Journal: BMC Oral Health

Article Title: Synergistic proanthocyanidin-copper oxygen-generating microneedle enhances anti-inflammatory activity in dental pulp stem cells and macrophage efferocytosis

doi: 10.1186/s12903-026-07870-1

Figure Lengend Snippet: A : Fluorescence staining of ROS levels in macrophages cultured in the leachate of each hydrogel group; ( B ): Semi-quantitative analysis of ROS fluorescence staining; ( C , D , E) : Expression levels of IL-6, IL-1β, and TNF-α in macrophages cultured in the leachate of each hydrogel group; ( F) : Statistical analysis of macrophage efferocytosis index by immunofluorescence staining; G : Statistical analysis of macrophage efferocytosis index by flow cytometry; ( H ): Immunofluorescence staining to detect macrophage efferocytosis levels (blue: nuclei of macrophages; green: cell membrane of macrophages; red: apoptotic hDPSCs); ( I ): Flow cytometry detection of macrophage efferocytosis levels (FITC-A: F4/80-labeled macrophages; PE-A: CellTracker-labeled apoptotic cells)

Article Snippet: Macrophages (RAW264.7) were incubated for 24 h, and 1 mL of DMEM containing 200 ng/mL LPS was added to induce inflammation for 24 h, then 5 × 10 4 stained apoptotic cells and samples of different groups were added and co-cultured for 24 h. Then, it was fixed with 4% paraformaldehyde for 20 min, washed with PBS, blocked with goat serum for 20 min at room temperature, and incubated with F4/80 antibody (Bosterbio, China) overnight at 4°C.

Techniques: Fluorescence, Staining, Cell Culture, Expressing, Immunofluorescence, Flow Cytometry, Membrane, Labeling

Journal: Stem Cells International

Article Title: Noggin Combined With Human Dental Pulp Stem Cells to Promote Skeletal Muscle Regeneration

doi: 10.1155/sci/2812390

Figure Lengend Snippet: Noggin antagonize BMP's inhibition of myogenic differentiation of DPSCs. (a) 3D protein structure of Noggin's competitive inhibition of BMP4 binding to BMPRIA. Red indicates Noggin. Gray indicates BMP4. Purple indicates BMPRIA. Cyan indicates a competitive site of Noggin binding to BMPRIA. (b) Protein expression of Pax7, Pax3, Eya2, and Desmin was assessed using a western blot when rescuing with BMP4. (c–e) Protein expression of p-Smad1/5/9 and Smad1 was assessed using western blot by Noggin treatment for different times (3, 6, and 9 h) and subsequently stimulated with BMP4 or control for 1 h (c), and semiquantitative analysis of the expression ratio of (d) p-Smad1/5/9/ β -actin, (e) p-Smad1/5/9/Smad1 were performed ( n = 4). (f, g) Relative mRNAs expression of (f) ID1 and (g) MSX1 were assessed using quantitative PCR ( n = 3). The differences were presented as ⁣ ∗ p < 0.05 and ⁣ ∗∗∗ p < 0.001. 100N = 100 ng/mL Noggin; 200N = 200 ng/mL Noggin. BMP, bone morphogenetic protein; BMPRIA, BMP4 to BMP-receptor I A; DPSCs, dental pulp stem cells; PCR, polymerase chain reaction.

Article Snippet: Antibodies against β -actin (abs830031; Absin Bioscience, Shanghai, China), Pax7 (ab187339), and Myf5 (ab125078) from Abcam; MyHC IIB (BF-F3) and MyHC IIA (SC-71) from Developmental Studies Hybridoma Bank (DSHB, ID, USA); p-Smad 1/5/9 (#13820) and Smad 1 (#6944) from Cell Signaling Technology (CST, MA, USA); and MyoD (#18943), Desmin (#60226), MRF4 (#11754), Pax3 (#21383), Six1 (#10709), Eya2 (#11314), ID1 (#18475), and MSX1 (#17678) from Proteintech (IL, USA) were used.

Techniques: Inhibition, Binding Assay, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 2. DHT suppresses kiss1 expression and promotes unfolded protein response (UPR) in DHT-induced PCOS rats. (a) Quantitative Real-time PCR (qPCR) analysis of kiss1 in the hypothalamus of DHT-induced PCOS (PCOS_DHT) and control (ctrl) rats after DHT exposure for 1 or 3 weeks. n = 9. ***P < 0.001, t-test. (b) Immunofluorescence labeling for kiss1 (green) in the hypothalamus of PCOS_DHT and ctrl rats after DHT exposure for 3 weeks. DAPI (Blue) was used for nuclear label. Scale bar, 20 μm. (c) qPCR of UPR pathway factors (ATF4, BIP, CHOP, PDI) in DHT and ctrl rats DHT exposure for 3 weeks. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, t-test. (d-e) Western blots for (d) and qPCR analysis of (e) kiss1 and PDI in DHT and ctrl rats after DHT exposure for 9 weeks. n = 3–6. ** P < 0.01, *** P < 0.001, t-test. qPCR data are relative mRNA levels normalized with that of GAPDH for kiss1(a) and PDI (e) or β-actin for other genes of interest (c and e). β-actin was used as a loading control for all western blots.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Immunofluorescence, Labeling, Western Blot

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 3. Kisspeptin mediates DHT's effects on endoplasmic reticulum (ER) stress in GT1–7 cells. (a) qPCR of kiss1 and UPR pathway factors (BIP, PDI, CHOP, ATF4, GADD34 and Xbp1s) in GT1–9 cells treated with DHT (10 nM), kp234 (a kiss1 inhibitor, 1 nM) or DMSO as the control (Ctrl) for 24 h. n = 3. ** P < 0.01, *** P < 0.001, t-test. (b-c) qPCR analysis of Kiss1, BIP, PDI, CHOP, ATF4, GADD34 and Xbp1s (b) and western blots for ATF4 and Bax (c) in GT1–9 cells transfected with siRNAs against kiss1 (two designs, sikiss1_1 or sikiss1_2) or non-silencing control siRNAs (NC) for 24 h. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, t-test. qPCR data are relative mRNA levels normalized with that of GAPDH. GAPDH was used as a loading control for all western blots. Error bars are smaller than symbol size except where shown.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Control, Western Blot, Transfection

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 4. Kisspeptin rescues DHT-induced ER stress in GT1–7 cells. (a-b) Western blots for ATF6, IRE1α, Perk, BIP p-eIF2α and eIF2α (a) and qPCR analysis of GADD34 and Xbp1s/Xbp1 (b) in GT1–7 cells 30 min after treatment with DHT (10 nM), Kp10 (a kiss1 peptide, 10 nM) or DMSO as control (ctrl). n = 3. ** P < 0.01, ***P < 0.001, t-test. (c-d) Western blots (c) or qPCR analysis (d) for ATF4, CHOP, PDI and XBP1 in GT1–7 cells 24 h after treatment with DHT (10 nM), Kp10 (10 nM), or DMSO as Ctrl. n = 3. ** P < 0.01, *** P < 0.001, t-test. (e) qPCR analysis of ATF4, BIP, PDI and CHOP in GT1–7 cells after treatment with thapsigargin (TG, an ER stress inducer, 20 nM), Kp10 (10 nM) or vehicle control (Ctrl). n = 3. *** P < 0.001, t-test. GAPDH was used as a loading control for all western blots. Error bars are smaller than symbol size except where shown.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Western Blot, Control

Journal: Cancer Biology & Medicine

Article Title: Migration and invasion inhibitory protein inhibits M2 macrophage polarization to suppress colorectal cancer progression through the STING–NFκB2–IL10 axis

doi: 10.20892/j.issn.2095-3941.2025.0282

Figure Lengend Snippet: MIIP modulates the STING–NFκB2–IL10 signaling pathway in CRC cells. Immunofluorescence staining showing the effects of MIIP knockdown and MIIP overexpression on dsDNA levels (A) and the distribution and expression of STING (B) in SW480 and SW620 cells. (C) Western blots showing the effects of MIIP knockdown and overexpression on STING, TRAF3, p-p100, and p52 levels. (D) ELISA results showing the effects of MIIP knockdown and overexpression on IL10 levels in the cell culture medium. (E and F) Effects of MIIP knockdown and/or STING knockdown on p52 expression (E) and IL10 levels in the cell culture medium (F). All data are presented as the means ± SDs ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001; scale bar, 50 μm.

Article Snippet: The primary antibodies used included mouse anti-dsDNA (1:1000, ab270732; Abcam, Cambridge, MA, USA), rabbit anti-NFKB2/p52 (1:200, 15503-1-AP; Proteintech, Wuhan, Hubei, China), rabbit anti-TMEM173/STING (1:200, 19851-1-AP; Proteintech, Wuhan, Hubei, China), and rabbit anti-IL-10 antibodies (1:100, 20850-1-AP; Proteintech, Wuhan, Hubei, China).

Techniques: Immunofluorescence, Staining, Knockdown, Over Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer Biology & Medicine

Article Title: Migration and invasion inhibitory protein inhibits M2 macrophage polarization to suppress colorectal cancer progression through the STING–NFκB2–IL10 axis

doi: 10.20892/j.issn.2095-3941.2025.0282

Figure Lengend Snippet: MIIP inhibits tumor growth and metastasis and M2 macrophage infiltration in a syngeneic CRC mouse model through the STING–NFκB2–IL10 axis. (A) Tumors in situ (indicated by green arrows) and liver metastatic nodules (indicated by black arrows) were observed in a syngeneic CRC mouse model. Statistics for the size of tumors in situ (B) and the number of metastatic foci (C). (D) Statistical analysis of IL10 levels in mouse blood. (E) Representative images of HE and IHC staining for SATB2, MIIP, STING, IL10, p52, and CD163 (green arrows) in tumor tissues from the different groups. The bar graphs show the percentages of high/low STING (F), IL-10 (G), and p52 (H) expression in the different groups. (I) Comparison of the number of CD163-positive cells among the different groups. All data are presented as the means ± SDs ( n = 3). ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; scale bar, 50 μm.

Article Snippet: The primary antibodies used included mouse anti-dsDNA (1:1000, ab270732; Abcam, Cambridge, MA, USA), rabbit anti-NFKB2/p52 (1:200, 15503-1-AP; Proteintech, Wuhan, Hubei, China), rabbit anti-TMEM173/STING (1:200, 19851-1-AP; Proteintech, Wuhan, Hubei, China), and rabbit anti-IL-10 antibodies (1:100, 20850-1-AP; Proteintech, Wuhan, Hubei, China).

Techniques: In Situ, Immunohistochemistry, Expressing, Comparison

Journal: Molecular Medicine Reports

Article Title: Downregulation of transgelin 2 promotes breast cancer metastasis by activating the reactive oxygen species/nuclear factor-κB signaling pathway

doi: 10.3892/mmr.2019.10643

Figure Lengend Snippet: Loss of TAGLN2 expression induces NF-κB activation. (A) Luciferase activity of an NF-κB-luciferase reporter plasmid in TAGLN2-overexpressing 293T cells was measured 48 h after stimulation with siRNA-TAGLN2 or scramble siRNA; luciferase activity was normalized to that of Renilla . Data were representative of three independent experiments. (B) Immunoblotting of IκB, p50, p65/RelA and TAGLN2 expression in MDA-MB-231 and MCF-7 cells transfected with pLKO.1-shRNA-TAGLN2 and pLKO.1-shRNA-scramble. The results of western blotting were representative of three independent experiments. (C) Cell migration was promoted in MDA-MB-231 cells transfected with pLKO.1-shRNA-TAGLN2 compared with cells treated with scramble shRNA. Treatment with 18 µM SN50 for 24 h reversed the effects of TAGLN2 downregulation on cell invasion. Data are representative of three independent experiments. *P<0.05 vs. scramble or as indicated. DMSO, dimethyl sulfoxide; NF-κB, nuclear factor-κB; shRNA, short hairpin RNA; siRNA, small interfering RNA; TAGLN2, transgelin 2.

Article Snippet: The membranes were probed with primary antibodies against PRDX1 (1:1,000; cat. no. EPR5433; Epitomics, Abcam, Cambridge, UK), FLAG (1:500; cat. no. F7425; Sigma-Aldrich, Merck KGaA), TAGLN2 (1:1,000; cat. no. 60044-1-lg; ProteinTech Group, Inc.), IκB (1:3,000; cat. no. 66418-1-lg; ProteinTech Group, Inc.), p50 (1:200; cat. no. 15506-1-AP; ProteinTech Group, Inc.), p65 (1:1,000; cat. no 10745-1-AP; ProteinTech Group, Inc.), MMP1 (1:1,000; cat. no. 54376; Cell Signaling Technology), MMP2 (1:1,000; cat. no. 4022; Cell Signaling Technology), CXCR4 (1:1,000; cat. no. ab181020; Abcam, Cambridge, UK), Lamin B1 (1:3,000; cat. no. 12987-1-AP; ProteinTech Group, Inc.) and GAPDH (1:3,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.) on the rocking table at 4°C overnight.

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Transfection, shRNA, Migration, Small Interfering RNA

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 5. Hyperlactylation of Ikzf1 at Lys164 is important for TH17 differentiation. (A) Radar diagram showing the top 15 DLPs in CD4+ T cells of EAU mice. (B) Enriched GO biological processes of up-regulated Kla proteins. (C and D) MS/MS spectra of the lactylated peptides of Ikzf1 at Lys164 and Lys373. (E) Immunoblot after immuno- precipitation (IP) assays showing increased lactylation of Ikzf1 in CD4+ T cells of EAU mice (n = 4 samples per group; **P < 0.01 by two-tailed unpaired Student’s t test). (F) Expression of Ikzf1 in CD4+ T cells of TH0 and TH17 groups measured through Western blotting (n = 4 samples per group; ns, no significance by two-tailed unpaired Student’s t test). (G) CD4+ T cells were transfected with the control virus (control), only sh-Ikzf1 (sh-Ikzf1), shIkzf1+oeWT-Ikzf1 (oeWT-Ikzf1), shIkzf1+oeK164R Ikzf1 (oeK164R-Ikzf1), and shIkzf1+oe K373R Ikzf1 (oeK373R-Ikzf1) adenovirus. Expression levels of Ikzf1 in different groups were measured through Western blotting (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (H) FCM analysis of the frequency of TH17 cells in different groups as described in (G) (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (I) Expression levels of IL-17 in the media of corresponding groups were tested by ELISA (n = 8 samples per group; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis test). m/z, mass/charge ratio.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Tandem Mass Spectroscopy, Western Blot, Immunoprecipitation, Two Tailed Test, Expressing, Transfection, Control, Virus, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 6. Ikzf1 K164la was up-regulated in CD4+ T cells of EAU mice. (A) Dot blot assays of Ikzf1-K164la antibody. (B) Ikzf1-K164la levels in TH0 and TH17 cells measured by Western blotting (n = 4 samples per group; **P < 0.01 by two-tailed unpaired Student’s t test). (C) Ikzf1-K164la levels in TH17 cells in response to DCA and rotenone treatment (n = 3 samples per group; *P < 0.05 by one-way ANOVA and Bonferroni post hoc test). (D) Ikzf1-K164la levels in CD4+ T cells of EAU mice at different time points (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (E) Ikzf1-K164la levels in CD4+ T cells of EAU mice in response to DCA and rotenone treatment (n = 3 to 4 samples per group; *P < 0.05 by one-way ANOVA and Bonferroni post hoc test).

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Dot Blot, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 7. CUT& Tag analysis reveals the transcriptional consequences of Ikzf1 under TH17 differentiation condition. (A) Binding density of WT Ikzf1 was visualized by deepTools. The heatmap presents the CUT& Tag counts on the different Ikzf1 binding peaks in CD4+ T cells between WT and K164R groups under TH17 induction con- dition, ordered by signal strength. (B) Genome-wide distribution of Ikzf1 binding peaks in CD4+ T cells of WT and K164R groups. (C) GO analysis of the decreased Ikzf1 binding peaks at candidate target genes. (D) Genome browser tracks of CUT& Tag signal at the representative target gene loci. (E) mRNA expression levels of IL-2, IL-4, Tlr4, and Runx1 measured using RT-qPCR (n = 4 samples per group; **P < 0.01 and ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (F) Luciferase activity of the IL-2 promoter–driven reporter vector was measured between the control, sh-Ikzf1, shIkzf1+oeWT-Ikzf1, shIkzf1+oeK164R-Ikzf1, and shIkzf1+oe K373R Ikzf1 groups (n = 8 samples per group; ***P < 0.001 by Kruskal-Wallis test). (G) Luciferase activity of the Runx1 promoter–driven reporter vector was measured between the control, sh-Ikzf1, shIkzf1+oeWT-Ikzf1, shIkzf1+oeK164R-Ikzf1, and shIkzf1+oe K373R Ikzf1 groups (n = 8 samples per group; ***P < 0.001 by one-way ANOVA and Dunnett’s T3 post hoc test). (H) FCM analysis of the frequency of TH17 cells in corresponding groups (n = 4 per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (I) Ex- pression levels of IL-17 in the media of corresponding groups tested by ELISA (n = 8 per group; *P < 0.05 and ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). 50UTR, 50 untranslated region.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Binding Assay, Genome Wide, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 8. Schematic diagram of the current study. Global lactylome reveals that Ikzf1 lactylation levels are up-regulated in the CD4+ T cells of EAU mice. Further exper- iments demonstrated that Ikzf1 K164 lactylation promotes TH17 differentiation by regulating IL-2, IL-4, Tlr4, and Runx1 expression.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Protein levels of YBX1 in 10 different types of tumors from the CPTAC database. B mRNA levels of YBX1 in 21 different types of tumors from the TCGA database. C–G Enrichment functional analysis of proteins interacting with YBX1 in ccRCC cell line 786-O through Co-IP pull-down and identified by mass spectrometry. C Mass spectrometry identification schematic. D Gene Ontology Biological Process (GO-BP) enrichment. E KEGG pathway enrichment. F UniPort annotation keywords. G Wiki Pathways enrichment analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A and B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Mass Spectrometry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot validation of YBX1 protein expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. B qRT-PCR validation of YBX1 mRNA expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. C Lactate production upon YBX1 knockdown in ACHN and 786-O cells. D ATP production detection upon YBX1 knockdown in ACHN and 786-O cells. E Real-time monitoring of extracellular acidification rate (ECAR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. F Quantitative analysis of glycolytic capacity and glycolytic reserve. G Real-time monitoring of oxygen consumption rate (OCR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. H Quantitative analysis of mitochondrial respiratory capacity and respiratory reserve. I Lactate dehydrogenase (LDH) activity detection upon YBX1 knockdown and overexpression in ACHN and 786-O cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in B – D , F , H and I . Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Biomarker Discovery, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A , B Effects of YBX1 overexpression on the proliferation of ACHN and 786-O cells. A Colony formation assay. B CCK-8 cell proliferation assay. C, D Effects of YBX1 overexpression on the migration and invasion of ACHN and 786-O cells. C Transwell cell migration assay. D Transwell cell invasion assay. E Detection of bioluminescence intensity of 8 pairs of tumors in nude mice using an in vivo imaging system. F Quantification analysis of bioluminescence intensity at the site of renal orthotopic tumors. G Statistical analysis of in situ tumor weight, expressed as the weight of the left minus the corresponding right kidney of nude mice. H Immunohistochemical staining and quantification to detect protein expression of YBX1, LDHA and Ki-67 in tumor tissues from control and YBX1 knockdown groups in nude mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , C , D , F , G and H . Two-way ANOVA with correction for multiple comparisons in B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Over Expression, Colony Assay, CCK-8 Assay, Proliferation Assay, Migration, Cell Migration Assay, Invasion Assay, In Vivo Imaging, In Situ, Immunohistochemical staining, Staining, Expressing, Control, Knockdown

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A, B Protein expression levels of YBX1 ( A ) and LDHA ( B ) in ccRCC from the CPTAC database. C, D Correlation of gene expression between YBX1 and LDHA in ccRCC from the TCGA database ( C ) and in an Asian population ( D ). E Western blot analysis of YBX1 and LDHA protein expression in tumor (T) and paired adjacent non-tumor (P) tissues from 27 ccRCC patients, showing representative images from 4 pairs of tissues. F Quantification of YBX1 and LDHA proteins in ccRCC and adjacent non-cancerous tissues from 27 cases using Image J software, normalized to β-actin. G Correlation of YBX1 and LDHA protein expression in tumor tissues from 27 ccRCC patients. H-M Immunohistochemical staining to assess the expression of YBX1 and LDHA in 63 pairs of ccRCC and adjacent tissues. H, I Representative immunohistochemical staining images and quantification of IHC staining using Image J software. J Correlation of quantified immunohistochemical staining of YBX1 and LDHA in tumor tissues of 63 ccRCC patients. K-M Levels of YBX1 and LDHA in ccRCC patients stratified by tumor size, T stage and Fuhrman grade. N , O Impact of YBX1 expression on overall survival (OS) ( N ) and recurrence free survival (RFS) ( O ) in ccRCC patients from the TCGA database. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , B , K , L and M . Spearman correlation statistics in C , D , G and J . Paired t -test in F and I. Log-rank test in N and O. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Expressing, Gene Expression, Western Blot, Software, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Identification of LDHA as a potential YBX1-interacting protein by IP/MS analysis. B Protein-protein interaction (PPI) network diagram of YBX1 and LDHA from the GeneMANIA and STRING databases. C Co-IP assay to verify the YBX1-LDHA interaction in ccRCC cell lines. D Immunofluorescence staining to examine the expression of YBX1 and LDHA in ACHN and 786-O cell lines, with confocal microscopy used to observe the co-localization of YBX1 and LDHA. E Molecular docking analysis of the YBX1-LDHA interaction. F Schematic diagram of GFP-tagged YBX1 structural domain peptide fragments. G 786-O cells transfected with GFP-tagged YBX1 or its truncation mutants for 48 h, followed by Co-IP using anti-GFP antibody. H Schematic representation of Flag-tagged LDHA domain protein peptides. I 786-O cells transfected with Flag-tagged LDHA or its truncation mutants for 48 h, followed by Co-IP using anti-Flag antibody. J Assessment of the impact on LDH activity after transfecting YBX1 or its truncated mutants into 786-O cells for 48 h. ** P < 0.01, **** P < 0.0001, ns: no significant difference. One-way ANOVA with correction for multiple comparisons in J. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Confocal Microscopy, Transfection, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A qRT-PCR analysis of LDHA mRNA expression levels in ACHN and 786-O cells following YBX1 knockdown and overexpression. B CUT&Tag peak plots and heatmap showing YBX1 binding intensity in ACHN cells. C Pie chart showing the distribution of YBX1 binding regions. D IGV plot showing the occupancy of YBX1 in the LDHA promoter region. E Motif diagram of YBX1 binding sequences from the JASPAR database. F Molecular docking analysis of YBX1 with four predicted binding sites (BS) in the LDHA promoter sequence. G The LDHA promoter sequence was divided into 3 fragments containing the predicted BS, followed by primer design and ChIP-qPCR to verify YBX1 binding to the LDHA promoter region. H The LDHA promoter sequence was divided into 4 fragments, and the BS-1 site that was mainly bound was mutated, the luciferase-tagged truncated plasmids were designed. After co-transfection of the target and control Renilla plasmids into ccRCC cells, YBX1 binding to the LDHA promoter region was verified using dual-luciferase reporter assays. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in A, G and H. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, Sequencing, ChIP-qPCR, Luciferase, Cotransfection, Control

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot analysis of LDHA protein expression levels in ACHN and 786-O cells after YBX1 knockdown and overexpression. B Dual-luciferase reporter assays evaluating the effects of YBX1 and LDHA on glycolysis-related signaling pathways in ccRCC cells co-transfected with target plasmids and control Renilla plasmids. C Western blot analysis of p-p65 (Ser536) and total p65 expression after overexpression of YBX1 and LDHA. D Immunohistochemical staining to detect protein expression of total p65 and p-p65 (Ser536) in tumor tissues from control and YBX1 knockdown groups in nude mice. Representative images and quantification. E, F Western blot ( E ) and qRT-PCR ( F ) analysis of LDHA knockdown efficiency in 786-O cells 72 h post-transfection with si- LDHA . G , H Western blot ( G ) and LDH activity ( H ) analysis of LDHA inhibition in 786-O cells treated with Oxamate (0-80 mM for 48 h). I , J Western blot analysis of p-p65 (Ser536) and total p65 in YBX1-overexpressing cells following LDHA knockdown ( I ) or Oxamate treatment (60 mM for 48 h) ( J ). K-N Lactate production ( K, L ) and cell proliferation ( M , N ) assays in YBX1-overexpressing cells after LDHA knockdown or Oxamate treatment. O Schematic diagram illustrating the YBX1-LDHA-NF-κB mechanism in ccRCC progression. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in B , D , F and H . One-way ANOVA with correction for multiple comparisons in K and L. Two-way ANOVA with correction for multiple comparisons in M and N. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Luciferase, Protein-Protein interactions, Transfection, Control, Immunohistochemical staining, Staining, Quantitative RT-PCR, Activity Assay, Inhibition