Journal: bioRxiv

Article Title: Targeting one-carbon metabolic vulnerabilities of metastasis with therapeutic potential

doi: 10.64898/2026.02.07.704548

Figure Lengend Snippet: ( A ) Schematic of the metastasis functional screening using molecular inhibitors in vivo. ( B, C, and D ) Gross pulmonary metastases from human melanoma A375sm (B), mouse melanoma B16F10 (C), and mouse rhabdomyosarcoma RMS14 (D) in response to molecular inhibitor in the experimental metastasis assay by tail vein injection. Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). Cal C, 100 nM calphostin C; Rho, 12nM Rho Inhibitor I; Rap, 10nM Rapamycin; H-89, 50nM PKA inhibitor; SB, 10µM p38 inhibitor SB203580; TSA, 300nM HDAC inhibitor Trichostatin A; MS, 10µM HDAC inhibitor MS-275; PD, 20µM MAPK inhibitor PD98059; LY, 10µM PI3K/AKT inhibitor LY294002. ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n = 10. ( E, F, and G ) The related cell viability of human melanoma A375sm (E), mouse melanoma B16F10 (F), and mouse rhabdomyosarcoma (G) cells, pretreated with molecular inhibitors. Data are represented as mean ± SEM of three independent experiments. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. ( H ) Gross pulmonary metastases from mouse melanoma B16F10 pretreated with 10µM PI3K/AKT inhibitor LY294002 at the indicated time (LY12, 12 hours, LY24, 24 hours and LY48, 48 hours). Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n=10. ( I ) Gross pulmonary metastases from mouse rhabdomyosarcoma RMS14 cells pretreated with 20µM MAPK inhibitor PD98059 at the indicated time (PD12, 12 hours, PD24, 24 hours and PD48, 48 hours). Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n=10.

Article Snippet: Calphostin C, Rho Inhibitor I, Rapamycin, PKA inhibitor H-89, p38 inhibitor SB203580, HADC inhibitor Trichostatin A and MS-275, MAPK inhibitor PD98059, PI3K/AKT inhibitor LY294002, Rottlerin, bisindoylmaleimide I (BMI), PKC β inhibitor I, and FDA-approved-Drug-Library (1562 compounds, HY-L022) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Functional Assay, In Vivo, Injection, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Targeting one-carbon metabolic vulnerabilities of metastasis with therapeutic potential

doi: 10.64898/2026.02.07.704548

Figure Lengend Snippet: (A) Schematic of microarray analysis to focus on the inhibition of tumor metastasis. (B) The significant differentiated gene expression patterns associated with the inhibition of tumor metastasis progression and the identification of metabolism pathways associated with tumor metastasis. The significantly expressed genes in B16F10 (a) or RMS14 (b) cells treated with inhibitors (Cal C, 100 nM calphostin; Rap, 10nM Rapamycin; 20µM MAPK inhibitor PD98059; LY, 10µM PI3K/AKT inhibitor LY294002) compared with DMSO control were filtered by a p-value of 0.05 and an absolute value of fold change of 1.5 in ANOVA analysis. a1, Cal C v.s. DMSO; a2, Rap v.s. DMSO; a3, LY24 v.s. DMSO; a4, LY48 v.s. DMSO in B16F10 cells. b1, Cal C v.s. DMSO; b2, Rap v.s. DMSO; b3, PD24 v.s. DMSO; b4, PD48 v.s. DMSO in RMS14 cells. ( C ) Gene signaling pathway (GO gene pathway) analysis revealed that top 15 biological pathways and functions within a given gene list from micrroarry analysis regulate one-carbon metabolism and de novo serine synthesis (SSP). Both p and FDR valuest were transformed to negative log (-log2) values. ( D ) Schematic of the one-carbon metabolism and De novo serine synthesis (SSP) pathways. ( E to H ) Validation of gene expression identified in cDNA microarray analysis by Western blot analysis. The human melanoma A375sm (E) and WM88 (F), mouse melanoma B16F10 (G), or mouse rhabdomyosarcoma RMS14 (H) cells were treated with molecular inhibitors. DMSO, as a mock control; Cal C, 100 nM calphostin C; Rho, 12nM Rho Inhibitor I; Rap, 10nM Rapamycin; SB, 10 µM p38 inhibitor SB203580; PD, 20 µM MAPK inhibitor PD98059; LY, 10 µM PI3K/AKT inhibitor LY294002; Rott, 5μM of rottlerin; BMI, 20 nM of bisindoylmaleimide I; Iβ, 20 nM of PKCβ inhibitor I. β -actin was used as a control.

Article Snippet: Calphostin C, Rho Inhibitor I, Rapamycin, PKA inhibitor H-89, p38 inhibitor SB203580, HADC inhibitor Trichostatin A and MS-275, MAPK inhibitor PD98059, PI3K/AKT inhibitor LY294002, Rottlerin, bisindoylmaleimide I (BMI), PKC β inhibitor I, and FDA-approved-Drug-Library (1562 compounds, HY-L022) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Microarray, Inhibition, Gene Expression, Control, Transformation Assay, Biomarker Discovery, Western Blot

Journal: bioRxiv

Article Title: Targeting one-carbon metabolic vulnerabilities of metastasis with therapeutic potential

doi: 10.64898/2026.02.07.704548

Figure Lengend Snippet: ( A ) Western blotting showed the expression of the indicated one-carbon and SSP metabolism pathway genes treated with different compounds, including identified new compounds comp4, comp7, and comp9. The cells were treated with 5 µM NCT503, 10 µM comp2, 10 µM comp3, 100 nM comp4, 10µM venetoclax, 10 µM comp6, 10 nM comp7, 30 nM CB-839, 10µM comp9, DMSO, 5 mM 2-DG, 1 mM BSO, 10 µM comp13, 10 µM comp14, 2 mM metf (Metformin) for 36 hours. ( B, C ) Seahorse analysis showed the effect of the different compounds on OCR (B) and ATP production (C). The cells were treated with 100 nM calphostin (Cal C), 10nM Rapamycin (Rap), 20µM MAPK inhibitor PD98059 (PD), 10µM PI3K/AKT inhibitor LY294002 (LY), 100 nM comp4, 5 µM NCT503, 10 nM comp7, 10µM comp9, 2 mM Metformin(Metf), 10 µM comp13, 10 µM comp14 and DMSO control. ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. ( D, E ) Representation of bioluminescence signals of tumor metastasis from before treatment (D) and treated for two weeks with indicated compouds (E) after A375sm cell transplantation in NSG mice tracked by of the In Vivo Imaging System (IVIS). C, DMSO control; NCT503, 20 mg/Kg; Comp2, 25 mg/Kg; Comp4, 30 mg/Kg; comp7, 3 mg/Kg; comp9, 10 mg/Kg; venetoclax, 12 mg/Kg. ( F ) Gross pulmonary metastases from mice transplanted with cells by tail vein injection and treated with different compounds. N=10. Graphs show the mean ± SEM. The p-value is shown by an unpaired t-test (two-tailed). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Calphostin C, Rho Inhibitor I, Rapamycin, PKA inhibitor H-89, p38 inhibitor SB203580, HADC inhibitor Trichostatin A and MS-275, MAPK inhibitor PD98059, PI3K/AKT inhibitor LY294002, Rottlerin, bisindoylmaleimide I (BMI), PKC β inhibitor I, and FDA-approved-Drug-Library (1562 compounds, HY-L022) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Western Blot, Expressing, Control, Transplantation Assay, In Vivo Imaging, Injection, Two Tailed Test

Journal: Inflammation

Article Title: Impaired BCAA Catabolism Contributes To Acute Lung Injury By Triggering Oxidative Stress and Inflammatory Response Via the MAPK Pathway

doi: 10.1007/s10753-025-02439-6

Figure Lengend Snippet: BCKA exacerbates LPS-induced macrophage inflammatory response by activating the MAPK pathway. ( A - B ) The protein level of p-ERK, ERK, p-JNK, JNK, p-p38, and p38 in primary peritoneal macrophages was detected using Western blot ( n = 5). ( C - D ) The supernatant of primary peritoneal macrophages was collected for the analysis of IL-1β and IL-6 ( n = 5). ( E ) Quantification of MDA in primary peritoneal macrophages ( n = 5). ( F ) Relative ROS level in lungs as assessed by DCF intensity ( n = 5). ( G ) Quantification of total SOD activity in primary peritoneal macrophages ( n = 5). * P < 0.05, ** P < 0.01, and *** P < 0.001. U0126: ERK inhibitor, SP600125: JNK inhibitor, SB203580: p38 inhibitor

Article Snippet: To investigate the effects of BCKA on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BCKA (500 μM) for 6 h. Similarly, to investigate the effects of BT2 on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BT2 (50 μM) for 6 h. To explore the mechanism by which BCAA metabolic dysregulation triggers inflammatory responses, we used MAPK inhibitors (U0126: ERK inhibitor, SP600125: JNK inhibitor, SB203580: p38 inhibitor; MedChemExpress) to intervene with macrophages.

Techniques: Western Blot, Activity Assay

Journal: Inflammation

Article Title: Impaired BCAA Catabolism Contributes To Acute Lung Injury By Triggering Oxidative Stress and Inflammatory Response Via the MAPK Pathway

doi: 10.1007/s10753-025-02439-6

Figure Lengend Snippet: The MAPK pathway is activated by BCKA to induce ALI. ( A ) Lung sections of mice were stained using H&E staining. Representative images of the staining are shown (Bar = 100 μm). ( B ) Inflammation score (based on H&E staining) expressed on a numerical scale ( n = 5). ( C ) BALF was collected and assayed for LDH activity ( n = 5). ( D ) Lung wet-to-dry ratio ( n = 5). ( E ) Total proteins in BALF ( n = 5). ( F ) Pulmonary function as assessed by lung compliance ( n = 5). ( G-H ) The mRNA expression of Il-1β and Il-6 in the lung tissue was determined by real-time PCR ( n = 5). ( I-K ) The numbers of total cells, macrophages, and neutrophils were determined in BALF ( n = 5). ( L ) MPO activity in the lungs ( n = 5). * P <0.05, ** P <0.01, and *** P <0.001.

Article Snippet: To investigate the effects of BCKA on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BCKA (500 μM) for 6 h. Similarly, to investigate the effects of BT2 on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BT2 (50 μM) for 6 h. To explore the mechanism by which BCAA metabolic dysregulation triggers inflammatory responses, we used MAPK inhibitors (U0126: ERK inhibitor, SP600125: JNK inhibitor, SB203580: p38 inhibitor; MedChemExpress) to intervene with macrophages.

Techniques: Staining, Activity Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Inflammation

Article Title: Impaired BCAA Catabolism Contributes To Acute Lung Injury By Triggering Oxidative Stress and Inflammatory Response Via the MAPK Pathway

doi: 10.1007/s10753-025-02439-6

Figure Lengend Snippet: Impaired BCAA catabolism contributes to ALI by triggering oxidative stress and inflammatory response via the MAPK pathway

Article Snippet: To investigate the effects of BCKA on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BCKA (500 μM) for 6 h. Similarly, to investigate the effects of BT2 on the inflammatory response of macrophages, primary macrophages were treated with LPS (100 ng/mL) with or without BT2 (50 μM) for 6 h. To explore the mechanism by which BCAA metabolic dysregulation triggers inflammatory responses, we used MAPK inhibitors (U0126: ERK inhibitor, SP600125: JNK inhibitor, SB203580: p38 inhibitor; MedChemExpress) to intervene with macrophages.

Techniques:

Journal: bioRxiv

Article Title: An APP-centered molecular gateway integrates innate immunity and retinoic acid signaling to drive irreversible metamorphic commitment

doi: 10.64898/2026.01.22.700939

Figure Lengend Snippet: (A) Settlement rate of inhibitor-treated larvae. The box plots with superimposed jitter plots display the larval settlement rate under various inhibitor treatments. The biofilm stimulus condition is used as the positive control. The concentration of each inhibitor is indicated on the horizontal axis. The data represent the settlement rate of larvae remaining attached out of 10 larvae across six independent biological replicates (total n = 60). Statistical significance among treatment groups was assessed using one-way ANOVA followed by Tukey’s HSD post hoc test, with grouping letters indicating significant differences ( p < 0.05); treatments sharing a letter are not significantly different. (B) Quantitative assessment of the functional hierarchy. The box plots with superimposed jitter plots show the Metamorphic Progression Scores (MPS) for larvae treated with various pharmacological inhibitors with or without all-trans retinoic acid (RA). The MPS was calculated based on the metamorphic stage reached by the larvae in the identical assays used for the settlement rate analysis in (A). The concentration of each inhibitor is indicated on the horizontal axis. The MPS represents the average metamorphic stage reached (0 = brachiolaria; 1 = early; 2 = middle; 3 = late; 4 = pre-juvenile; 5 = juvenile). Statistical significance among the treatment groups was assessed using one-way ANOVA followed by Tukey’s HSD post hoc test ( *p < 0.05; n.s., not significant). A significant RA-dependent rescue condition (a statistically significant increase in MPS compared with the inhibitor-alone condition) is highlighted in grey, establishing the functional hierarchy of the pathways relative to the RA commitment signal. (C) Representative image illustrating pathway functional hierarchy. Images show representative larval morphology under the control, inhibitor-only, and inhibitor + RA conditions. These images specifically represent the high-concentration inhibitor treatments (MyD88 inhibitor: 50 µM; MAPK inhibitors: 10 µM; IKKβ and HSP90AA1 inhibitors: 1 µM). MyD88 inhibition completely blocks the behavioral decision of settlement. JNK and p38 inhibition caused a distinct early-stage arrest (low MPS), and the effects of their inhibition were significantly rescued by RA co-treatment. In contrast, ERK inhibition arrested metamorphosis at the middle stage, and this block was not rescued by exogenous RA. Similarly, IKKβ and HSP90AA1 inhibition arrested metamorphosis at later stages, and this block was not rescued by exogenous RA, functionally placing all three pathways (ERK, IKKβ, and HSP90AA1) downstream of the RA commitment signal. Scale bar: 200 µm. Inhibitors used: T6167923 (MyD88 inhibitor), IKK-16 (IKKβ inhibitor), U0126 (ERK inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 inhibitor), and Luminespib (HSP90AA1 inhibitor).

Article Snippet: The inhibitors were dissolved in DMSO and applied at the indicated concentrations: the MyD88 inhibitor T6167923 (5 or 50 μM; MedChemExpress), the IKKβ inhibitor IKK-16 (0.1 or 1 μM; MedChemExpress), and MAPK inhibitors SP600125 (JNK), SB202190 (p38), and U0126 (ERK) (1 or 10 μM; MedChemExpress or FUJIFILM Wako Pure Chemical Corporation), and the HSP90AA1 inhibitors luminespib (0.1 or 1 μM; Chemscene).

Techniques: Positive Control, Concentration Assay, Functional Assay, Control, Inhibition, Blocking Assay