Journal: Cells

Article Title: Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

doi: 10.3390/cells8040345

Figure Lengend Snippet: The SHP-1 and SHP-2 in complexes containing γ-tubulin. ( I ) The expression profile of SHP-1 and SHP-2 in selected mouse tissues and BMMCs: A gel-based RT-PCR analysis of mouse SHP-1 ( Ptpn6 ) and SHP-2 ( Ptpn11 ) is shown. Mouse spleen and heart served as positive controls for SHP-1 and SHP-2, respectively. ( II ) The extracts from BMMCs precipitated with immobilized Abs specific to SHP-1 ( A , D , G ), SHP-2 ( B , E , H ), or γ-tubulin sequence 434–449 ( C , F , I ): The blots were probed with Abs to γ-tubulin (γ-Tb), SHP-1, and SHP-2. The load ( lane 1 ), the immobilized Abs not incubated with cell extracts ( lane 2 ), the precipitated proteins ( lane 3 ), and the carriers without Abs and incubated with cell extracts ( lane 4 ).

Article Snippet: To prepare C-terminally enhanced green fluorescent protein (EGFP)-tagged mouse SHP-1 (gene Ptpn6 ; RefSeq ID: NM_013545.3), the coding sequence without a stop codon was amplified from the C-terminally Myc-DDK-tagged Ptpn6 (tv1) (OriGene Technologies, Rockville, MD, USA; MR209258) by PCR using the forward 5′-GCTC GAATTC ATGGTGAGGTGGTTTC-3′ and reverse 5′-AGC GTCGAC CTTCCTCTTGAGAGAACCT-3′ primers.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Incubation

Journal: Cells

Article Title: Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

doi: 10.3390/cells8040345

Figure Lengend Snippet: The generation of SHP-1 deficient cell lines. ( A ) A schematic diagram of Ptpn6 with sites targeted by guide RNA (sgRNA) sequences: The targeted sites (blue) and protospacer-adjacent motifs (PAM; red) on the gene (18.01 kb) containing 16 exons are shown. The defined domains are indicated. ( B ) The PCR amplification of genomic DNA from the control cells (BMMC) and SHP-1-deficient cell lines (SHP-1_KO1, SHP-1_KO2, SHP-1_KO3) with primers flanking the deleted region: The template is not present in the control sample. Due to the large size of the deleted region (approx. 6 kb), no amplification was found in the control BMMCs. The amplification of short fragments (approx. 560 bp) was detected in SHP-1-deficient clones. ( C ) The SHP-1 protein levels in BMMCs and SHP-1-deficient cell lines analyzed by an immunoblotting of whole-cell lysates: GCP2 served as the loading control.

Article Snippet: To prepare C-terminally enhanced green fluorescent protein (EGFP)-tagged mouse SHP-1 (gene Ptpn6 ; RefSeq ID: NM_013545.3), the coding sequence without a stop codon was amplified from the C-terminally Myc-DDK-tagged Ptpn6 (tv1) (OriGene Technologies, Rockville, MD, USA; MR209258) by PCR using the forward 5′-GCTC GAATTC ATGGTGAGGTGGTTTC-3′ and reverse 5′-AGC GTCGAC CTTCCTCTTGAGAGAACCT-3′ primers.

Techniques: Amplification, Clone Assay, Western Blot

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Repression of HNF1α aggravates hepatic fibrogenesis in both DMN and BDL models. (A , B) Adenovirus carrying shRNA against HNF1α (shHNF1α) or negative control (shNC) was injected into rats prior to DMN administration (A) and BDL treatment (B) , and 2 weeks later the expression of HNF1α and α-SMA in the fibrotic livers was analyzed by immunohistochemistry. Hematoxylin and eosin (HE) and Sirius red staining were used to examine pathological alterations and collagen deposition. (C) Semi-quantitative analysis of Sirius red staining in the fibrotic livers from AdshHNF1α or AdshNC-treated rats ( n = 10 rats in each group). (D) mRNA levels of HNF1α , α-SMA and COL1A1 in the livers were detected by real-time PCR Scale bar, 100 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: shRNA, Negative Control, Injection, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: HNF1α overexpression attenuates hepatic fibrosis. (A , B) A single dose of adenovirus carrying human HNF1α gene (HNF1α) or control virus (GFP) was injected into rats after DMN injection (A) or BDL operation (B). The fibrotic livers were analyzed at 4 weeks after DMN treatment or 3 weeks after BDL. The expression of HNF1α and α-SMA was assessed by immunohistochemistry. HE and Sirius red staining were used to examine pathological alterations and collagen deposition. (C) Semi-quantitative analysis of Sirius red staining in the fibrotic livers from AdHNF1α or AdGFP-treated rats ( n = 6 rats in each group). (D) mRNA levels of HNF1α , α-SMA and COL1A1 in the livers were detected by real-time PCR. Scale bars, 100 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: Over Expression, Control, Virus, Injection, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Anti-fibrotic effect of HNF1α depends on the transcriptional activation of SHP-1. (A) Transcript level of HNF1α and SHP-1 in primary rat hepatocytes treated with AdshHNF1α or AdshNC. (B) Correlation between the mRNA levels of HNF1α and SHP-1 in human liver tissues. Each data point represents an individual sample, and the correlation coefficient (r) is shown. (C) A schematic representation of the promoter region of SHP-1 , the potential cis -acting elements for HNF1α (arrow), mutation sites and the fragment amplified in ChIP-PCR. (D) The nested deletion analysis shows the transactivation effect of HNF1α on rat SHP-1 promoter. (E) HNF1α occupancy at the SHP-1 loci detected by ChIP-PCR in freshly isolated hepatocytes. (F) Suppression of SHP-1 reverses the anti-fibrotic effect of HNF1α. AdshSHP-1 or AdshNC was simultaneously delivered with AdHNF1α into DMN-treated rats. Collagen deposition and the expression of HNF1α, SHP-1 and α-SMA were detected in the livers. (G) Hydroxyproline content was assayed in the fibrotic livers ( n = 9 rats in each group). Scale bars, 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: Activation Assay, Mutagenesis, Amplification, Isolation, Expressing

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Crosstalk between HSCs and hepatocytes in vitro . (A) A schematic representation of co-culture experiments with primary HSCs and hepatocytes isolated from rats. (B) Suppression of HNF1α in hepatocytes enhances the activation of HSCs. Endogenous HNF1α level in primary rat hepatocytes pretreated with AdshHNF1α or AdshNC was detected by western blot (left). mRNA levels of α-SMA and COL1A1 in HSCs were assessed by RT-PCR (right). (C) mRNA level of α-SMA and COL1A1 in HSCs co-cultured with AdshHNF1α- or AdshNC-treated hepatocytes. Antibody against IL-6, TNFα or TGFβ1 was added into the co-culture to block the corresponding cytokine. (D) Hepatocytes overexpressing HNF1α attenuates the activation of HSCs. Expression of exogenous human HNF1α and endogenous rat HNF1α in hepatocytes treated with AdHNF1α or AdGFP analyzed by western blot is shown in the top panel; mRNA levels of α-SMA and COL1A1 in HSCs are shown in the bottom panels. (E) Western blot analysis of HNF1α and SHP-1 in hepatocytes co-cultured with quiescent or activated HSCs for 48 h. Antibodies against TNFα, IL-6 and control IgG were used to block the cytokines in co-culture. (F) Expression of HNF1α and SHP-1 in hepatocytes transfected with miRNA inhibitors and then co-cultured with quiescent or activated HSCs for 48 h.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: In Vitro, Co-Culture Assay, Isolation, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Blocking Assay, Expressing, Control, Transfection

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: NCAPD3 contributes to cell proliferation in LC cells. ( A - B ) NCAPD3 expression was raised in LUSC patients with different cancer stages using the UALCAN database. ( C ) Survival analysis of LUSC patients according to the mRNA expression of the NCAPD3 using the log-rank test in Kaplan-Meier Plotter. ( D ) Protein expression levels of NCAPD3 in LC cell lines were detected by Western blotting. ( E ) Western blot assay was used to measure the knockdown and over-expression efficiency in LC cells. ( F ) CCK-8 assay and ( G ) colony formation assay were applied to estimate the influences of knockdown and over-expression of NCAPD3 on the proliferation of H1975 and HCC827, respectively. ** P < 0.01 , vs. normal group; ▲ P < 0.05 , ▲▲ P < 0.01 , vs. si-NC group or vector group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Expressing, Western Blot, Knockdown, Over Expression, CCK-8 Assay, Colony Assay, Plasmid Preparation

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: NCAPD3 inhibits apoptosis and promotes migration and invasion in LC cells. ( A ) Cell apoptosis in H1975 transfected with NCAPD3 siRNA and HCC827 transfected with NCAPD3 overexpression plasmids was assessed by Flow cytometry. ( B ) The levels of cell apoptosis-associated proteins were determined by Western blotting in transfected H1975 and HCC827 cells. ( C ) Cell migration and invasion in LC cell lines H1975 and HCC827 were assessed by Transwell assay. Scale bar, 100 μm. ( D ) The protein expression levels of EMT-related targets were detected through western blotting after transfection in H1975 and HCC827 cells. ▲ P < 0.05 , ▲▲ P < 0.01 , vs. si-NC group or vector group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Migration, Transfection, Over Expression, Flow Cytometry, Western Blot, Transwell Assay, Expressing, Plasmid Preparation

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: NCAPD3 induces aerobic glycolysis in LC cells. ( A ) Single-gene GSEA analysis of NCAPD3. ( B ) Glucose uptake in NCAPD3-knockdown H1975 cells and NCAPD3-over-expression HCC827 cells was measured with flow cytometry by using the fluorescent glucose analog 2-NBDG. ( C ) Lactate production was measured in LC cells with NCAPD3-knockdown or NCAPD3-over-expression using the Lactic Acid (LA) Content Assay Kit. ( D ) The extracellular acidification rate (ECAR) of NCAPD3-knockdown H1975 cells and NCAPD3-over-expression HCC827 cells was also monitored. ( E ) The ATP levels in NCAPD3-knockdown H1975 cells and NCAPD3-over-expression HCC827 cells were quantified using the ATP Assay Kit. ( F ) The protein levels of NCAPD3 and aerobic glycolysis-related targets HK2, PKM2, GLUT1, and LDHA were determined by Western blotting. ▲ P < 0.05 , ▲▲ P < 0.01 , vs. si-NC group or vector group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Knockdown, Over Expression, Flow Cytometry, ATP Assay, Western Blot, Plasmid Preparation

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: NCAPD3 regulated aerobic glycolysis in LC cells through the MEK/ERK/LDHA signaling pathway. ( A ) The protein levels of MEK/ERK pathway-associated targets were detected by western blotting in NCAPD3-knockdown H1975 cells and NCAPD3-over-expression HCC827 cells. ( B ) Cell proliferation in HCC827 cells treated with U0126 (50 µM) and NCAPD3 overexpression plasmids in alone or combination was assessed by a colony formation assay. ( C ) Glucose uptake in HCC827 cells treated with U0126 (50 µM) and NCAPD3 overexpression plasmids in alone or combination was assessed by Flow cytometry. ( D ) The lactate levels in HCC827 cells treated with U0126 (50 µM) and NCAPD3 overexpression plasmids in alone or combination were assessed by the Lactic Acid (LA) Content Assay Kit. ( E ) The protein expression levels of HK2, PKM2, GLUT1, LDHA, p-MEK, MEK, p-ERK, and ERK in HCC827 cells treated with U0126 (50 µM) and NCAPD3 overexpression plasmids in alone or combination were detected through western blotting. ▲ P < 0.05 , ▲▲ P < 0.01 , vs. vector group. # P < 0.05 , ## P < 0.01 , vs. Oe-NCAPD3 group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Western Blot, Knockdown, Over Expression, Colony Assay, Flow Cytometry, Expressing, Plasmid Preparation

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: Histone lactylation-NCAPD3 promotes aerobic glycolysis, cell proliferation, and migration in LC cells. ( A ) Western blotting reveals the global lactylation and H3K18la levels of NCAPD3-knockdown H1975 cells and NCAPD3-over-expression HCC827 cells. ( B ) The protein expression levels of NCAPD3 in H1975 and HCC827 cells with treatment of a series of concentrations of 2-DG or oxamate. ( C - D ) Proliferation and migration ( E ) of HCC827 cells with or without NCAPD3 over-expression treated with oxamate (20 mM) were evaluated by CCK-8, colony formation, and transwell assays, respectively. ( F - G ) Proliferation and migration ( H ) of H1975 cells with or without NCAPD3 knockdown treated with lactate (5 mM) were evaluated by CCK-8, colony formation, and transwell assays, respectively. ▲ P < 0.05 , ▲▲ P < 0.01 , vs. vector or si-NC group. ## P < 0.01 , vs. oxamate + vector or lactate + si-NC group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Migration, Western Blot, Knockdown, Over Expression, Expressing, CCK-8 Assay, Plasmid Preparation

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: NCAPD3 knockdown inhibited tumor growth of H1975 by MEK/ERK/LDHA axis in vivo. ( A ) Representative images of xenograft tumors in H1975-bearing tumor mice. ( B ) Tumor volume and ( C ) weight in mice. ( D ) Immunohistochemical (IHC) staining results showing the NCAPD3, Ki67, and LDHA protein expressions in tumor tissues of mice. Scale bar, 100 μm. ( E ) Western blot assays showing the global lactylation and H3K18la levels, the expression of Ki67, NCAPD3, Ki67, and LDHA, as well as MEK and ERK phosphorylation levels in tumor tissues of mice. ▲▲ P < 0.01 , vs. si-NC group

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: Knockdown, In Vivo, Immunohistochemical staining, Immunohistochemistry, Western Blot, Expressing, Phospho-proteomics

Journal: Cancer Cell International

Article Title: NCAPD3 contributes to lung cancer progression through modulated lactate-induced histone lactylation and MEK/ERK/LDHA axis

doi: 10.1186/s12935-025-03814-x

Figure Lengend Snippet: Schematic diagram illustrating effects of NCAPD3 in promoting lung cancer progression. NCAPD3 promotes cell proliferation and metastasis of LC cells by increasing LDHA and lactate production. Then, lactate-induced histone lactylation activates MEK/ERK pathway. Hence, it is probable that there is a synergistic reaction between histone lactylation and NCAPD3 in Lung cancer cell metabolism

Article Snippet: Next, the membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against NCAPD3 (1:1000, DF9413, Affinity, Jiangsu, China), Bax (1:1000, AF0120, Affinity), Bcl-2 (1:1000, AF6139, Affinity), Caspase-3 (1:1000, AF6311, Affinity), PCNA (1:2000, 10205-2-AP, proteintech), E-cadherin (1:1000, 3195T, Cell Signaling Technology), Vimentin (1:1000, ab20346, Abcam), MMP-2 (1:1000, 66366-1-Ig, proteintech, Proteintech Group, Inc, China), HK2 (1:1000, BF0283, Affinity), PKM2 (1:1000, AF5234, Affinity), GLUT1 (1:1000, AF5462, Affinity), LDHA (1:1000, bs-34202R, Bioss), p-MEK (1:1000, AF8035, Affinity), MEK (1:1000, AF6385, Affinity), p-ERK (1:1000, AF1015, Affinity), ERK (1:1000, 4695s, Cell Signaling Technology), Pan Kla (1:1000, PTM-1401, PTM-biolab, Hangzhou, China), H3K18la (1:1000, A18807, ABclonal Technology Co., Ltd., Wuhan, China), Histone H3 (1:1000, AF0863, Affinity), GAPDH (1:5000, D190090, Sangon, China), and β-actin (1:5000, 81115-1-RR, proteintech) overnight at 4 °C.

Techniques: