Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 1 Deletion of ATR in Purkinje cells leads to locomotor dysfunction and learning defects in mice. a Sagittal brain sections of the control (Ctr) and ATR-PCΔ (PCΔ) brains were stained with cresyl violet (Nissl staining). Upper panel shows the complete cerebellum. The magnified view of the frames in the upper and mid panels are shown in the lower panels, respectively. The images are representative from 3 to 4 mice of each genotype analyzed. DCN Deep Cerebellar Nuclei, ML Molecular Layer, PCL Purkinje Cell Layer, GCL Granular Cell Layer. b Sagittal sections of 3-month-old control and ATR-PCΔ mice were stained with DAPI, Calbindin and GFAP antibodies to label nuclei, Purkinje cells and Bergmann glia, respectively. ML Molecular Layer, PCL Purkinje Cell Layer, GCL Granular Cell Layer. c Quantification of total Purkinje cell number compared to control at the indicated age. The data is represented as fold changes compared to the control group. The number of mice is indicated within the bar. Error bars indicate SD. Student’s t-test (unpaired, two- tailed) is performed for the statistical analysis. The p values are indicated in the graphs. d Quantification of the thickness of the molecular layer of the cerebellum of mice at the indicated age. The number of mice is indicated within the bar. Student’s t-test (unpaired, two-tailed) is performed for the statistical analysis. The p values are indicated in the graphs. e The rotarod performance of 4–9-month-old mice on five consecutive days. Error bars indicate SEM. P values: day 2, p = 0.014; day 3, p = 0.002; day 5, p = 0.028; group comparison p = 0.034. f The rotarod performance of 18–20-month-old mice on five consecutive days. Error bars indicate SEM. P values: day 1 p = 0.025; day 5, p = 0.024, group comparison p = 0.041. g The beam walking performance was tested in the 18–20-month-old group. The latencies on 1 cm width beam platform on three consecutive days are shown. Error bars indicate SEM. p = 0.072. h The number of hindlimb slips during the beam walking performance test. Error bars indicate SEM. P values: day 2, p = 0.015; group comparison, p = 0.017. Two-way repeated measures ANOVA with Sidak’s multiple comparisons test or MWU within individual days was performed for the statistical analysis in e–h. *p < 0.05, **p < 0.01. The number of mice (n) tested are indicated within the graph legend. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Control, Staining, Two Tailed Test, Comparison

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 3 ATR deletion in excitatory neurons of the mouse forebrain (ATR-FBΔ) leads to developments of epileptic signs. a Dorsal views of the brains from 3-month-old control (Ctr) and ATR-FBΔ (FBΔ) mice. Ob Olfactory Bulb, Ctx Cortex, Cb Cerebellum. b Coronal brain sections of 3-month-old control and ATR-FBΔ brains were stained with Nissl. The upper panel shows the complete half hemisphere and the lower panel displays the magnified images of the hippocampal regions. The images are representative from 3 to 4 mice per genotype analyzed. Ctx Cortex, HC Hippocampus, Amy. Amygdala, DG Dentate Gyrus, CA Cornu Ammonis, H Hilus. c Hippocampal astrogliosis in ATR-FBΔ mice. Coronal sections from control and ATR-FBΔ littermates at 10–12 months old after epileptic seizures were stained with DAPI, GFAP and NeuN to label nuclei, astrocytes and post-mitotic neurons, respectively. Right panels show magnifications of the dentate gyrus. Neurons are leaving the dentate gyrus (arrows) and expression of GFAP is markedly increased. CA Cornu Ammonis, DG Dentate Gyrus, H Hilus, GL Granular Layer. The graph shows the quantification of GFAP-positive cells within the hippocampal area from at least 2–3 sections per animal. The number of mice used is indicated within the bar. Data are presented as mean values of ± SD. Student’s t-test (unpaired, two- tailed). p = 0.013. *p < 0.05. d Mossy fiber sprouting in the hippocampus of 10-month-old epileptic ATR-FBΔ mice. Coronal sections of brains were stained with DAPI, ZnT-3 and SMI312 antibodies to label nuclei, mossy fibers and axonal neurofilaments, respectively. White arrows indicate the mossy fiber sprouting in IML. Neurofilaments follow the same pattern of ZnT-3 staining. The magnified images of the white rectangles on the DG and CA3 areas are shown in the right panels. The images are representative from 3 mice per genotype analyzed. CA Cornu Ammonis, DG Dentate Gyrus, SPMF Suprapyramidal Mossy Fibers, IPMF Infrapyramidal Mossy Fibers, H Hilus, GL Granular Layer, ML Molecular Layer, IML Inner Molecular Layer. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Control, Staining, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 4 The CA3 region is susceptible to epileptiform activity in the ATR-deleted hippocampus. a Deletion of ATR increases the amplitude of the preictal epileptiform discharges (PEDs). Example 30-second sections of 40-minute extracellular field potential recordings of PEDs in CA3. Zoom-in representative PEDs (0.5–5000 Hz) in population field activity band (PFA, 1–100 Hz) and fast ripple activity band (FRA, 200–500 Hz). b PEDs show higher instantaneous power in ATR-FBΔ (FBΔ) CA3 slices compared to controls (Ctr). Color-coded raster plots of the PED timing in all recorded slices. Colors represent the maximum instantaneous power of each PED in 1–100 Hz band. The slices are arranged in descending order, by the first slice having the highest number of detected PEDs. The slices containing PEDs with a power-level beyond 0.4 µV2 (high-activity PEDs) are designated with a black square next to them. The ratio of slices with high-activity versus those with low-activity PED instantaneous power is increased after ATR deletion (right panel); Chi-square test (one- tailed), p = 0.012. c The instantaneous power of the whole field potential signal in 1–100 Hz band, computed within non-overlapping 1-min bins. The mean (solid line) ± SEM (shaded area). d Same as (c), but only for isolated PEDs (peak values) within 5-min bins. A one-tailed permutation test method of Cohen is used to compare the results of the two groups at each bin, with p < 0.05 (see Methods). e ATR-deletion leads to an increase in the power spectrum of PEDs over 1–100 Hz band. Power spectral density (PSD) of the PED events (solid lines), together with that of the baseline (i.e. non-PED epochs; dotted lines). f The summed baseline-normalized power presents an increase in the power of PEDs over 1–100 Hz shown in (e). Data presented as mean ± SEM. g ATR-deletion induces stronger fast ripples within PEDs. Same as (f), but for the fast ripple band (200–500 Hz). h Same as (f), but for the gamma band (30–90 Hz). i ATR-deletion increases the rate of spikes within PEDs, as detected in the multiple unit activity signal (MUA, > 500 Hz). Data are obtained from 16 slices of 4 control and 18 slices of 4 ATR-FBΔ mice and presented as mean ± SEM. Two-tailed t-test for (e–i): p = 0.026 for (e) and (f), p = 0.023 for (g), p = 0.027 for (h), and p = 0.016 for (i). *p < 0.05. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Activity Assay, One-tailed Test, Isolation, Control, Two Tailed Test

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 7 ATR deletion in neurons does not cause activation of the DDR. a Western blot analysis of the hippocampus of 3-month-old control (Ctr) and ATR- FBΔ (FBΔ) mice for indicated proteins. The DDR activation is controlled by HCT116 cells without (−) or with (+) tBHP for 15 min. GAPDH serves as loading control. Four mice (numbers on the top of lanes) of the indicated genotypes were analyzed. b Immunostaining of ATR-PCΔ cerebellum with antibodies against Calbindin (for PCs) and γH2AX (DNA damage marker, white arrows). The γH2AX signal is controlled by the cortical sections of mice after 4 Gy ionizing irradiation (IR). The images are representative from 3 to 4 mice per genotype analyzed. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Activation Assay, Western Blot, Control, Immunostaining, Marker, Irradiation

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 8 ATR deletion upregulates presynaptic proteins SYT2 and PROT. a Western blot analysis of the indicated proteins in whole-cell extract (S1) and synaptosome (P2) fractions of 3-month-old hippocampus of control (Ctr) and ATR-FBΔ (FBΔ) mice. GAPDH controls loading. Note, GAD67 expression is controlled by a separate control GAPDH below. The right panel shows the quantification of the indicated proteins after normalization with GAPDH. The data is represented as relative fold change in protein expression in P2 fraction, compared to control mice. N = 3 mice for each genotype. The vertical dashed line in the graph indicates control mice. Data are mean values ± SD. Student’s t-test (unpaired, one-tailed for ATR, SYT2 and PROT, two-tailed for others). P value: ATR, p = 0.025; SYT, p = 0.001; PROT, p = 0.029. *p < 0.05, **p < 0.01. b ATR interacts with SYT2 and PROT in vitro. Murine neuroblastoma cells (N2a) were transfected with Tomato-SYT2 or Tomato-PROT treated with or without with 2 µM ATR inhibitor VE-821. SYT2 and PROT were immunoprecipitated with Tomato antibody and blotted by indicated antibodies. The experiment was repeated four times. c Immunoprecipitation of protein extract from hippocampi and cerebella using antibodies as indicated. IP against IgG serves as control. Histone H3 serves as loading control for the inputs. IgG light chain was used to control the amount of antibodies used for IP. Note, an overloading of IgG in IP-IgG lanes correlates with unspecific binding signals in these samples. The number indicates individual mice. The experiment was repeated twice. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Western Blot, Control, Expressing, One-tailed Test, Two Tailed Test, In Vitro, Transfection, Immunoprecipitation, Binding Assay

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of MRE11 phosphorylation kinetics at MRE11S676S678. ( A ) MRE11pS676pS678 antibody immunoprecipitate the phosphorylated form of MRE11 after ionizing radiation (IR). Western blot (MRE11) of immunoprecipitated MRE11 using an antibody against MRE11S676S678 (nonP) site and the corresponding phosphorylated MRE11pS676pS678 (pSpS) sites from control and ATLD2 lymphoblastoid cells treated with 0 or 10 Gy IR. Unbound sample from each immunoprecipitation was run in parallel and also immunoblotted for total MRE11 (GeneTex). ( B ) Dose-dependent increase of MRE11 phosphorylation following IR. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control and A-T lymphoblastoid cells treated with 0, 2, 5 or 10 Gy IR. ATLD2 cells were also run as a negative control (0 and 10 Gy). Western blot for the MRN complex proteins, MRE11, RAD50 and NBS1. ( C ) Increasing MRE11 phosphorylation over 2 h post 5 Gy. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control lymphoblastoid cells either untreated (−) or harvested at 0.6, 2, 4 or 10 h post 5 Gy IR. ATLD2 lymphoblastoid cells were either untreated (−) or harvested at 2 and 10 h post 5 Gy IR and run as a negative control. ( D ) MRE11 phosphorylation in response to DNA double strand break inducing agents. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control cells treated with either IR, camptothecin (CPT), hydrogen peroxide (H2O2), etoposide (ETOP), cisplatin (CISP) or methlymethanesulfonate (MMS).

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of ATM signalling, cell survival and chromosomal aberrations in the cell line expressing non-phosphorylatable MRE11S676AS678A. ( A ) Stable MRN complex in WT and non-phosphorylatable mutant MRE11 corrected cell lines. Western blot of immunoprecipitated MRE11 from control (MCR5) and ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines. Also immunoblotted for NBS1 and RAD50. ( B ) ATM signalling in the non-phosphorylatable mutant MRE11 cell line is comparable to the WT corrected cell line. Total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines were extracted 30 min post 5 Gy (+) or left as unirradiated controls (−) and western blotted for ATM S1981 autophosphorylation and total ATM, as well as the ATM kinase substrates, SMC1 and KAP1. Tubulin was immunoblotted as loading control. ( C ) The non-phosphorylatable mutant MRE11 cell line displays increased cellular sensitivity to IR. Plot of percent survival after 0, 1, 2, 3, 4 and 5 Gy IR. NFF (control), ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), A-T, ATLDS676A (S676A) and ATLDS678A (S678A) cell lines were treated with increasing doses of IR and percent survival assessed by clonogenic cell survival assay. The mean ± standard deviation is plotted from 3 independent experiments. ( D ) Non-phosphorylatable mutant MRE11 cell line is unable to correct radiation induced chromosomal aberrations. Aberrations (chromatid breaks, chromosome breaks and interchanges) were scored from Giemsa stained metaphases in 2 Gy irradiated NFF (control), A-T, ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), ATLDS676A (S676A) and ATLDS678A (S678A) cell lines. Induced chromosomal aberrations (ICA) were totalled and divided by number of metaphases as indicated in parentheses.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clonogenic Cell Survival Assay, Standard Deviation, Staining, Irradiation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Schematic graph of animal sensitization and challenge. B Histological analysis of lung tissues stained with HE staining or PAS staining. C Histology score of HE staining and PAS staining. Statistical significance was determined with Mann-Whitney test. D mRNA expression of Bhlhe40 was assayed by RT-qPCR. Statistical analysis was performed by two-tailed Student’s t-test. E Western blot analysis of BHLBE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. F Immunohistochemistry staining was performed to detected the level of BHLHE40 in lung tissue. Statistical analysis was performed by two-tailed Student’s t-test. G Double immunofluorescence staining for BHLHE40 (red) and F4/80 (green) in lung tissues. The number of double positive (F4/80 + BHLHE40 + ) cells was quantified. Statistical analysis was performed by two-tailed Student’s t-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Immunohistochemistry, Double Immunofluorescence Staining, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice in asthma group received lentiviral delivery of LV- shBhlhe40 or LV-shNC. B RT-qPCR tested the mRNA expression of Bhlhe40 in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Representative western blot and quantitative analysis of BHLHE40 expression in lung tissues. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative HE images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. E Representative PAS images in lung tissue sections and quantitative analysis of histologic scoring. Statistical analysis was performed by Kruskal-Wallis test. F Total protein concentrations in BALF in OVA-induced asthma. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 8 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A ELISA forYM1 in BALF. B – E RT-qPCR assay showed the mRNA expression of Ym1 , Fizz1 , Cd206 , Arg1 in lung tissues. F The protein level of CD206 and ARG1 in lung tissues. G Quantitative analysis of CD206 protein level. H Quantitative analysis of ARG1 protein level. I Western blot assessed the level of BHLHE40, CD206, and ARG1 in monocyte-derived alveolar macrophages (Mo-AMs). J Quantitative analysis of BHLHE40 protein level in Mo-AMs. K Quantitative analysis of CD206 protein level in Mo-AMs. L Quantitative analysis of ARG1 protein level in Mo-AMs. N = 8 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Derivative Assay, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Bone marrow derived macrophages (BMDMs) were isolated from BALB/c mice, and then stimulated with IL-4 to induce M1 polarization. Lentivirus infection was conducted before IL-4 treatment. B The expression level of Bhlhe40 was determined by RT-qPCR. C Western blot analysis of BHLBE40 in BMDMs. D – G The mRNA expression level of Ym1 , Fizz1 , Cd206 , and Arg1 in BMDMs. H The protein level of CD206 and ARG1 in BMDMs. I Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. N = 4 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Derivative Assay, Isolation, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A , B Nrtn expression level was determined by RT-qPCR and western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. C Dual-luciferase reporter assay. N = 3 biological replicates. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in BMDMs. Statistical analysis was performed by two-tailed Student’s t-test. E RT-qPCR showed the mRNA expression of Fizz1 , Ym1 , Arg1 , and Cd206 . Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F The protein level of CD206 and ARG1 was assayed by western blot. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. G Immunofluorescence analysis of ARG1 expression in BMDMs, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 3-4 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Immunofluorescence, Cell Counting, Standard Deviation

Journal: Communications Biology

Article Title: BHLHE40 contributes to allergic asthma progression in mice through NRTN downregulation in macrophages

doi: 10.1038/s42003-025-08288-1

Figure Lengend Snippet: A Mice were stimulated with thioglycolate and IL-4 by intraperitoneal injection, and then physiological peritoneal macrophages were obtained. B Validation by ChIP-qPCR analysis of BHLHE40 binding to Nrtn in peritoneal macrophages. Statistical analysis was performed by two-tailed Student’s t-test. C RT-qPCR examined the mRNA level of Bhlhe40 in peritoneal macrophages. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. D Representative immunoblot and quantitative analysis of BHLHE40 expression. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. E Immunofluorescence analysis of ARG1 expression in peritoneal macrophages, and the quantification of ARG1 positive cell count. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. F Peritoneal macrophages were infected with LV- shBhlhe40 and LV- shNrtn , and then were subjected to immunofluorescence staining for ARG1. The number of ARG1 positive cells was quantified. Statistical analysis was performed by One-way ANOVA with Tukey’s post-test. Error bars represent standard deviation. N = 4 biological replicates.

Article Snippet: For double immunofluorescence staining, slides were incubated with primary antibody against BHLHE40 (1:500, NB100-1800SS, Novus), and F4/80 (1:50, Sc-377009, Santa cruz) at 4 °C overnight.

Techniques: Injection, Biomarker Discovery, ChIP-qPCR, Binding Assay, Two Tailed Test, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Cell Counting, Infection, Staining, Standard Deviation