Journal: Clinical Cancer Research

Article Title: Redirecting B7-H3.CAR T Cells to Chemokines Expressed in Osteosarcoma Enhances Homing and Antitumor Activity in Preclinical Models

doi: 10.1158/1078-0432.CCR-23-3298

Figure Lengend Snippet: RNA-seq and multiplex analyses identify chemokines produced by primary OS samples. A, Expression of 28 chemokines by RNA-seq (FPKM shown; n = 85 OS samples); screen cutoff shown at 1 FPKM. B, Expression of 28 chemokines by protein secretion ( n = 8 patient samples); screen cutoff shown at 3 × 10 3 integrated pixel density. Green bars in A and B denote ligands deemed positive in both RNA and protein screens; error bars indicate SEM. C, Chemokine receptors (ligands in parentheses) CXCR2 (IL8), CXCR3 (CXCL10), CXCR6 (CXCL16), CCR7 (CCL21), and CCR8 (CCL18) as expressed endogenously and detected by flow cytometry on activated T cells ( n = 5 healthy donors; error bars indicate SEM). D, ELISA demonstrating detection of IL8 and CXCL16 in patient transport media specimens ( n = 6 patient tumor samples, 1 normal bone sample; error bars indicate SEM).

Article Snippet: After 24 hours, the supernatant was harvested, and ELISA was performed using Duoset ELISA kits for IL8 (R&D Systems) or CXCL16 (R&D Systems) according to the manufacturer’s instructions and quantified using a TeCan plate reader (Tecan Group Ltd., Mannedorf, Switzerland) at 450 and 570 nm.

Techniques: RNA Sequencing Assay, Multiplex Assay, Produced, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: LINK‐A overexpression reduces adaptive thermogenesis in HFD‐fed mice by remodeling the regional inflammatory microenvironment a) Heatmap of inflammatory factor mRNA expression levels in the mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice detected by qRT‐PCR. per group n = 6. b–d) Representative images of H&E‐stained sections and IHC of mammary glands(b). The IL‐1β(c) or CXCL16(d) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001. e–g) Representative images of H&E‐stained sections and IHC of scWAT(e). The IL‐1β(f) or CXCL16(g) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. h–i) Mouse plasma concentrations of IL‐1β(h) and CXCL16(i) were measured by ELISA. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. j) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in BMSCs‐derived beige adipocytes treatment using IL‐β or/and CXCL16 cytokines by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. k) Schematic diagram of the effect of inflammatory factors IL‐1β and CXCL16 on adipose thermogenesis. l–m) Following one week of dietary intervention, IL‐1β (l) and Cxcl16 (m) mRNA expression levels of the mammary gland in WT or LINK‐A KI mice were detected by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001.

Article Snippet: The concentrations of IL‐1β and CXCL16 in mouse plasma were measured using the Mouse IL‐1β ELISA kit (absin, abs520001) and the Mouse CXCL16 ELISA kit (R&D Systems, DY503).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: LINK‐A overexpression induces inflammatory factor expression by stabilizing HIF1α through the HFD‐induced HB‐EGF a) Schematic diagram of HRE site at CXCL16 promoters(up) and HIF1α binds the CXCL16 promotor region by HIF1α ChIP‐seq database analysis(down). b) CXCL16 ChIP‐qPCR primer sequences(blue) and HRE site sequences (yellow). c) The interaction between HIF1α and CXCL16 promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, **** p <0.0001. d) IL‐1β ChIP‐qPCR primer sequences(blue) and HRE site sequences(yellow). e) The interaction between HIF1α and IL‐1β promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, ** p <0.01. f) Immunoblot analysis of HIF1α protein levels in the mammary gland of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. The HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. g) qRT‐PCR analysis of Hif1α mRNA in mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. h) The interaction between HIF1α and CXCL16 promoter (Pro) in MCF‐10A was verified by a Dual‐Luciferase reporter assay. The CXCL16 Pro mutant (mut) site contains HRE(b). Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ** p <0.01. i) The interaction between HIF1α and IL‐1β promoter in MCF‐10A was verified by a Dual‐Luciferase reporter assay. The IL‐1β Pro mut site contains HRE(d). Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, *** p <0.001. j) The mRNA levels of CXCL16 , IL‐1β in MCF‐10A empty vector (EV) or HIF1α overexpression (OV‐HIF1α) cells were detected by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, two‐way ANOVA, **** p <0.0001. k) The mRNA levels of CXCL16 , IL‐1β in MCF‐10A shControl (shCtrl) or shHIF1α (shHIF1α#1, shHIF1α#2) cells by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, two‐way ANOVA, ns = no significance, **** p <0.0001. l) qRT‐PCR analysis of Hb‐egf mRNA in mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001. m) The protein level of HIF1α in MCF‐10A EV or LINK‐A overexpression (OV‐ LINK‐A ) cells with or without HB‐EGF stimulation was detected by immunoblot analysis, and the HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. n) The mRNA level of HIF1α in MCF‐10A EV or LINK‐A overexpression cells with or without HB‐EGF stimulation was detected by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance. o,p) The concentrations of IL‐1β(o), and CXCL16(p) in the culture medium were measured by ELISA. Data presented as mean ± SEM, pooled data from six independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. q) Schematic diagram of LINK‐A promotes the expression of IL‐1β and CXCL16 by stabilizing their transcription factors HIF1α.

Article Snippet: The concentrations of IL‐1β and CXCL16 in mouse plasma were measured using the Mouse IL‐1β ELISA kit (absin, abs520001) and the Mouse CXCL16 ELISA kit (R&D Systems, DY503).

Techniques: Over Expression, Expressing, ChIP-sequencing, ChIP-qPCR, Western Blot, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: Activation of the LINK‐A /HB‐EGF/HIF1α pathway in mammary cells reduces adipocyte thermogenesis a) Schematic representation of bone marrow mesenchymal stem cells (BMSCs)‐derived beige adipocytes treated with MCF‐10A cells conditioned media: LINK‐A overexpressing MCF‐10A and control cells were stimulated with HB‐EGF, and the conditioned media was obtained to treat beige adipocytes. b) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in adipocytes treated with different conditioned media by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. c–f) The mRNA levels of thermogenic genes in adipocytes treated with different conditioned media by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. g) Schematic representation of BMSCs‐derived beige adipocytes treated with MCF‐10A cells conditioned media: IL‐1β inhibitor or/and CXCL16 inhibitors add to the conditioned media of HB‐EGF‐stimulated LINK‐A overexpression MCF‐10A cells, respectively, and the conditioned media was obtained to treat beige adipocytes. h) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in adipocytes treated with different conditioned media by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. i–l) The mRNA levels of thermogenic genes in adipocytes treated with different conditioned media by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, *** p <0.001, **** p <0.0001. m) Schematic diagram of LINK‐A affecting adipocyte thermogenesis.

Article Snippet: The concentrations of IL‐1β and CXCL16 in mouse plasma were measured using the Mouse IL‐1β ELISA kit (absin, abs520001) and the Mouse CXCL16 ELISA kit (R&D Systems, DY503).

Techniques: Activation Assay, Derivative Assay, Control, Western Blot, Quantitative RT-PCR, Over Expression

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: ASO drug inhibiting LINK‐A attenuates obesity and metabolic disorders in mice a) Schematic diagram of the mouse mammary gland in situ LINK‐A LNAs injection strategy. b) LINK‐A levels in mice mammary glands injected with LINK‐A LNAs in situ for two weeks by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. c) Body weight changes in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, ns = no significance, **** p <0.0001. d,e) GTT(d) and ITT(e) of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, * p <0.05, *** p <0.001. f) The HIF1α protein levels in the mammary gland of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were detected by immunoblot analysis, and the HIF1α protein levels were quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. g,h) The plasma concentrations of IL‐1β(g), and CXCL16(h) in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were measured by ELISA. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. i–k) Representative images of H&E‐stained sections and IHC of the mammary gland (nipple)(i), mammary gland (duct)(j), and scWAT(k), and the IL‐1β or CXCL16 protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. l) HFD‐KI treated with LINK‐A LNAs or Scr LNAs for two weeks were placed at 4 °C, 48 h, and their protein levels of UCP1, P‐PKA, PKA, P‐HSL, HSL in scWAT were detected by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. m) HFD‐KI treated with LINK‐A LNAs or Scr LNAs for two weeks were placed at 4 °C, 48 h, and their mRNA levels of thermogenic genes in scWAT were detected by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01.

Article Snippet: The concentrations of IL‐1β and CXCL16 in mouse plasma were measured using the Mouse IL‐1β ELISA kit (absin, abs520001) and the Mouse CXCL16 ELISA kit (R&D Systems, DY503).

Techniques: In Situ, Injection, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: The LINK‐A expression was positively correlated with the expression of inflammatory cytokines in overweight people a–e) Representative images of H&E‐stained sections and IHC of breast tissue microarrays(a) of patients with overweight, and the inflammatory factor levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, n = 12, chi‐square test, ns = no significance, **** p <0.0001. f–i) Correlation between IL‐1β , CXCL16 , LINK‐A and BMI. Data presented as mean ± SEM, n = 45, Pearson chi‐square test. * p <0.05, ** p <0.01, *** p <0.001. j–m) Correlation between BMI, IL‐1β , CXCL16 , LINK‐A , and HIF1α protein. Data presented as mean ± SEM, n = 24, Pearson chi‐square test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. n) Article General Mechanism Diagram.

Article Snippet: The concentrations of IL‐1β and CXCL16 in mouse plasma were measured using the Mouse IL‐1β ELISA kit (absin, abs520001) and the Mouse CXCL16 ELISA kit (R&D Systems, DY503).

Techniques: Expressing, Staining

Journal: Cell reports

Article Title: LHPP-mediated inorganic pyrophosphate hydrolysis-driven lysosomal acidification in astrocytes regulates adult neurogenesis.

doi: 10.1016/j.celrep.2023.112975

Figure Lengend Snippet: Figure 5. LHPP KO alleviates the stress-related decline in adult hippocampal neurogenesis (A) Experimental design used to study the proliferation of hippocampal neural stem cells in vivo. (B and C) Representative images of BrdU staining of the dorsal and ventral hippocampi of 12-week-old male WT and LHPP KO mice under stress-naive conditions or after exposure to CUS (left). Statistical analysis of BrdU-positive cells (n = 8–10; right). Scale bar: 100 mm. (D) Experimental design used to study the differentiation of hippocampal neural stem cells in vivo. (E and F) Representative images of BrdU and NeuN staining of the dorsal and ventral hippocampi of 12-week-old male WT and LHPP KO mice under stress-naive conditions or after exposure to CUS (left). Statistical analysis of BrdU+/NeuN+ cells (n = 8; right). Scale bar: 100 mm. (G) Representative images of C/EBPb staining of the hippocampi of 12-week-old male WT and LHPP KO mice under stress-naive conditions or after exposure to CUS (left). Quantification of the percentage of C/EBPb-stained area between the dentate gyrus and CA1 (n = 5; right). Scale bar: 100 mm. (H) The contents of CCL12, CXCL9, and CXCL16 in dentate gyrus lysates from 12-week-old male WT and LHPP KO mice exposed to CUS by ELISA (n = 6). The data are shown as the mean ± SD or individual points. Statistical analyses were performed by two-way ANOVA with Sidak’s post hoc multiple comparisons test (B, C, and E–G) or two-tailed Student’s t test (H). *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figures S9 and S10.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Hippocampi National human brain bank for development and function of PUMC http://anatomy.sbm.pumc.edu.cn/brainbank/ Chemicals, peptides, and recombinant proteins Inorganic pyrophosphatase (yeast) New England Biolabs #M2403S EZview Red ANTI-FLAG M2 Affinity Gel beads Sigma #F2426 Dextran, Fluorescein and Tetramethylrhodamine ThermoFisher #D1951 DAPI Sigma #D9542 CCL3 PeproTech #250-09 CCL5 PeproTech #250-07 CCL12 PeproTech #250-04 CXCL2 PeproTech #250-15 CXCL9 PeproTech #250-18 CXCL16 PeproTech #250-28 BrdU Abcam #ab142567 Plicamycin MedChemExpress #HY-A0122 Bafilomycin A1 MedChemExpress #HY-100558 MG-132 MedChemExpress #HY-13259 Cell lysis reagent Sigma #C2978 RIPA lysis buffer Solarbio #R0020 protease inhibitor cocktail Bimake #B14002 Native Mouse IgG protein Abcam #ab198772 Critical commercial assays Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit R&D Systems #MMA00 Mouse CCL4/MIP-1 beta Quantikine ELISA Kit R&D Systems #MMB00 Mouse/Rat CCL5/RANTES Quantikine ELISA Kit R&D Systems #MMR00 Mouse CCL8/MCP-2 DuoSet ELISA R&D Systems #DY790 Mouse CCL12/MCP-5 Quantikine ELISA Kit R&D Systems #MCC120 Mouse CXCL1/KC Quantikine ELISA Kit R&D Systems #MKC00B Mouse CXCL2/MIP-2 Quantikine ELISA Kit R&D Systems #MM200 Mouse CXCL9/MIG Quantikine ELISA Kit R&D Systems #MCX900 Mouse CXCL16 DuoSet ELISA R&D Systems #DY503 Bio-Plex Pro Mouse Chemokine Panel 31-Plex Bio-Rad #12009195 Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex Bio-Rad #M60009RDPD Pierce BCA Protein Assay, Reagent A Thermo Fisher #23228 Pierce BCA Protein Assay, Reagent B Thermo Fisher #23224 Lysosome Enrichment Kit for Tissues and Cultured Cells Thermo Fisher #89839 Mitochondria Isolation Kit for Cultured Cells Thermo Fisher #89874 Minute Cytoplasmic & Nuclear Extraction Kit Invent Biotechnologies #SC-003 Pyrophosphate assay kit Abcam #ab112155 ATP assay kit Abcam #ab83355 Lipid peroxidation assay kit Abcam #ab118970, Ferric Reducing Antioxidant Power Assay Kit Abcam #ab234626 Tyramide SuperBoost kits Thermo Fisher #B40922 Intracellular pH Calibration Buffer Kit Thermo Fisher #P35379 Yefluor 488 EdU Imaging Kits Yeasen #40275ES60 Syn-PER Synaptic Protein Extraction Reagent Thermo Fisher #87793 Deposited data Raw and analyzed data This paper SRA: PRJNA732103 (Continued on next page) Cell Reports 42, 112975, August 29, 2023 15

Techniques: In Vivo, BrdU Staining, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: CXCR6 promotes dermal CD8 + T cell survival and transition to long-term tissue residence

doi: 10.1101/2023.02.14.528487

Figure Lengend Snippet: ( A ) Quantification of CXCL16 in vaccinia virus (VACV) infected skin at various days post infection (d.p.i) by scarification; n=3–5 per group. ( B ) Representative immunofluorescence images of skin 14 d.p.i. with VACV. Scale bar = 100μm. ( C ) The percentage of OT-1 T cells in the epidermis after infection with OVA 257–264 -expressing VACV (VACV-OVA) calculated from immunofluorescent images. Data are cumulative of at least two experiments with n=5 (day 14) or n=6 (day 20 and 42–45). ( D ) Experimental design where 15,000 WT and 15,000 CXCR6 −/− OT-1 T cells were transferred to K14-VEGFR3-Ig mice and the following day infected with VACV-OVA via skin scarification. ( E ) Quantification of the ratio of WT and CXCR6 −/− OT-1 T cells in the spleen and skin of K14 VEGFR3-Ig mice 49 d.p.i. with VACV-OVA. Data are representative from at least two experiments with n=4. ( F ) Experimental design where congenically distinct 15,000 WT Kaede + OT-1 T cells and 15,000 CXCR6 −/− Kaede + OT-1 T cells were co-transferred to C57Bl/6J mice and infected the following day with VACV-OVA via skin scarification. Kaede expressing cells in skin were photoconverted 6 d.p.i. and harvested 1 day later from skin and draining lymph node (dLN). ( G ) Representative flow of skin and dLN. ( H ) The ratio of photoconverted WT Kaede red + to photoconverted CXCR6 −/− Kaede red + in skin and dLN following photoconversion. Data are representative from at least two experiments with n=5. Statistical significance determined using unpaired student’s t test ( A,C ) or paired student’s t test ( E,H ). Error bars represent SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: Ears were homogenized and CXCL16 concentration was quantified using Mouse CXCL16 DuoSet ELISA (R&D Systems) as per the manufacturers protocol.

Techniques: Virus, Infection, Immunofluorescence, Expressing