Journal: Cell reports

Article Title: Identification of CCL3 as a Schwann cell chemotactic factor essential for nerve regeneration.

doi: 10.1016/j.celrep.2025.115322

Figure Lengend Snippet: Figure 2. CCL3 is a chemoattractant for Schwann cells (A) Rat SC migration in Boyden chambers in response to no factors (control), 3% serum (serum), or 10 or 50 ng/ml CCL3 diluted in 0.1% bovine serum albumin/ PBS (n = 8 independent experiments).

Article Snippet: Bridge fibroblasts, bridge macrophages, bridge CM, mouse macrophage cell line CM (from cells cultured at 20% O2,1.5% O2 alone or treated with scrambled short interfering RNA (Scr siRNA) or CCL3 siRNAs 1–3), recombinant rat CCL3 at 10 and 50 ng/ml (R&D systems) and recombinant human CCL3 at 50 ng/ml (R&D systems) were used to test the role of macrophage derived CCL3 in SCmigration.

Techniques: Migration, Control

Journal: Cell reports

Article Title: Identification of CCL3 as a Schwann cell chemotactic factor essential for nerve regeneration.

doi: 10.1016/j.celrep.2025.115322

Figure Lengend Snippet: Figure 4. CCL3 ablation impairs Schwann cell migration and axonal regrowth following injury (A and B) Representative confocal images of immunostained longitudinal sections of control and CCL3 KO mouse sciatic nerves at Day 7 after transection to detect SCs (eGFP, green, PLP- eGFP mice), axons (NF, white), and nuclei (Hoechst, blue). Dashed lines indicate the region of the nerve bridge. Scale bars, 300 mm. (C and D) Quantification of SC area (C) and axonal area (D) (n = 7 control mice, n = 8 CCL3 KO mice). (E and F) (E) Representative images of SC cords (eGFP, green) from the bridge showing organized, aligned cords of SCs in control mice, which are more disorganized in CCL3 KO animals. Scale bar, 100 mm, quantified in (F) (n = 169 SC cords from 7 control mice; n = 251 SC cords from 8 CCL3 KO mice). (G) Representative confocal images of longitudinal sections of control and CCL3 KO injured sciatic nerves at Day 5 following injury, immunostained for blood vessels (CD31, magenta). Scale bar, 300 mm. (H) Quantification of blood vessel (BV) area in the nerve bridge (n = 7 control mice, n = 7 CCL3 KO mice). Data are presented as mean ± SEM. For (C), (D), and (H), an unpaired two-tailed Student’s t test was used. For (F), an unpaired two-tailed Welch’s t test was used. *p < 0.05; **p < 0.01; ns, not significant. See also Figure S4.

Article Snippet: Bridge fibroblasts, bridge macrophages, bridge CM, mouse macrophage cell line CM (from cells cultured at 20% O2,1.5% O2 alone or treated with scrambled short interfering RNA (Scr siRNA) or CCL3 siRNAs 1–3), recombinant rat CCL3 at 10 and 50 ng/ml (R&D systems) and recombinant human CCL3 at 50 ng/ml (R&D systems) were used to test the role of macrophage derived CCL3 in SCmigration.

Techniques: Migration, Control, Two Tailed Test

Journal: Translational Psychiatry

Article Title: Methylphenidate enhances neuronal differentiation and reduces proliferation concomitant to activation of Wnt signal transduction pathways

doi: 10.1038/s41398-018-0096-8

Figure Lengend Snippet: a The time line of cell culturing, treatment with either MPH (1 nM–100 μM), GBR12909 (selective dopamine transporter inhibitor; 1 nM–100 μM) or R-spondin 1 (R-spo1; Wnt activator, 100 ng/ml) and proliferation measurements. b The time line of cell culturing, inhibiting Wnt-signaling by treating cells with Dkk1 (LRP inhibitor; 200 ng/ml) half an hour before MPH treatment and proliferation measurements. c Mean cell index (impedance measured by xCELLigence) values as % of control ± s.e.m. at 16 h after MPH treatment (1 nM–100 µM) showing significant decrease in cell proliferation. d Mean of BrdU positive cells as percentage of control ± s.e.m. at 16 h after MPH treatment (1 nM–100 µM) resulted in lower cell proliferation. Blocking Wnt-signaling using Dkk1 enhanced significantly cell proliferation. Similarly, inhibiting Wnt-signaling with Dkk1 30 min before MPH treatment resulted in increased proliferation. As expected, activation of Wnt-signaling by R-spondin 1 reduced proliferation of the cells. Moreover, the selective dopamine transporter inhibitor GBR12909 did not affect SH-SY5Y cell proliferation. n = 5 independent experiments; N = 18302 counted cells; Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Moreover, the selective dopamine transporter inhibitor GBR12909 did not affect SH-SY5Y cell proliferation. n = 5 independent experiments; N = 18302 counted cells; Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001 Fig. 4 Influence of MPH treatment on SH-SY5Y cells differentiation and the involvement of Wnt-signaling. a The time line of cell culturing, treatment with either MPH (1 nM–100 μM), GBR12909 (selective dopamine transporter inhibitor; 1 nM–100 μM) or R-spondin 1 (R-spo1; Wnt activator, 100 ng/ml) and neurite outgrowth determination via ‘Neurite outgrowth staining kit’ (Thermo Fisher) for differentiation evaluation. b The time line of cell culturing, inhibiting Wnt-signaling by treating cells with Dkk1 (LRP inhibitor; 200 ng/ml) half an hour before MPH treatment and differentiation evaluation. c Representative neurite outgrowth staining of vehicle, MPH (100 μM), GBR12909 (10 μM), Dkk1 (200 ng/ml), Dkk1 + MPH (100 μM) and R-spondin 1 (100 ng/ml) treated SH-SY5Y cells.

Techniques: Cell Culture, Control, Blocking Assay, Activation Assay, MANN-WHITNEY

Journal: Translational Psychiatry

Article Title: Methylphenidate enhances neuronal differentiation and reduces proliferation concomitant to activation of Wnt signal transduction pathways

doi: 10.1038/s41398-018-0096-8

Figure Lengend Snippet: a The time line of cell culturing, treatment with either MPH (1 nM–100 μM), GBR12909 (selective dopamine transporter inhibitor; 1 nM–100 μM) or R-spondin 1 (R-spo1; Wnt activator, 100 ng/ml) and neurite outgrowth determination via ‘Neurite outgrowth staining kit’ (Thermo Fisher) for differentiation evaluation. b The time line of cell culturing, inhibiting Wnt-signaling by treating cells with Dkk1 (LRP inhibitor; 200 ng/ml) half an hour before MPH treatment and differentiation evaluation. c Representative neurite outgrowth staining of vehicle, MPH (100 µM), GBR12909 (10 µM), Dkk1 (200 ng/ml), Dkk1 + MPH (100 µM) and R-spondin 1 (100 ng/ml) treated SH-SY5Y cells. Scale bar = 50–100 µM. d Neurite outgrowth was significantly increased after MPH treatment, which was inhibited significantly by Dkk1 treatment. Similar to MPH, R-spondin 1 treatment increased significantly cell differentiation. However, the selective dopamine transporter inhibitor, GBR12909, caused a significant decrease in SH-SY5Y cell differentiation, while the higher dose of 100 µM induced cell death. n = 3–5 independent experiment; N = 2172 measured cells; Mann–Whitney test, ** p < 0.05, *** p < 0.001 vs. control

Article Snippet: Moreover, the selective dopamine transporter inhibitor GBR12909 did not affect SH-SY5Y cell proliferation. n = 5 independent experiments; N = 18302 counted cells; Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001 Fig. 4 Influence of MPH treatment on SH-SY5Y cells differentiation and the involvement of Wnt-signaling. a The time line of cell culturing, treatment with either MPH (1 nM–100 μM), GBR12909 (selective dopamine transporter inhibitor; 1 nM–100 μM) or R-spondin 1 (R-spo1; Wnt activator, 100 ng/ml) and neurite outgrowth determination via ‘Neurite outgrowth staining kit’ (Thermo Fisher) for differentiation evaluation. b The time line of cell culturing, inhibiting Wnt-signaling by treating cells with Dkk1 (LRP inhibitor; 200 ng/ml) half an hour before MPH treatment and differentiation evaluation. c Representative neurite outgrowth staining of vehicle, MPH (100 μM), GBR12909 (10 μM), Dkk1 (200 ng/ml), Dkk1 + MPH (100 μM) and R-spondin 1 (100 ng/ml) treated SH-SY5Y cells.

Techniques: Cell Culture, Staining, Cell Differentiation, MANN-WHITNEY, Control

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 2. Ovulation rate declines with increasing body weight but is improved by treatment with ER stress inhibitors. (A) Hematoxylin and Eosin-stained ovary sections from 14-week-old gonadotropin-treated lean (+/+ or +/bbb) and obese Blobby (bbb/bbb) mice. Arrows indicate unruptured follicles, which are prevalent in ovaries from obese mice. Representative examples from n=4 mice per genotype are shown. Panels a-c are higher magnifications of the indicated follicles of the Blobby mouse. (B) The number of ovulated oocytes in 14-week-old lean and obese mice treated with saline vehicle (Veh.), salubrinal (Sal) or BGP-15 i.p. once per day for 4 days. Values are means+s.e.m. (lean mice+Veh, n=19; lean+Sal, n=5; lean+BGP-15, n=10; obese mice+Veh, n=22; obese+Sal, n=16; obese+BGP-15, n=14). Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test; P<0.0001. Right panel: the number of ovulated oocytes in oviducts is not reduced in bbb/bbb mice prior to the onset of obesity, at 6 weeks of age (+/+; +/bbb: n=11; bbb/bbb n=4; Student’s t-test).

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Staining, Saline

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 5. Treatment with salubrinal or BGP-15 induces mtDNA replication and normalizes mitochondrial membrane potential (ΔΨm) and autophagy in oocytes from obese mice. Obese mice (bbb/bbb) or lean littermates (+/+ or +/bbb) were treated with vehicle (Veh), salubrinal (Sal) or BGP-15 i.p. once daily for 4 days, and oocytes were collected from the oviducts. (A) Live oocytes stained with JC-1, where red fluorescence indicates high ΔΨm, and green indicates low ΔΨm. (B) Ratio of red to green fluorescence, an indicator of mitochondrial activity. Data are presented as means+s.e.m., n=6-30 oocytes from four mice per group. (C) Live oocytes were assessed for autophagic vacuoles, visualized as green fluorescence. Hoechst 33342 (blue). (D) Autophagy levels were quantified as the sum total of green fluorescence within each oocyte. Data are presented as means+s.e.m., n=8-10 oocytes from three mice per group. (E) mtDNA copy number in individual oocytes collected from treated mice. Data are presented as means+s.e.m.; n=19-38 oocytes from six mice per group over three independent experiments. (F) Representative examples of oocytes from three or four mice per treatment group immunostained for TFAM or DRP1 (green) and Hoechst 33342 (blue). Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test, P<0.0001.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Membrane, Staining, Fluorescence, Activity Assay

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 6. Oocytes from obese Blobby mice exhibit impaired embryo development that is restored by treatment with salubrinal or BGP-15. Obese Blobby (bbb/bbb) mice or lean littermates (+/+ or +/bbb) at 14 weeks of age (except where indicated) were treated with vehicle (Veh; saline), salubrinal (Sal) or BGP-15 daily for 4 days. Ovulated oocytes were collected from oviducts 16 h following hCG injection and fertilized in vitro. (A) At 4 h after in vitro fertilization, many degenerate oocytes (arrows) from untreated obese mice were observed. (B) The percentage of viable oocytes presented as means+s.e.m. Embryo development was assessed on day 2 and day 5 following IVF and is presented as the mean percentage of embryos exhibiting appropriate (‘on-time’) development+s.e.m. (C) Two-cell embryos developed from viable oocytes by day 2. (D) Blastocysts developed from two-cell embryos by day 5. Lean, n=24; lean+Sal, n=7; lean+BGP- 15, n=6; Blobby, n=15; Blobby+Sal, n=6; Blobby+BGP-15, n=7 pools of oocytes from animals within same group. [C,D, right-hand panels are identical assessments of oocytes from 6-week-old (non-obese) mice. +/+, n=13; Blobby, n=10.] (E) Blastocysts were collected at day 5 and the mtDNA copy number of individual blastocysts was measured by using quantitative PCR. Data are expressed as mean copy number+s.e.m.; lean, n=22 blastocysts; lean+Sal, n=10 blastocysts; lean+BGP-15, n=7 blastocysts; Blobby, n=14 blastocysts; Blobby+Sal, n=10 blastocysts; Blobby+BGP-15, n=7 blastocysts collected from three independent IVF experiments. Different letters indicate significant differences by one-way ANOVA, Tukey’s post hoc test; P<0.0001.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Saline, Injection, In Vitro, Real-time Polymerase Chain Reaction

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 7. Altered fetal outcomes of oocytes from obese Blobby mice are alleviated after treatment with salubrinal or BGP-15. Obese mice (bbb/bbb) and lean littermates (+/+) were treated with vehicle (Veh; saline), salubrinal (Sal) or BGP-15 for 4 days, followed by the isolation of oocytes from oviducts and fertilization by using IVF. Blastocysts were collected at day 5 of culture and transferred to non-obese pseudo-pregnant recipient mice with six blastocysts per uterine horn. On embryonic day 14.5, fetuses were collected and weighed (A). Liver (B), heart (C) and kidney (D) were dissected and the relative mtDNA copy number normalized to β-actin nuclear DNA was determined. Data are presented as means+s.e.m.; lean+Veh, n=18 fetuses from six surrogates; Blobby+Veh, n=20 fetuses from six surrogates; Blobby+Sal, n=22 fetuses from six surrogates; Blobby+BGP-15, n=15 fetuses from four surrogates. (E) Liver mtDNA samples (n=4 fetuses from each treatment group) were analyzed by using next generation sequencing, and rearrangements were identified. Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test; P<0.01.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Saline, Isolation, Next-Generation Sequencing

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: LPS induction established inflammation model in MAC-T cells. ( A ) MAC-T cell viability was monitored by CCK-8 assay after LPS treatment with different concentration. ( B – F ) Relative expression level of IL-1β , IL-8 , IL-6 , TNF-α and TLR4 were evaluated by qRT-PCR after LPS treatment with different concentration, respectively. ( G ) IL-1β and IL-8 protein expression were detected by Western blot after LPS treatment with different concentration. ( H , I ) Blot bands of IL-1β and IL-8 were digitized for optical density (OD) value using Image-Pro Plus 6.0, respectively. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: CCK-8 Assay, Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: CTH expression and H 2 S production are repressed in LPS-induced inflammatory process. ( A ) Location analysis of CBS, CTH, and CK18 proteins in the MAC-T cells (400×). ( B ) H 2 S production in supernatant was monitored by Micro H 2 S Content Assay Kit after LPS treatment with different concentration. ( C , D ) Relative expression level of CTH and CBS were evaluated by qRT-PCR after LPS treatment with different concentration, respectively. ( E ) CTH and CBS protein expression were detected by Western blot after LPS treatment with different concentration. ( F , G ) Blot bands of CTH and CBS were digitized for OD value using Image-Pro Plus 6.0, respectively. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: CTH/H 2 S rescued cell survival during inflammation process. ( A , B ) Relative expression level of CTH and CBS were evaluated by qRT-PCR after LPS, BCA and/or NaHS treatment, respectively. ( C ) CTH and CBS protein expression were detected by Western blot after LPS, BCA and/or NaHS treatment, respectively. ( D , E ) Blot bands of CTH and CBS were digitized for OD value using Image-Pro Plus 6.0, respectively. ( F ) H 2 S production in supernatant was monitored by Micro H 2 S Content Assay Kit after LPS, BCA and/or NaHS treatment, respectively. ( G ) Cell viability was monitored by CCK-8 assay after LPS, BCA and/or NaHS treatment, respectively. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: H 2 S repressed inflammatory cytokines expression. ( A , B ) Relative expression level of IL-8 and IL-1β were evaluated by qRT-PCR after LPS, BCA and/or NaHS treatment, respectively. ( C ) IL-8 and IL-1β protein expression were detected by Western blot after LPS, BCA and/or NaHS treatment, respectively. ( D , E ) Blot bands of IL-8 and IL-1β were digitized for OD value using Image-Pro Plus 6.0, respectively. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: Blocking IL-8 signaling significantly increased H 2 S synthesis and CTH/CBS expression. ( A ) Cell viability after IL-8 receptor antagonist SB225002 treatment with different concentration was monitored by CCK-8 assay. ( B ) H 2 S production in supernatant was monitored by Micro H 2 S Content Assay Kit after LPS, SB225002 and/or NaHS treatment, respectively. ( C , D ) Relative expression levels of CTH and CBS were evaluated by qRT-PCR after LPS, SB225002 and/or NaHS treatment, respectively. ( E ) CTH and CBS protein expression were detected by Western blot after LPS, SB225002 and/or NaHS treatment, respectively. ( F , G ) Blot bands of CTH and CBS were digitized for OD value using Image-Pro Plus 6.0, respectively. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: Blocking Assay, Expressing, Concentration Assay, CCK-8 Assay, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: CTH/H 2 S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells

doi: 10.3390/ijms231911822

Figure Lengend Snippet: SB225002 treatment inhibited IL-8 and IL-1β expression. ( A , B ) Relative expression level of IL-8 and IL-1β were evaluated by qRT-PCR after LPS, SB225002 and/or NaHS treatment, respectively. ( C ) IL-8 and IL-1β protein expression were detected by Western blot after LPS, SB225002 and/or NaHS treatment, respectively. ( D , E ) Blot bands of IL-8 and IL-1β were digitized for OD value using Image-Pro Plus 6.0, respectively. ( F ) The interaction of CTH/H 2 S and IL-8, different colored arrows represent different pathways. The different lowercase letters above the bars indicate a significant difference in different treatment groups ( p < 0.05).

Article Snippet: Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [ , , ] and cell viability test results, and adjusted according to the reagent situation.

Techniques: Expressing, Quantitative RT-PCR, Western Blot