11. (B) The bar graph shows the relative ratio of Keap1 against Nrf2 co-immunoprecipitated with the Myc antibody in cells treated with curcumin (CCM) or not. (C) HEK293 cells were transiently co-transfected with the ARE-Luc reporter and TK-Renilla plasmids along with the Myc-p62 wild-type or mutant (S349A) plasmid. After treatment with either DMSO (−) or 10 μM curcumin (+) for 12 h, the cells were assayed for the luciferase activity. Data shown are mean ± S.E. of three independent experiments and were analyzed using the Student’s t-test. (*p < 0.05, ***p < 0.001).

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: Curcumin activates Nrf2 through PKCδ-mediated p62 phosphorylation at Ser351.

doi: 10.1038/s41598-021-87225-8

Figure Lengend Snippet: Figure 8. Schematic diagram showing the mechanism of Nrf2 activation by curcumin. Curcumin induces the phosphorylation of p62 at S351 by PKCδ activation. Phosphorylated p62 interferes the association of Nrf2 with Keap1, stabilizing Nrf2. Accumulated Nrf2 moves into nucleus and induces the expression of its downstream genes such as GST-mu1, NQO1, HO1, and p62 etc. Increased p62 takes part in the stabilization of Nrf2 again, thus forming a positive-feedback loop.

Article Snippet: Anti-Nrf2 rat monoclonal antibody (14596) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 6. Rbm24 directly regulates Notch1 mRNA stability in the adult NSPCs. (A-B) Representative images of GFAP/Rbm24/Notch1 (A), DCX/Rbm24/Notch1 (B) co-staining in the SVZ from 2-month-old CTL mice. Scale bar, 20 μm, zoom scale bar, 10 μm. (C-D) Immunoblot (C) and quantification (D) of Notch1 protein in the SVZ from 2-month-old CTL and UKO mice (n = 6 mice for each group). (E) Quantification of Notch1 mRNA in the neurospheres from 2-month-old CTL and UKO mice (n = 3 mice for each group). (F) RT-PCR analysis of Notch1 mRNA in input and Rbm24-antibody IP in the SVZ from 2-month-old CTL mice. (G) Quantification of Notch1 mRNA in adult NSPCs from 2-month-old CTL and UKO mice with actinomycin D administration (n = 3 mice for each group). (H) Quantification of luciferase activities for 3’-UTR fragments of Notch1 in 293T cells between pcRbm24 and pcDNA3.1 condition (n = 4 for each group). (I) Schematic diagram showing the effects of Rbm24 deficiency on Notch1. Data are presented as mean ± SEM. *p < 0.05; ***p < 0.001; n.s., not significant. LV: lateral ventricle; NSPCs: neural stem/progenitor cells.

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Luciferase

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 7. Rbm24 overexpression ameliorates adult neurogenesis in the SVZ of PD mice. (A-C) Immunoblot (A) and quantification of Rbm24 (B) and Notch1 (C) protein in the SVZ from 7-month-old WT and PD mice (n = 6 mice for each group). (D) Schematic diagram showing the lentivirus injection into the lateral ventricle. (E) Timeline of BrdU (short-term) and lentivirus injection in 7-month-old WT and PD mice. (F-G) Immunoblot (F) and quantification (G) of Rbm24 and Notch1 protein in the SVZ from 7-month-old OEC-PD and OER-PD mice (n = 6 mice for each group). (H) Quantification of Rbm24, Notch1, Heyl mRNA in the SVZ from 7-month-old OEC-PD and OER-PD mice (n = 6 mice for each group). (I-K) Representative images (I) and quantification of GFAP+/BrdU+ (J), GFAP+ (K) cells in the SVZ from 7-month-old WT, PD, OEC-PD, and OER-PD mice (n = 4 mice for each group). Scale bar, 50 μm. (L-N) Representative images (L) and quantification of DCX+/BrdU+ (M), DCX+ (N) cells in the SVZ from 7-month-old WT, PD, OEC-PD, and OER-PD mice (n = 4 mice for each group). Scale bar, 50 μm. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. LV: lateral ventricle.

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Over Expression, Western Blot, Injection

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 9. Notch1 inhibition blocks the effects of Rbm24 overexpression on SVZ-OB pathway neurogenesis and olfactory performance in PD mice. (A) Timeline of DAPT, BrdU and lentivirus injection in 7-month-old PD mice. (B-C) Immunoblot (B) and quantification (C) of NICD protein levels in the SVZ from 7-month-old OER-PD mice treated with vehicle or DAPT (n = 6 mice for each group). (D-F) Representative images (D) and quantification of GFAP+/BrdU+ (E), GFAP+ (F) cells in the SVZ

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Inhibition, Over Expression, Injection, Western Blot

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A-D ) Wild-type, Bim −/− , and Bid −/− MEFs ( panel B ) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D ), Δψm (24 h, panel C ) and cell death measurements (24 h, panel A ). *p<0.05, significantly different from wild-type cells. ( E-H ) Human Jurkat T cells, stably depleted of BIM or BID by RNAi ( panel F; note: black triangles denote crop marks ), were exposed to heat shock (44°C for 1 h) in a humidified incubator (5% CO 2 –95% air). As before, the cells were then transferred to a 37°C incubator and later collected for MOMP, caspase-3 activation, PARP cleavage (24 h, panel H ), Δψm (24 h, panel G) and cell death measurements (24 h, panel E ). ( I ) Wild-type, Bim −/− , and Bid −/− MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Activation Assay, Stable Transfection, Staining

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: Wild-type and Bax −/− Bak −/− DKO cells were exposed to ( A-C ) heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air), or ( D and E ) UV irradiation (4 min) on a transilluminator. The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panels C and E ), Δψm (24 h, panel B ) and cell death measurements (24 h, panels A and D ). cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from wild-type cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Irradiation, Activation Assay

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A-C ) Vector control MEFs and those expressing a DN-caspase-9 (C287A) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel C ), Δψm (24 h, panel B ) and cell death measurements (24 h, panel A ). ( D ) Wild-type, Bim −/− , and DN-caspase-9 MEFs were exposed to heat shock as described above, but were left in culture afterwards for 72 h, after which the plates were stained with crystal violet. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet; *p<0.05, significantly different from vector control cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Plasmid Preparation, Expressing, Activation Assay, Staining

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: ( A ) Cartoon illustrating the interactions of BH3-only proteins with multidomain proapoptotic (BAX and BAK) and antiapoptotic (MCL-1, BCL-2, and BCL-X L ) BCL-2 family members. BH3-only proteins can function as BAX/BAK activators (BIM, tBID, PUMA), or as sensitizers (NOXA, BAD, BMF, BIK, and HRK) that displace activators from antiapoptotic family members. ( B-D ) Wild-type and Mcl-1 −/− MEFs ( panel B, inset ) were exposed to heat shock (44°C for 1–1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel D ), Δψm (24 h, panel C ) and cell death measurements (24 h, panel B ). *p<0.05, significantly different from wild-type cells. ( E and F ) Wild-type MEFs were preincubated with DMSO (control) or ABT-737 (250 nM) for 1 h and subsequently exposed to heat shock (44°C for 1.5 h) in a humidified incubator (5% CO 2 –95% air). The cells were then transferred to a 37°C incubator and later collected for MOMP (16 h), caspase-3 activation, PARP cleavage (24 h, panel F ) and cell death measurements (24 h, panel E ). *p<0.05, cells treated with ABT-737 were significantly different from those treated with DMSO. cPARP, cleaved PARP; cCasp3, cleaved/active caspase-3; S/N, supernatant; Pel, mitochondrial pellet.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

doi: 10.1371/journal.pone.0084388

Figure Lengend Snippet: According to the canonical pathway, heat shock induces formation of a RAIDD-caspase-2 complex that activates caspase-2. Following cleavage by caspase-2, truncated Bid (tBID) then stimulates BAX/BAK-dependent MOMP, cytochrome c release, and formation of the Apaf-1/caspase-9 apoptosome complex, which in turn activates the effector caspase-3. In our hands, however, loss of BID or inhibition of the apoptosome provides only modest short-term protection at lower exposures (1 h, 44°C), whereas loss of BIM profoundly inhibits cell death and facilitates long-term protection. In the context of heat shock, BIM induces MOMP and loss of Δψm in a BAX/BAK-dependent manner (and may be responsible for triggering caspase-3 activation in the absence of detectable mitochondrial injury). Thus, BIM appears to mediate an alternative (and perhaps dominant) pathway to heat shock-induced apoptosis.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): BAX (CS 2772); BAK (CS 3814); BID (CS 2002), cleaved caspase-3 (CS 9662); total caspase-3 (CS 8G10); hCaspase-9 (CS 9502); β-actin (CS 4970); cytochrome c (CS 11940); PARP (CS 9542); and cleaved PARP (CS 9544).

Techniques: Inhibition, Activation Assay

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 2. Expression of mouse CST gene in various tissues. (A) Northern blot of CST mRNAs. First, 20 mg total RNA from indicated organs was electrophoresed, blotted, and hybridized with a digoxigenin-labeled (±) strand RNA probe made from the full-length mouse CST cDNA. Lane 1, kidney; lane 2, brain; lane 3, testis; lane 4, heart; lane 5, skeletal muscle; lane 6, small intestine; lane 7, stomach; lane 8, liver; lane 9, lung; lane 10, spleen. (B) RT-PCR analysis of CST and actin mRNAs. Next, 1 mg total RNA from indicated organs was reverse-transcribed with a random primer. The resulting cDNAs were separately amplified by PCR using a set of CST primers, 50-GGGTTTCCTGAGATGAC-30 (nucleotides 1±17 in Fig. 1) and 50-TAGTGCGCGTTGTAGCT-30 (nucleotides 634±650) and actin primers, 50-TTACCAACTGGGACGACATG-30 (nucleotides 149±168 in [16]) and 50-AGGAGCCAGAGCAGTAATCT-30 (nucleotides 869±888). The PCR products were separately electrophoresed, blotted, and hybridized with CST and actin probes. The observed PCR products showed the predicted sizes of 650 (CST) and 740 bases (actin). Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4, kidney; 5, stomach; 6, small intestine; 7, lung; 8, liver; 9, spleen; 10, testis. (C) CST activity. Whole homogenates from indicated organs were assayed for CST activity and protein concentration as described in Materials and methods. 1, kidney; 2, brain; 3, testis; 4, heart; 5, skeletal muscle; 6, small intestine; 7, stomach; 8, liver; 9, lung; 10, spleen.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Activity Assay, Protein Concentration

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 3. Multiple forms of mouse CST cDNA with 50-UTR unique sequences. Seven types of unique DNA sequences on the 50 end of mouse CST cDNAs are represented by boldface letters. The 50-terminus of the homologous sequence is indicated by arrows. The translation-initiation codon ATG is boxed and indicated by arrowheads. In forms A, B2, and C, type a, b, and c sequences are underlined with continuous lines and type d sequence is underlined with broken lines.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Sequencing

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 5. RT-PCR analysis of tissue-specific use of alternative exon 1. (A) Position of each primer and probe. Boxes represent exons. Symbols for exons are the same as in Fig. 4. Black boxes are coding exons. Total RNAs from various tissues were reverse transcribed using GSP1 primer. After isolation of the first cDNA strand, PCR was performed using a combination of a common antisense-primer 2A, corresponding to exon 2, and seven sense-primers relating to the seven exons 1, aS, bS, cS, dS, eS, fS, or gS. The PCR products were subjected to Southern hybridization with a probe EX2, corresponding to exon 2. (B) RNA from indicated organs was examined. PCR was performed using sense-primer aS (lane a), bS (lane b), cS (lane c), dS (lane d), eS (lane e), fS (lane f ), or gS (lane g). Minor bands in lanes a and b are represented by asterisks in small intestine.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, Hybridization

Journal: JCI Insight

Article Title: Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

doi: 10.1172/jci.insight.87680

Figure Lengend Snippet: Immunofluorescence of (A) retinaldehyde dehydrogenase 2 (RALDH2), p75 neurotrophin receptor (p75NTR), and PDGFRβ; (B) β3-tubulin and RALDH2; (C and D) p75NTR; (E) p75NTR and CD21; (F) CD21 and CXCL13; and (G) CD3ε, CD21, and B220 in aged kidneys after ischemic reperfusion injury (IRI). Arrows indicate TLT localization. Arrowheads indicate the localization of the B cell area. Aged kidneys were analyzed 14 (B and C), 30 (A), and 45 (D–G) days after IRI. (H) Retinoic acid (RA) induces p75ntr mRNA expression in C3H10T1/2 mouse embryonic fibroblasts and FACS-sorted PDGFRβ+ cells (n = 3 per group). The data are presented as dot plots (mean ± SD). (I) Activation-induced cytidine deaminase (Aid) mRNA levels of kidneys 45 days after various ischemic time IRI in young and aged mice (n = 4 per group) and correlation with TLT size (n = 16, aged mice only). The expression levels were normalized to those of Gapdh and expressed relative to those of controls or young mouse kidney at day 0 (IRI). *P < 0.001 versus controls. A 2-tailed Student’s t test was used to analyze data from FACS-sorted PDGFRβ+ cells; 1-way ANOVA with Tukey’s post-hoc analysis was used for other experiments. Correlation was determined by Pearson’s correlation analysis. The box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. Scale bars: (A, C, and D) 100 μm, (E and G) 50 μm, (B and F) 10 μm.

Article Snippet: The following primary antibodies were used in the mouse immunohistochemistry and immunofluorescence experiments: anti-αSMA (catalog C6198; Sigma-Aldrich), -AID (catalog 4959; Cell Signaling), -GFP (catalog ab13970; Abcam), -Ki67 (catalog 14-5698; eBioscience), -CD45 (catalog 14-0451; eBioscience), –CD11c-PE (catalog 12-0114; eBioscience), –PDGFR-β (catalog 14-1402; eBioscience), –β3tubulin-FITC (catalog A488-435L; Covance), -Cre (catalog 69050-3; Novagen), –LYVE-1 (catalog ab14917; Abcam), -CD21 (catalog ab75985; Abcam), –ER-TR7 (catalog T-2109; BMA Biomedicals), -CXCL13 (catalog AF470; R&D Systems), -CCL19 (catalog AF880; R&D Systems), -CCL21 (catalog AF457; R&D Systems), - p75NTR (catalog AF1157; R&D Systems), -CD3ε (catalog 550275; BD PharMingen), -B220 (catalog 557390; BD PharMingen), –PD-1 (catalog 114102; BioLegend), -CD4 (catalog 100726; BioLegend), -CD8 (catalog 100425; BioLegend), -PNAd (catalog 120801; BioLegend), and -ALDH1A2 (catalog HPA010022; Sigma-Aldrich) antibodies.

Techniques: Immunofluorescence, Expressing, Activation Assay

Journal: JCI Insight

Article Title: Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

doi: 10.1172/jci.insight.87680

Figure Lengend Snippet: (A–L) Histological analyses of aged human kidney samples. (A and B) Periodic acid-Schiff (PAS) staining and immunofluorescence analysis of (C) CD20 and CD3ε; (E) Ki67 and CD3ε/CD20 (arrowheads indicate double-positive cells); (G) CXCL13 and CD21; (J) CXCL13 and CD45; (K) CD21, α-smooth muscle actin (αSMA), and CD45; and (L) p75 neurotrophin receptor (p75NTR) and CD21, and immunohistochemical analysis of (D) peripheral lymph node addressin (PNAd); (F) CD21 (arrowheads indicate the localization of CD21+ follicular dendritic cell [FDC] networks); (H) CD20, activation-induced cytidine deaminase (AID), CD21, and retinaldehyde dehydrogenase 2 (RALDH2) (serial sections); and (I) RALDH2 (arrowheads indicate the localization of B cell follicles). The outlined region in (H) is magnified in (I). Arrows indicate TLT localization. Scale bars: (A, D, H, K, and L) 50 μm, (B, C, and F) 100 μm, (E, G, I, and J) 10 μm. (M) Quantitative analysis of TLT frequencies in human samples (n = 56; young = 13, aged = 43). #P < 0.05 (Pearson’s χ2 test).

Article Snippet: The following primary antibodies were used in the mouse immunohistochemistry and immunofluorescence experiments: anti-αSMA (catalog C6198; Sigma-Aldrich), -AID (catalog 4959; Cell Signaling), -GFP (catalog ab13970; Abcam), -Ki67 (catalog 14-5698; eBioscience), -CD45 (catalog 14-0451; eBioscience), –CD11c-PE (catalog 12-0114; eBioscience), –PDGFR-β (catalog 14-1402; eBioscience), –β3tubulin-FITC (catalog A488-435L; Covance), -Cre (catalog 69050-3; Novagen), –LYVE-1 (catalog ab14917; Abcam), -CD21 (catalog ab75985; Abcam), –ER-TR7 (catalog T-2109; BMA Biomedicals), -CXCL13 (catalog AF470; R&D Systems), -CCL19 (catalog AF880; R&D Systems), -CCL21 (catalog AF457; R&D Systems), - p75NTR (catalog AF1157; R&D Systems), -CD3ε (catalog 550275; BD PharMingen), -B220 (catalog 557390; BD PharMingen), –PD-1 (catalog 114102; BioLegend), -CD4 (catalog 100726; BioLegend), -CD8 (catalog 100425; BioLegend), -PNAd (catalog 120801; BioLegend), and -ALDH1A2 (catalog HPA010022; Sigma-Aldrich) antibodies.

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Activation Assay

Journal: Cell reports

Article Title: PPARγ Interaction with UBR5/ATMIN Promotes DNA Repairto Maintain Endothelial Homeostasis

doi: 10.1016/j.celrep.2019.01.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat Anti-ORC2 Monoclonal Antibody, Unconjugated, Clone 3G6 , Cell Signaling Technology , Cat#4736, RRID:AB_2157716.

Techniques: Control, Recombinant, In Vitro, Transfection, Flow Cytometry, Single Cell Gel Electrophoresis, Mass Spectrometry, Sequencing, Real-time Polymerase Chain Reaction, Cloning, Plasmid Preparation, Software