Journal: bioRxiv

Article Title: Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

doi: 10.64898/2026.04.16.718871

Figure Lengend Snippet: A-B. Pseudo-colored images of HeLa cells expressing ratioCitron1Low ( A ) and ratioCitron1High ( B ) in cytosol before and after addition of citrate. Scale bar: 100 µm. C-D. Δ R / R and Δ F / F values of ratioCitron1Low ( C ) and ratioCitron1High ( D ) versus time upon the addition of citrate. HeLa cells were incubated with 4 μM digitonin for 10 mins before the experiment and a final concentration of 20 mM citrate was added at t = 0 (ratioCitron1Low n = 16, ratioCitron1High n = 19, mean ± SD). E. Time courses of Δ R / R ₀ of ratioCitron1 variants (ratioCitron1Low ( n = 20), ratioCitron1High ( n = 10), ratioCitron1Low-con ( n = 5), ratioCitron1High-con ( n = 5)) expressed in HeLa cells upon the citrate titration. HeLa cells expressing ratioCitron1 variants were treated with digitonin and citrate titration was carried out. The final concentration of citrate is 100 μM at 0 min, 1 mM at 5 min, 10 mM at 10 min (mean ± SD). F-G. Δ R / R values of ratioCitron1 variants expressed in cytosol ( F ) and in mitochondria (mito-ratioCiton1) ( G ) of HeLa cells upon the addition of the ACLY inhibitor of BMS-303141. H. Δ R / R values of ratioCitron1 variants expressed in mitochondria (mito-ratioCitron1) of HeLa cells upon the addition of MPC inhibitor UK-5099 (final concentration: 25 μM). Δ F/F = ( F on - F off )/ F off , and F with excitation 405 nm or 470 nm and emission 518/45 nm. Δ R / R = ( R on - R off )/ R off , and R = ( F with excitation 470 nm and emission 518/45 nm) / ( F with excitation 405 nm and emission 518/45 nm).

Article Snippet: For imaging the treatment with MPC inhibitor UK-5099 (MedChemExpress) and ACLY inhibitor BMS-303141 (MedChemExpress), Hank’s balanced salt solution (HBSS; Nacalai Tesque, 09735-75) and 10 mM HEPES (Nacalai Tesque, 177557-94) was used as imaging buffer.

Techniques: Expressing, Incubation, Concentration Assay, Titration

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: GOLM1 interacts with ACLY and affects the phosphorylation of ACLY within macrophage (A) Venn diagram of identified proteins in Huh7, HIEC6, and NCI-H295R cells using IP-MS. (B-C) Validation of GOLM1 protein interactions with ACLY in the Thp1 (B) cells and BMDMs (C) by IP and western blot assay. (D) Immunofluorescence staining of GOLM1 and ACLY in Thp1 nc cells. Red, Glom1; green, ACLY; blue, DAPI. Shown are 2D-pictures (middle) of 3D-images (left), which were created using 5 overlaying 1.0 μm z-sections. (E and F) WB bands (E) and quantitative analysis (F) of the P-ACLY and GOLM1 protein expression levels in nucleus of Thp1 nc and Thp1 −/− cells. n = 3 independent experiments. (G) Assessment of P-ACLY content in the nucleus by immunofluorescence staining of P-ACLY and DAPI. Quantitative analysis was conducted using Coloc plugin in Image J. (H) WB analysis of P-ACLY, ACLY and GOLM1 protein contents in the nuclear and cytoplasmic fractions of siNC - and siGolm1 -Thp1 cells. n = 3 independent experiments. (I) WB analysis of P-ACLY, ACLY and GOLM1 protein contents in the nuclear and cytoplasmic fractions of vector- and Golm1 +/+ -Thp1 cells. n = 3 independent experiments. The data are presented as the means ± SEMs. The results presented in (F) were analyzed via two-tailed Student's t test. **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: HEK293T cells were transfected with Acly or Golm1 targeting siRNA (Sangon Biotech, Shanghai) and the desired plasmid using jetPRIME (101000046, Polyplus Transfection). siRNA was mixed with the transfection reagent in serum-free medium, incubated for 10 min, and added to cells at 50–60 % confluency.

Techniques: Phospho-proteomics, Protein-Protein interactions, Biomarker Discovery, Western Blot, Immunofluorescence, Staining, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: GOLM1 binds with ACLY and antagonizes the phosphorylation of ACLY by PKA (A) Schematic representation showing structural domains of Acly and the truncation mutants. Acly -A, amino acids 1–620; Acly -B, amino acids 621–1101; Acly -S455mut, with serine mutated to alanine at position 455. (B-D) IP and immunoblot analysis of the interaction between the GOLM1 and the ACLY-A, ACLY-B, and ACLY-S455mut in HEK293T cells transfected with the corresponding plasmids for 48 h. (E) IP and immunoblot analysis of the interaction between ACLY and PKA in Thp1 nc and Thp1 −/− cells. (F) IP and immunoblot analysis of the interaction between ACLY and PKA in siNC - and siGolm1 -HEK293T cells transfected with HA- Acly and His- Pka for 48 h. (G) IP and immunoblot analysis of the interaction ACLY, PKA, and GOLM1 in HEK293T cells transfected with Flag- Golm1 in a concentration-dependent manner followed by co-transfection with HA- Acly and His- Pka for 48 h. All blots are representative of 3 independent experiments.

Article Snippet: HEK293T cells were transfected with Acly or Golm1 targeting siRNA (Sangon Biotech, Shanghai) and the desired plasmid using jetPRIME (101000046, Polyplus Transfection). siRNA was mixed with the transfection reagent in serum-free medium, incubated for 10 min, and added to cells at 50–60 % confluency.

Techniques: Phospho-proteomics, Western Blot, Transfection, Concentration Assay, Cotransfection

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: Macrophage-specific deletion of Golm1 suppresses tumor growth relying on the elevated Nucleic P-ACLY (A) Schematic diagram of Thp1 −/− cell culture and inhibition of ACLY expression treatments. (B) WB analysis of the indicated protein expression levels in the nucleus of each treated group. n = 3 independent experiments. (C) The establishment of the different treated Thp1-Huh7 mixture tumor model on nude mice. (D) Proliferation curves of subcutaneous models involving Huh7 cells cocultured with different treated Thp1 cells. n = 5 nude mice per group. (E and F) Representative images (E) and statistical analysis (F) of subcutaneous tumor at the experimental endpoint in groups of nude mice. Scale bar = 20 mm. n = 5 nude mice per group. (G and H) WB bands (G) and quantitative analysis (H) of the indicated protein expression levels in subcutaneous tumors assayed by WB. n = 3 independent experiments. The data are presented as the means ± SEMs. The results presented in (D, F, and H) were analyzed via one-way ANOVA followed by Tukey's multiple comparisons test. ns, not statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: HEK293T cells were transfected with Acly or Golm1 targeting siRNA (Sangon Biotech, Shanghai) and the desired plasmid using jetPRIME (101000046, Polyplus Transfection). siRNA was mixed with the transfection reagent in serum-free medium, incubated for 10 min, and added to cells at 50–60 % confluency.

Techniques: Cell Culture, Inhibition, Expressing

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: Nucleic P-ACLY within Golm1 -knockout macrophage regulates Efemp1 expression through H3K27 acetylation (A) Total histone H3 acetylation levels in Thp1 nc and Thp1 −/− cells determined by Histone H3 acetylation detection Kit. n = 9 replicated wells. (B-C) WB detecting (B) and quantitative analysis (C) of the protein levels of various acetylated histone H3. n = 3 independent experiments. (D) PCA plot of CUT&Tag-seq data of experiments with Thp1 nc and Thp1 −/− cells. n = 3 samples/group. (E) Pie chart showing the distribution of H3K27ac peaks across annotated genomic regions in Thp1 nc and Thp1 −/− cells. (F) Heatmap showing the genomic occupancy of H3K27ac from −3 kb flanking TSS to + 3 kb flanking TES in Thp1 nc or Thp1 −/− cells. The x-axis represents the position of reads relative to the TSS, and the y-axis represents the read density. (G) CUT&Tag-seq volcano plot of different gene peaks between Thp1 nc and Thp1 −/− cells. (H) RNA-seq volcano plot of DEGs between BMDMs wt and BMDMs mko cells. n = 3 mice/group. (I) Overlap of differentially expressed genes identified by RNA-seq and CUT&Tag-seq. Secreted proteins with statistically significant differences are listed below. (J) IGV snapshot of Thp1 nc and Thp1 −/− cells showing the H3K27ac CUT&Tag signals at the Efemp1 genomic loci. (K) H3K27ac occupancy in the genome of Efemp1 analysed by ChIP-qPCR. n = 4 duplicates/group. (L) Relative expression of Efemp1 genes in Thp1 nc and Thp1 −/− cells determined by qPCR. n = 6 duplicates/group. The data are presented as the means ± SEMs. The results presented in (A, C, K, and L) were analyzed via two-tailed Student's t test. ns, not statistically significant. **p < 0.01; ***p < 0.001.

Article Snippet: HEK293T cells were transfected with Acly or Golm1 targeting siRNA (Sangon Biotech, Shanghai) and the desired plasmid using jetPRIME (101000046, Polyplus Transfection). siRNA was mixed with the transfection reagent in serum-free medium, incubated for 10 min, and added to cells at 50–60 % confluency.

Techniques: Knock-Out, Expressing, RNA Sequencing, ChIP-qPCR, Two Tailed Test