Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: PLX3397 attenuates astrocytic scar formation and increases the inflammatory response following SCI. (A) Representative images of astrocytic scars in sagittal sections of the spinal cord at 14 days after spinal cord injury (SCI). In SCI mice, astrocytes surrounding the lesion area packed tightly together as an astrocytic scar barrier, and this astrocytic front was less prominent in mice with PLX3397 treatment. Scale bars: 50 μm. (B) In the SCI mice, the spread of CD68 + inflammatory cells was restricted by a dense glial scar, whereas the boundary of the PLX3397-treated mice was disrupted, and a significant infiltration of CD68 + cells was observed. Scale bars: 50 μm. (C) aCasp3/NeuN costaining in sagittal sections indicating apoptotic neurons. Scale bars: 50 μm. (D) PLX3397 treatment increased the lesion size (calculated as GFAP-negative staining area). Microglia elimination increased the concentration of the proinflammatory cytokines (E) TNF-α, (F) IL-6, and (G) IL-1β at 14 days post-injury (dpi). (H) The proportion of NeuN + /aCasp3 + cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test for E–G; unpaired t -test for D and H). Data are expressed as the mean ± SD. The experiments were repeated six times. aCasp3: Activated caspase-3; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; IL: interleukin; NeuN: neuronal nuclei; PLX3397: colony-stimulating factor 1 receptor inhibitor; SCI: spinal cord injury; TNF-α: tumor necrosis factor-α.

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: Negative Staining, Concentration Assay

Journal: Neural Regeneration Research

Article Title: Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

doi: 10.4103/1673-5374.355978

Figure Lengend Snippet: Effect of Argon preconditioning on caspase-3 cleavage. (A) Evaluation of caspase-3 cleavage by western blotting. Densitometric analysis of western blots of caspase-3 cleavage, normalized against uncleaved caspase-3. Between-group comparisons were performed with a one-way analysis of variance using the post hoc Holm Sidak test ( n = 6; mean ± SD; * P < 0.05, ** P < 0.01, ** *P < 0.001). (B) Representative western blot image of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on caspase-3 cleavage.

Article Snippet: For normalization, blots were re-probed with p44/42 MAPK (mitogen-activated protein kinase, ERK1/2) antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 9102, RRID: AB_330744), NF-κB p65 (D14E12) (rabbit, 1:1000, Cell Signaling Technology, Cat# 8242, RRID: AB_10859369), Akt antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 9272, RRID: AB_329827), caspase-3 antibody (rabbit, 1:4000, Cell Signaling Technology, Cat# 9662, RRID: AB_331439), β-actin antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 4967, RRID: 330288), and NFE2L2 (rabbit, 1:1000, Abcam, Cat# ab62352, RRID: AB_944418).

Techniques: Western Blot, Inhibition

Journal: Neural Regeneration Research

Article Title: Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

doi: 10.4103/1673-5374.355978

Figure Lengend Snippet: Effect of Argon preconditioning on caspase-3 cleavage. (A) Evaluation of caspase-3 cleavage by western blotting. Densitometric analysis of western blots of caspase-3 cleavage, normalized against uncleaved caspase-3. Between-group comparisons were performed with a one-way analysis of variance using the post hoc Holm Sidak test ( n = 6; mean ± SD; * P < 0.05, ** P < 0.01, ** *P < 0.001). (B) Representative western blot image of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on caspase-3 cleavage.

Article Snippet: After protein transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Schwalbach, Germany), the membranes were blocked with 5% skim milk in Tween20/PBS (TBST) or BSA (bovine serum albumin) and incubated in the recommended dilution of protein-specific antibody (p44/42 MAP kinase (phospho-ERK1/2 [extracellular-signal regulated kinase]) (Thr202/Tyr204), rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9101, RRID: AB_331646; phospho-NF-κB (Ser536) (93H1), rabbit, 1:1000, Cell Signaling Technology, Cat# 3033, RRID: AB_331284; phospho-Akt (protein kinase B) antibody (Ser473), rabbit, 1:1000, Cell Signaling Technology, Cat# 9271, RRID: AB_329825; Bax antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2772, RRID: AB_10695870; Bcl-2 antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2876, RRID: AB_2064177; and cleaved caspase-3 antibody (Asp175), rabbit, 1:500, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188; NF-E2-related factor 2 (Nrf2) antibody [nuclear factor erythroid 2-related factor 2, phosphor-S40], rabbit, 1:60000, Abcam, Cat# ab76026, RRID: AB_1524049).

Techniques: Western Blot, Inhibition

Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Western Blot, Staining

Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: Macrophages promote anti-androgen resistance in prostate cancer bone disease

doi: 10.1084/jem.20221007

Figure Lengend Snippet: Neutrophils contribute minimally to anti-androgen resistance. Data related to . (A) Representative H&E staining of MycCaP-Bo bone metastasis sample. Circled area indicates the tumor region. Red arrows indicate newly formed bone matrix; black arrow indicates bone absorption area. (B) Representative TRAP staining of MycCaP-Bo bone metastasis. Red arrows indicate TRAP + osteoclasts. (C) Representative image of Iba1 (red) and F4/80 (green) IF staining in bone metastasis lesion showing the specificity of Iba1 as the macrophage marker. (D) Representative images of Ki-67 staining of bone metastasis lesions with treatment of vehicle (Veh) or enzalutamide (Enz) at indicated time points. (E) Quantification of Ki-67 staining in bone metastasis lesions with treatment of vehicle or enzalutamide at indicated time points. (F) Representative images of cleaved caspase-3 staining of the same samples as in D and E. (G) Quantification of cleaved caspase-3 staining in the same samples as in D and E. (H) Gating strategy for identification of F4/80 + macrophages, CCR2 + Inflam-Monos, and neutrophils in bone metastasis. (I) Representative flow cytometry dot plots showing macrophage depletion using liposomal clodronate, shown as the percentage of F4/80 + cells (gated cells) in CD45 + total cells. (J) Relative growth of spontaneous tumor in HiMyc mice under vehicle or enzalutamide combined with the treatment of liposome PBS (L-PBS) or L-Clod following the schematic diagram on top. (K) Quantification showing the percentage of neutrophils (gated as CD45 + CD11b + Ly-6G + , shown as in ) in total CD45 + cells from bone metastasis samples collected on day 21 with DT and Glu-DT (control toxin) treatment ( n = 6). (L) Quantification of relative tumor growth on day 14 after treatments (normalized to day 0) showing that neutrophil depletion using anti-Ly-6G Ab did not affect anti-androgen response in vivo. MycCaP-Bo bone metastasis received daily treatment of vehicle or enzalutamide plus isotype (Iso) or neutrophil-depleting Abs (Anti-Ly-6G, 200 mg/mouse, i.p. injection, twice a week; n = 6). (M) Relative cell number of MycCaP-Bo cells upon 4 d of indicated treatments revealed that IL-23 did not affect enzalutamide response in vitro. Enzalutamide pre-treated MycCaP-Bo cells were further treated with normal medium (Ctrl), IL-23 alone (100 ng/ml), enzalutamide alone (Enz, 1 mM), and enzalutamide plus IL-23 (Enz+IL-23), followed by MTT assay on day 4 of treatments ( n = 4). (N) Relative cell number of MycCaP-Bo cells upon 4 d of indicated treatments revealed that MDSC-conditioned medium did not affect enzalutamide response in vitro. Enzalutamide–pre-treated MycCaP-Bo cells were further treated with normal culture (Ctrl), MDSC-conditioned medium alone (CM), enzalutamide alone (Enz, 1 mM), and enzalutamide plus enzalutamide-primed MDSC-conditioned medium (Enz+Enz-CM), followed by MTT assay on day 4 of treatments ( n = 4). Data are mean ± SEM; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. ANOVA was used in E and G, and two-tailed unpaired Student’s t test was used in J–N. Scale bar = 100 μm.

Article Snippet: Sections with thickness of 5 μm were dewaxed by histoclear, antigen-retrieved in citrate buffer (pH 6.0), blocked with 5% normal goat-serum for 1 h at room temperature, and incubated with primary Abs against Iba1 (EPR16588, 1:500, #ab178846; Abcam), or cleaved caspase-3 (5A1E, 1:100, #9664S; Cell Signaling Technology) overnight at 4°C.

Techniques: Staining, Marker, Flow Cytometry, In Vivo, Injection, In Vitro, MTT Assay, Two Tailed Test

Journal: Human Reproduction Open

Article Title: Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients

doi: 10.1093/hropen/hoad005

Figure Lengend Snippet: Assessment of the tumor-infiltration mimicking model (TIM). Histological and immunohistochemical (CD43, Caspase-3 and Ki67) analyses of ovarian tissue (OT) ( A ), HL60 cells embedded in 1% agarose ( B ), tumor formation by HL60 cells in human ovarian cortex tissue ( C ), and leukemic tumor eradication efficiency in TIMs by photo-dynamic therapy (PDT) purging with OR-141 (OR)/OR-loaded niosomes (ORN) with or without the light exposure ( D – G ). In the light-exposed OR-treated TIM, the HL60 cells display characteristics of apoptosis, but there are still some Ki67-positive cancer cells after post-PDT incubation time. In the light-exposed ORN-treated TIM, the HL60 cells were disintegrating, as evidenced by the diffuse brownish area in the CD43 staining due to the HL60 cell debris and the lack of Ki67 protein expression (×400 magnification). This was confirmed by the detection of sialophorin gene amplification in ovarian tissues (OTs) (negative control), TIMs (positive control) and ORN-treated TIMs ( H1 ), and droplet-digital (dd)PCR quantification ( H2 ) (* P < 0.01) (n = 4).

Article Snippet: Caspase-3 , Polyclonal (rabbit) , 1:200 in TBS + 4% NGS+0.4% BSA , Human tonsil , 9661S; Cell Signaling Technology, Beverly, MA, USA.

Techniques: Immunohistochemical staining, Incubation, Staining, Expressing, Amplification, Negative Control, Positive Control

Journal: Human Reproduction Open

Article Title: Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients

doi: 10.1093/hropen/hoad005

Figure Lengend Snippet: Analysis of the follicle population before and after xenografting. Follicle density ( A1 ) and proportion ( A2 ) in pre-graft, untreated (unt)-graft, and OR-141-loaded niosomes (ORN)-graft ovarian tissue (OT) fragments. Primordial and growing follicles from pre-graft ( B1, C1 ), unt-graft ( B2, C2 ), and ORN-graft groups ( B3, C3 ). Ki67-positive granulosa cells (brown stain) in preantral follicles in pre-graft ( D1 ), unt-graft ( D2 ), and ORN-graft OT ( D3 ) fragments and proportion of Ki67-positive follicles in the three different groups ( D4 ). Lack of caspase-3 staining in pre-graft ( E1 ), unt-graft ( E2 ), and ORN-graft OT ( E3 ) samples (n = 5).

Article Snippet: Caspase-3 , Polyclonal (rabbit) , 1:200 in TBS + 4% NGS+0.4% BSA , Human tonsil , 9661S; Cell Signaling Technology, Beverly, MA, USA.

Techniques: Staining