Journal: Cellular immunology

Article Title: Lnc-17Rik promotes the immunosuppressive function of Myeloid-Derived suppressive cells in esophageal cancer.

doi: 10.1016/j.cellimm.2023.104676

Figure Lengend Snippet: Fig. 2. Lnc-17Rik regulates the suppressive function of MDSC in vitro. (A) qRT-PCR of Lnc-17Rik in Lnc-17Rik knockdown (kdLNC) and exogenous Lnc-17Rik treated (oeLNC) MDSC. (B) qRT-PCR of Arg-1, NOS2, NOX2 and COX2 in Lnc-17Rik knockdown (kdLNC) and exogenous Lnc-17Rik treated (oeLNC) MDSC. (C) Immunoblotting analyses of Arg-1, NOS2, NOX2 and COX2 in Lnc-17Rik knockdown (kdLNC, left) and exogenous Lnc-17Rik treated (oeLNC, right) MDSC. (D-F) Arg- 1, H2O2 and NO in Lnc-17Rik knockdown (kdLNC, left) and exogenous Lnc-17Rik (oeLNC, right) treated MDSC. (G) Flow cytometry of ROS in Lnc-17Rik knockdown (kdLNC) and exogenous Lnc-17Rik (oeLNC) treated MDSC. (H-I) IFN-γ analyses in the supernatants of OT-I CD8+ and OT-II CD4+T cells in the presence of Lnc-17Rik knockdown (kdLNC, left) and exogenous Lnc-17Rik treated (oeLNC, right) MDSC. Error bars in A, B, D-F, H-I represent standard deviations from 3 independent measurements. Two-tailed, paired T test was used in A, B, D-F, H-I. *P < 0.05; **p < 0.05; ***p < 0.001. NS, no significant.

Article Snippet: Rabbit NOS2 and COX2, Arg-1 and gp91-phox antibodies from Proteintech (Rosemont, HQ). anti-V5 from Thermo Fisher Scientific (Pittsburgh, PA). β-actin antibody from Santa Cruz (Santa Cruz, CA).

Techniques: In Vitro, Quantitative RT-PCR, Knockdown, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Cellular immunology

Article Title: Lnc-17Rik promotes the immunosuppressive function of Myeloid-Derived suppressive cells in esophageal cancer.

doi: 10.1016/j.cellimm.2023.104676

Figure Lengend Snippet: Fig. 6. Lnc-17Rik promotes the enrichment of WDR5 in the promoter regions of Arg-1, NOS2, NOX2 and COX2. (A) ChIP-PCR of H3K4me3 enrichment region in the promoter of Arg-1, NOS2, NOX2 and COX2 in gain- and loss- of function MDSCs. The enrichment is expressed relative to chromatin input (%input). Data were normalized to the percent of pre-IP input for each sample. (B) Immunoblot of WDR5 and LAP in MDSCs after different concentration of exogenous Lnc-17Rik transfected into MDSCs. (C) ChIP-PCR of WDR5 enrichment region in the promoter of Arg-1, NOS2, NOX2 and COX2 in gain- and loss- of function MDSCs. The enrichment is expressed relative to chromatin input (%input). Data were normalized to the percent of pre-IP input for each sample. Two-tailed, paired T test was used in A, C. *P < 0.05; **p < 0.05; ***p < 0.001. NS, no significant.

Article Snippet: Rabbit NOS2 and COX2, Arg-1 and gp91-phox antibodies from Proteintech (Rosemont, HQ). anti-V5 from Thermo Fisher Scientific (Pittsburgh, PA). β-actin antibody from Santa Cruz (Santa Cruz, CA).

Techniques: Western Blot, Concentration Assay, Transfection, Two Tailed Test

Journal: Cellular immunology

Article Title: Lnc-17Rik promotes the immunosuppressive function of Myeloid-Derived suppressive cells in esophageal cancer.

doi: 10.1016/j.cellimm.2023.104676

Figure Lengend Snippet: Fig. 7. Human Lnc-17Rik has similar function with murine Lnc-17Rik in esophageal cancer. (A, B) QRT-PCR and immunoblotting analyses of Arg-1, NOS2, NOX2 and COX2 in Lnc-17Rik knockdown and overexpression human MDSC-like cells. (C) RNA-FISH of Lnc-17Rik in the monocytes of human esophageal cancer tissues. (D) QRT-PCR of Lnc-17Rik in CD3-HLA-DR-CD33+CD11b+CD14+and CD3-HLA-DR-CD33+CD11b+CD15+MDSCs isolated from peripheral blood of tumor patients. (E) Immunostaining and RNA-FISH in the peripheral blood CD3-HLA-DR-CD33+CD11b+ MDSCs in the patients with esophageal cancer. Scale bar = 100 µM. Two-tailed, paired T test (mean ± SEM) was used in A and E; *P < 0.05; **p < 0.05; ***p < 0.001. NS, no significant.

Article Snippet: Rabbit NOS2 and COX2, Arg-1 and gp91-phox antibodies from Proteintech (Rosemont, HQ). anti-V5 from Thermo Fisher Scientific (Pittsburgh, PA). β-actin antibody from Santa Cruz (Santa Cruz, CA).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Isolation, Immunostaining, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: E0703 targets ERβ to facilitate the upregulation of GLI3, thereby alleviating irradiation-induced DNA damage on lymphocytes

doi: 10.1186/s11658-025-00821-8

Figure Lengend Snippet: Stimulation of ATM-CHK2 signaling triggered by E0703 is mediated by GLI3. RNA sequencing in AHH-1 cells indicated a significant change in GLI3 triggered by E0703 upon IR damage ( A ). The reduced levels of the GLI3 protein in AHH-1 cells, spleen, and peripheral blood caused by IR were rescued by E0703 as shown by western blot ( B–F ), cell immunofluorescence ( C ), and tissue immunofluorescence ( D ). Lentiviruses were used to knock down GLI3 in AHH-1 cells ( G ). With γH2AX staining ( H ) and Calcein AM/propidium iodide staining ( I ), the radioprotective effect of E0703 was found to be mediated by GLI3. Stimulation of ATM-CHK2 signaling triggered by E0703 was mediated by GLI3 as shown by western blot ( J ). GLI3 overexpression ( K ) was found to restore the stimulation of ATM-CHK2 signaling ( L ) and alleviate DNA damage ( M ) after IR damage. Gray values for three independent bands were obtained in western blot; fluorescence intensities of five independent cells were determined in GLI3 or γH2AX staining; spleens of three mice in each group were subjected to immunofluorescence; and cells in five independent plates were subjected to Calcein AM/PI double staining. Representative images are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 indicated significant differences. ns, no significance

Article Snippet: Antibodies against γH2AX (no. ab26350, Abcam, 1:1000); β-actin (no. R380624, ZEN-BIOSCIENCE, 1:1000); ATR (no. 13934, Cell Signaling Technology, 1:1000); CHK1 (no. ab32531, Abcam, 1:1000); P53 (no. 345567, ZEN-BIOSCIENCE, 1:1000); phosphorylated CHK1 (p-CHK1; no. ab278717, Abcam, 1:1000); phosphorylated ATR (p-ATR; no. ab316925, Abcam, 1:1000); DNA-PKcs (no. ab32566, Abcam, 1:1000); KU70 (no. 200995, ZEN-BIOSCIENCE, 1:1000); KU80 (no. 201004, ZEN-BIOSCIENCE, 1:1000); phosphorylated DNA-PKcs (p-DNA-PKcs; no. ab124918, Abcam, 1:1000); RAD51 (no. ab133534, Abcam, 1:1000); ATM (no. ab199726, Abcam, 1:1000); CHK2 (no. ab109413, Abcam, 1:1000); phosphorylated ATM (p-ATM; no. ab36810, Abcam, 1:1000); phosphorylated CHK2 (p-CHK2; no. ab85743, Abcam, 1:1000); GLI3 (no. 28272-1-AP, Proteintech, 1:1000); ERβ (no. GTX70174, GeneTex, 1:1000); ERα (no. GTX127978, GeneTex, 1:1000); GAPDH (no. 60004-1-Ig, Proteintech, 1:1000); eIF4G1 (no. ab2609, Abcam, 1:1000); eIF4A1/A2/A3 (no. A24293, Abclonal, 1:1000); and eIF4E (no. ab33766, Abcam, 1:1000) were incubated with the membrane at 4 °C overnight, respectively.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Knockdown, Staining, Over Expression, Fluorescence, Double Staining

Journal: Cellular & Molecular Biology Letters

Article Title: E0703 targets ERβ to facilitate the upregulation of GLI3, thereby alleviating irradiation-induced DNA damage on lymphocytes

doi: 10.1186/s11658-025-00821-8

Figure Lengend Snippet: ERβ serves as a direct target of E0703 to transactivate the gene expression of GLI3 . CETSA ( A ), DARTS ( B ), and molecular docking ( C and D ) assays were adopted to analyze the binding of E0703 to ERβ and ERα. SPR analysis of the binding affinity of E0703 to the ERβ protein, with the apparent equilibrium dissociation constant (Kd) calculated ( E ). Protein expression of ERβ was detected by cell immunofluorescence ( F ) and western blot ( G ). Using selective antagonists of ERβ (PHTPP) and ERα (AZD9496), cell viability ( H ) and DNA damage ( I ) improved by E0703 were demonstrated to be mediated by ERβ. ERβ enrichment on the promoter region of GI3 could be triggered by E0703 ( J ), which significantly promoted the protein expression of GLI3 and triggered ATM-CHK2 signaling ( K ). Stimulation of ATM-CHK2 signaling triggered by E0703 was also mediated by ERβ ( L ). Cells in three independent plates were subjected to Calcein AM/PI double staining assay; fluorescence intensities of three independent cells were obtained in γH2AX staining assay; and gray values for three independent bands were obtained in western blot. Representative images are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 indicated significant differences. ns, no significance

Article Snippet: Antibodies against γH2AX (no. ab26350, Abcam, 1:1000); β-actin (no. R380624, ZEN-BIOSCIENCE, 1:1000); ATR (no. 13934, Cell Signaling Technology, 1:1000); CHK1 (no. ab32531, Abcam, 1:1000); P53 (no. 345567, ZEN-BIOSCIENCE, 1:1000); phosphorylated CHK1 (p-CHK1; no. ab278717, Abcam, 1:1000); phosphorylated ATR (p-ATR; no. ab316925, Abcam, 1:1000); DNA-PKcs (no. ab32566, Abcam, 1:1000); KU70 (no. 200995, ZEN-BIOSCIENCE, 1:1000); KU80 (no. 201004, ZEN-BIOSCIENCE, 1:1000); phosphorylated DNA-PKcs (p-DNA-PKcs; no. ab124918, Abcam, 1:1000); RAD51 (no. ab133534, Abcam, 1:1000); ATM (no. ab199726, Abcam, 1:1000); CHK2 (no. ab109413, Abcam, 1:1000); phosphorylated ATM (p-ATM; no. ab36810, Abcam, 1:1000); phosphorylated CHK2 (p-CHK2; no. ab85743, Abcam, 1:1000); GLI3 (no. 28272-1-AP, Proteintech, 1:1000); ERβ (no. GTX70174, GeneTex, 1:1000); ERα (no. GTX127978, GeneTex, 1:1000); GAPDH (no. 60004-1-Ig, Proteintech, 1:1000); eIF4G1 (no. ab2609, Abcam, 1:1000); eIF4A1/A2/A3 (no. A24293, Abclonal, 1:1000); and eIF4E (no. ab33766, Abcam, 1:1000) were incubated with the membrane at 4 °C overnight, respectively.

Techniques: Gene Expression, Binding Assay, Expressing, Immunofluorescence, Western Blot, Double Staining, Fluorescence, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: E0703 targets ERβ to facilitate the upregulation of GLI3, thereby alleviating irradiation-induced DNA damage on lymphocytes

doi: 10.1186/s11658-025-00821-8

Figure Lengend Snippet: E0703 triggers the interaction between GLI3 and eIF4G1 upon IR damage ( A , B ). Colocalization of GLI3 and eIF4G1 was determined by cell ( C ) and spleen tissue ( D ) immunofluorescence assays. E0703 triggered the formation of the eIF4F complex upon IR damage as shown by the Co-IP assay ( E , F ). Cells in three independent plates were subjected to co-staining of GLI3 and eIF4G1, and spleens from three mice in each group were subjected to immunofluorescence. Representative images are shown

Article Snippet: Antibodies against γH2AX (no. ab26350, Abcam, 1:1000); β-actin (no. R380624, ZEN-BIOSCIENCE, 1:1000); ATR (no. 13934, Cell Signaling Technology, 1:1000); CHK1 (no. ab32531, Abcam, 1:1000); P53 (no. 345567, ZEN-BIOSCIENCE, 1:1000); phosphorylated CHK1 (p-CHK1; no. ab278717, Abcam, 1:1000); phosphorylated ATR (p-ATR; no. ab316925, Abcam, 1:1000); DNA-PKcs (no. ab32566, Abcam, 1:1000); KU70 (no. 200995, ZEN-BIOSCIENCE, 1:1000); KU80 (no. 201004, ZEN-BIOSCIENCE, 1:1000); phosphorylated DNA-PKcs (p-DNA-PKcs; no. ab124918, Abcam, 1:1000); RAD51 (no. ab133534, Abcam, 1:1000); ATM (no. ab199726, Abcam, 1:1000); CHK2 (no. ab109413, Abcam, 1:1000); phosphorylated ATM (p-ATM; no. ab36810, Abcam, 1:1000); phosphorylated CHK2 (p-CHK2; no. ab85743, Abcam, 1:1000); GLI3 (no. 28272-1-AP, Proteintech, 1:1000); ERβ (no. GTX70174, GeneTex, 1:1000); ERα (no. GTX127978, GeneTex, 1:1000); GAPDH (no. 60004-1-Ig, Proteintech, 1:1000); eIF4G1 (no. ab2609, Abcam, 1:1000); eIF4A1/A2/A3 (no. A24293, Abclonal, 1:1000); and eIF4E (no. ab33766, Abcam, 1:1000) were incubated with the membrane at 4 °C overnight, respectively.

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: E0703 targets ERβ to facilitate the upregulation of GLI3, thereby alleviating irradiation-induced DNA damage on lymphocytes

doi: 10.1186/s11658-025-00821-8

Figure Lengend Snippet: The formation of the eIF4F complex triggered by E0703 is mediated by GLI3. Knockdown of GLI3 with a lentivirus inhibited the formation of the eIF4F complex triggered by E0703 ( A , B ), and overexpression of GLI3 rescued the dissociation of the eIF4F complex caused by IR ( C , D ). Using a selective antagonist of eIF4G1 (SBI0640756), activation of ATM-CHK2 signaling ( E ), inhibition of DNA damage ( F ), and improvement of cell survival ( G ) obtained with E0703 treatment upon IR damage were shown to be mediated by eIF4F complex formation. Gray values for three independent bands were obtained by western blot, and cells in five independent plates were subjected to Calcein AM/PI double staining assay. Representative images are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001 indicated significant differences

Article Snippet: Antibodies against γH2AX (no. ab26350, Abcam, 1:1000); β-actin (no. R380624, ZEN-BIOSCIENCE, 1:1000); ATR (no. 13934, Cell Signaling Technology, 1:1000); CHK1 (no. ab32531, Abcam, 1:1000); P53 (no. 345567, ZEN-BIOSCIENCE, 1:1000); phosphorylated CHK1 (p-CHK1; no. ab278717, Abcam, 1:1000); phosphorylated ATR (p-ATR; no. ab316925, Abcam, 1:1000); DNA-PKcs (no. ab32566, Abcam, 1:1000); KU70 (no. 200995, ZEN-BIOSCIENCE, 1:1000); KU80 (no. 201004, ZEN-BIOSCIENCE, 1:1000); phosphorylated DNA-PKcs (p-DNA-PKcs; no. ab124918, Abcam, 1:1000); RAD51 (no. ab133534, Abcam, 1:1000); ATM (no. ab199726, Abcam, 1:1000); CHK2 (no. ab109413, Abcam, 1:1000); phosphorylated ATM (p-ATM; no. ab36810, Abcam, 1:1000); phosphorylated CHK2 (p-CHK2; no. ab85743, Abcam, 1:1000); GLI3 (no. 28272-1-AP, Proteintech, 1:1000); ERβ (no. GTX70174, GeneTex, 1:1000); ERα (no. GTX127978, GeneTex, 1:1000); GAPDH (no. 60004-1-Ig, Proteintech, 1:1000); eIF4G1 (no. ab2609, Abcam, 1:1000); eIF4A1/A2/A3 (no. A24293, Abclonal, 1:1000); and eIF4E (no. ab33766, Abcam, 1:1000) were incubated with the membrane at 4 °C overnight, respectively.

Techniques: Knockdown, Over Expression, Activation Assay, Inhibition, Western Blot, Double Staining

Journal: Cell Death & Disease

Article Title: Slc25a21 in cisplatin-induced acute kidney injury: a new target for renal tubular epithelial protection by regulating mitochondrial metabolic homeostasis

doi: 10.1038/s41419-024-07231-2

Figure Lengend Snippet: A Representative immunofluorescence image of co-staining of SLC25A21 with Mitotracker revealed the mitochondrial localization of SLC25A21 in TKPTS cells. Green presents SLC25A21; red presents Mitotracker; blue presents DAPI. Scale bars, 20 µm. B The rate of oxygen consumption was assessed using the Seahorse XF-96 Extracellular Flux Analyzer in Slc25a21-OE and normal control (NC) cells after cisplatin (10 µM) treatment for 24 h ( left ). OLIGO oligomycin, FCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, ROT rotenone, AA antimycin A. Oxygen consumption rate for basal and maximal respiration was analyzed ( right ). n = 6, each group, and values are reported as mean ± SEM. C Relative ATP production in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition. n = 6, each group, and values are reported as mean ± SEM. D Heatmap showing mRNA expression of the 13 mitochondrially encoded genes in Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition, determined by RT-qPCR ( n = 10). Data are presented as Z-score. *indicates Slc25a21 AAV9 vs NC AAV9 , #indicates Slc25a21 AAV9 +Cisplatin vs NC AAV9 +Cisplatin. E Representative western blotting for protein expression levels of enzymes involved in mitochondrial OXPHOS in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition. NDUFB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8, ND1 NADH-ubiquinone oxidoreductase chain 1, SDHB Succinate dehydrogenase (ubiquinone) iron-sulfur subunit, CYTB Cytochrome b; UQCRC2 Cytochrome b-c1 complex subunit 2, COX1 Cytochrome c oxidase subunit 1, COX IV cytochrome c oxidase subunit 4 isoform 1, ATP5A1 ATP synthase subunit alpha, ATP6 ATP synthase subunit a. F Representative western blotting for PGC1α and TFAM protein expression levels of enzymes associated with mitochondrial biogenesis in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition. G The mtDNA copy number in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition measured by qRT-PCR. n = 8–10, each group, and values are reported as mean ± SEM. H Representative western blotting for Porin and CypD protein expression levels of enzymes associated with mitochondrial integrity in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition. CypD: cyclophilin D. I Relative fold change of MitoSOX detected in Slc25a21-OE and NC cells after cisplatin (10 µM) treatment for 24 h. n = 3, each group, and values are reported as mean ± SEM. J Representative immunohistochemistry images of 8-OHdG expression in the kidneys of Slc25a21 AAV9 and NC AAV9 mice after cisplatin or saline addition ( left ) and semi-quantitative IOD analysis of 8-OHdG expression ( right ). n = 7–9, each group, and values are reported as mean ± SEM. Scale bars, 50 µm. IOD integral optical density. In each case, data were presented as means ± SEM and statistical significance is assessed by One-way ANOVA analysis of variance of Tukey’s multiple comparisons test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; # indicates P < 0.05; ## indicates P < 0.01; ### indicates P < 0.001; #### indicates P < 0.0001; NS indicates not significant. See also Fig. .

Article Snippet: The primary antibodies used in this study included: SLC25A21 antibody (Thermo Fisher Scientific, PA5-101672; RRID: AB_2851106); NGAL antibody (Abcam, ab63929; RRID: AB_1140965); Mouse KIM-1 Antibody (R&D Systems, AF1817; RRID: AB_2116446); BAX (Cell Signaling Technology, 2772; RRID: AB_10695870); Cleaved Caspase3 (Cell Signaling Technology, 9664; RRID: AB_2070042); P53 (Cell Signaling Technology, 9282; RRID: AB_331476); β-Actin Rabbit mAb (High Dilution) (ABclonal, AC026; RRID: AB_2768234); α-Tubulin Mouse mAb (ABclonal, AC012; RRID: AB_2768341); Mouse anti HA-tag mAb (ABclonal, AE008; RRID: AB_2770404); NDUFB8 Rabbit mAb (ABclonal, A19732; RRID: AB_2938519); SDHB Rabbit mAb (ABclonal, A1809; RRID: AB_2861883); UQCRC2 Rabbit mAb (ABclonal, A4366; RRID: AB_2863250); COXIV Rabbit pAb (ABclonal, A6564; RRID: AB_2767158); ATP5A1 Rabbit mAb (ABclonal, A11217; RRID: AB_2861524); Anti-mtTFA antibody - Mitochondrial Marker (Abcam, ab47517; RRID: AB_945799); PGC1α Rabbit mAb (ABclonal, A20995; RRID: AB_3096101); ACAA2 Rabbit mAb (ABclonal, A0664; RRID: AB_3096102); DHTKD1 Rabbit pAb (ABclonal, A8369; RRID: AB_2769161); Citrate synthetase Rabbit mAb (ABclonal, A23371; RRID: AB_3096103); OGDH Rabbit mAb (ABclonal, A22163; RRID: AB_3096104); SUCLG2 Rabbit pAb (ABclonal, A19872; RRID: AB_2862784); ACO2 Rabbit mAb (ABclonal, A4524; RRID: AB_2863288); MT-ND1 Rabbit mAb (ABclonal, A9743; RRID: AB_3096106); ATP6 Rabbit pAb (ABclonal, A8193; RRID: AB_2768510); MT-CYB Rabbit pAb (ABclonal, A17966; RRID: AB_2861768); COX1 Rabbit mAb (ABclonal, A23123; RRID: AB_3096107); Membrane Integrity WB Antibody Cocktail (Abcam, ab110414; RRID: AB_2687585).

Techniques: Immunofluorescence, Staining, Control, Saline, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry