primary antibodies for lin28b  (Cell Signaling Technology Inc)

Journal: Molecular oncology

Article Title: The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

doi: 10.1002/1878-0261.13477

Figure Lengend Snippet: Fig. 2. LIN28B promotes Group 3 medulloblastoma viability and proliferation. (A) Relative levels of LIN28B protein in Group 3 MB cell lines D556, HDMB03, D341, and D425 and patient-derived cells BT52 and BT52CTC. (B) CellTiter- Glo assay demonstrates a significant reduction in viability in shLIN28B cells compared with shctl cells. LIN28B levels in HDMB03, D341, and D425 cells treated with either control shRNA (shctl) or LIN28B shRNA (shLIN28B-1) demonstrate reduction in LIN28B following shRNA transfection (see Fig. S2 for experiments using a 2nd shRNA (shLIN28B-5)). (C) BrdU incorporation analysis using immunofluorescence in Group 3 MB cell lines demonstrates a significant decrease in BrdU- positive cells following LIN28B knockdown (n = 3 for each group). (D) In D341 and (E), HDMB03 shLIN28B-1 knockdown cells LIN28B rescue leads to restoration of cell viability. Western blot of corresponding cell lysates demonstrating rescue of LIN28B protein in knockdown cells (LV105 overexpression plasmid control; LVLIN28B LIN28B expression plasmid) is shown below (unpaired t-test P-value: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3 for each group). Error bars represent SEM.

Article Snippet: Primary antibodies against LIN28B (#11965), PBK (#4942), Phospho-PBK (#4941), LIN28A (#8641), Ku80 (#2753), PARP (#9542), phospho-Rb (pRb) (Ser780) (#9307), Rb (D20) (#9313), histone H3 (D1H2) (#4499), phosphohistone H3 (pHH3) (Ser10) (D7N8E) (#53348), HMGA2 (#8179), and Kras (#53270) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Derivative Assay, Glo Assay, Control, shRNA, Transfection, BrdU Incorporation Assay, Knockdown, Western Blot, Over Expression, Plasmid Preparation, Expressing

Journal: Molecular oncology

Article Title: The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

doi: 10.1002/1878-0261.13477

Figure Lengend Snippet: Fig. 5. LIN28 inhibitor 1632 is not toxic to normal astrocytes and other nontumor cells. (A) Dose-dependent reduction in mouse astrocyte viability following treatment with 1632. IC50 calculated from nonlinear fit (regression) analysis for this replicate is demonstrated (error bar represents SEM calculated from six technical replicates; additional experiments used to establish range reported in text, average calculated from three biological replicates). (B) H&E, LIN28B, and cleaved caspase-3 staining of orthotopic brain slices derived from D341 cells treated with either DMSO or 250 lM 1632. Percentage of cleaved caspase-3 (CC3)-positive cells in the tumor and nontumor areas are shown (scale bar = 174 lm). Error bars represent SEM.

Article Snippet: Primary antibodies against LIN28B (#11965), PBK (#4942), Phospho-PBK (#4941), LIN28A (#8641), Ku80 (#2753), PARP (#9542), phospho-Rb (pRb) (Ser780) (#9307), Rb (D20) (#9313), histone H3 (D1H2) (#4499), phosphohistone H3 (pHH3) (Ser10) (D7N8E) (#53348), HMGA2 (#8179), and Kras (#53270) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Derivative Assay

Journal: Molecular oncology

Article Title: The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

doi: 10.1002/1878-0261.13477

Figure Lengend Snippet: Fig. 6. LIN28B inhibition prolongs survival and decreases tumor size in vivo. (A) Survival plots of mice orthotopically injected with D341 or D425 cells stably transfected with shctl or shLIN28B [log-rank (Mantel-Cox) test P-value: ***P < 0.001; ****P < 0.0001]. (B) Immunohistochemistry demonstrating LIN28B, Ki-67, and cleaved caspase-3 (CC3) staining in D425 orthotopic tumor sections 3 weeks after injection (tumor area encircled with black line; %-positive cells indicated in each figure; scale bar on bottom left = 100 lm). (C) HDMB03 cell flank tumors harvested from NSG mice after six doses of intratumor-injected vehicle (ethanol) or 1632 treatment. Scale with inch demarcation is shown for size comparison (also scale bar on bottom left of panel C = 1 inch). (D) Tumor volume change following 1632 treatment compared with vehicle control. Tumors were derived from either HDMB03 or D341 cells. Unpaired t-test P-value: **P < 0.05 (n = 10 for control; n = 11 for 1632). (E) Immunoblots of vehicle or 1632 treated HDMB03 xenograft flanks demonstrate reduction in LIN28B, PBK, c-MYC KRAS, phospho-Rb, and phospho- histone H3 after 1632 treatment. Error bars represent SEM.

Article Snippet: Primary antibodies against LIN28B (#11965), PBK (#4942), Phospho-PBK (#4941), LIN28A (#8641), Ku80 (#2753), PARP (#9542), phospho-Rb (pRb) (Ser780) (#9307), Rb (D20) (#9313), histone H3 (D1H2) (#4499), phosphohistone H3 (pHH3) (Ser10) (D7N8E) (#53348), HMGA2 (#8179), and Kras (#53270) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, In Vivo, Injection, Stable Transfection, Transfection, Immunohistochemistry, Staining, Comparison, Control, Derivative Assay, Western Blot

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a Representative IHC staining of Lin28B in Cohort I TMA. Scale bars: 200 μm. IHC immunohistochemistry. b IHC score of Lin28B as in panel a . c Representative western blotting for Lin28B expression in pairs of tumor and para-tumor tissues from patients with breast cancer. β-actin served as a loading control. T tumor; N para-tumor. d IHC score of Lin28B stratified by breast cancer subtypes in Cohort II TMA. TN triple-negative subtype. e IHC staining of Lin28B in different clinical stages of breast tumors. Scale bars: 200 μm. f IHC staining of Lin28B in breast tumors with different metastatic abilities. Scale bars: 200 μm. g Kaplan–Meier survival plot of breast cancer patients based on Lin28B expression in TMA Cohort II. h Kaplan–Meier survival plot of breast cancer patients of different subtypes based on Lin28B expression in TMA Cohort II and III. i The graphs showed the measured tumor volumes in mice inoculated with indicated cells ( n = 10). j A similar experiment to panel i . Tumor weights are shown ( n = 9). k , l Representative H&E stained sections of lung metastasis and metastasis incidence summary in mice from experiments in g . Scale bars: 200 μm. In i , data were presented as mean ± SEM. A two-sided log-rank test was used for statistical analysis of ( g ) and ( h ), one-way ANOVA for ( b ) and ( d ), and two-way ANOVA for ( i ), followed by multiple comparisons, two-tailed Student’s t -test for ( j ). For panels ( b ), ( d ), and ( j ), boxes represent data within the 25th to 75th percentiles. Whiskers depict the range of all data points. Horizontal lines within boxes represent mean values. For panels ( e ) and ( f ), experiments were repeated three times independently with similar results; data from one representative experiment are shown. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Immunohistochemistry, Western Blot, Expressing, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a Niche-characteristic genes were detected by qRT-PCR in the pre-metastatic lung of tumor-bearing mice at 3 weeks after tumor inoculation. GAPDH served as an internal control ( n = 6 mice in each group). FN Fibronectin. b Representative flow cytometry plots of CD11b + Ly6G + Ly6C + neutrophils gated on CD45 + lived cells in the pre-metastatic lung at 3 weeks after tumor inoculation. c The proportions and numbers of neutrophils as detected in panel b ( n = 6 mice in each group). d IF images and quantitation of Ly6G staining in the pre-metastatic lung of tumor-bearing mice at 3 weeks after tumor inoculation ( n = 6 random microscopic fields (RMFs) from 6 mice in each group). Scale bar, 100 μm. e Representative IF images for Ly6G co-localization with CD45, CD11b, and Ly6C in the pre-metastatic lung at 3 weeks after tumor inoculation. Scar bar: 100 um. f Schematic illustration of neutrophil depletion in tumor-bearing mice. The blue arrow indicates neutrophil depletion in the early phase, which starts on day 14 after tumor inoculation until the primary tumors were resected for consecutive six times, while the sky blue arrow indicates neutrophil depletion in the late phase, which begins on day 35 just after primary tumors were removed for consecutive six times. g The growth kinetics of primary tumors in mice treated with the indicated antibody ( n = 8 mice in each group). h H&E and quantification of lung metastasis in 4TO7-Lin28B mice in which the neutrophils were depleted in the early phase of tumor growth as illustrated in Fig. 2f ( n = 10 RMFs from six mice each group). Scale bars: 200 μm. i Quantification of CXCLs in BALF and serum of tumor-bearing mice at 3 weeks after tumor inoculation ( n = 6 mice in each group). Data were presented as mean ± SEM. P values were calculated using two-tailed Student’s t -test for ( a ), ( c ), ( d ), ( h ), and ( i ), two-way ANOVA followed by multiple comparisons for ( g ). * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Quantitative RT-PCR, Flow Cytometry, Quantitation Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a Quantification of N1/N2 marker genes in neutrophils collected from the pre-metastatic lung at 3 weeks after tumor inoculation. GAPDH served as an internal control ( n = 3 mice in each group). b Neutrophils were purified from the pre-metastatic lung at 3 weeks after tumor inoculation and the cells were subjected to in vitro culture for supernatant collection. ELISA assay was conducted to quantify the cytokine expression ( n = 6 mice in each group). c Quantification of indicated cytokines in BALFs of tumor-bearing mice at 3 weeks after tumor inoculation ( n = 6 mice in each group). d The neutrophils purified from the bone marrow of naïve mice were cultured in vitro in the presence of cytokines as indicated. Twenty-four hours later, the mRNA levels of N1/N2 markers of neutrophils were detected by qRT-PCR. GAPDH served as an internal control ( n = 3 culturing experiments). e Schematic illustration for inhibiting N2 conversation of lung neutrophils by intratracheally injecting with anti-IL-6 Ab, anti-IL-10 Ab, or both combined into 4TO7-Lin28B tumor-bearing mice. f Neutrophil phenotypes were detected in mice treated as indicated in panel e ( n = 3 mice in each group). g Schematic diagram of N2-conversion inhibition upon the influence of lung metastasis. h Image and quantification of lung metastasis of mice treated as indicated in g ( n = 8 RMFs from three mice each group). Scale bar: 200 μm. i Schematic diagram for inducing N2 conversion upon lung metastasis. j Image and quantification of lung metastasis of mice treated as indicated in i ( n = 8 RMFs from three mice each group). Scale bar: 200 μm. Data were presented as the mean ± SEM, and p values were calculated using a two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance. For panels ( b ) and ( c ), boxes represent data within the 25th to 75th percentiles. Whiskers depict the range of all data points. Horizontal lines within boxes represent mean values. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Marker, Purification, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Quantitative RT-PCR, Inhibition, Two Tailed Test

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a PD-L1 and PD-L2 expression of neutrophils in the pre-metastatic lung ( n = 6 mice in each group). b The experiment was the same as in panel a, except that CD80, CD86, OX40L, and ICOSL were measured. c Expression of PD-L1 and PD-L2 on the indicated cells in the pre-metastatic lung ( n = 6 mice in each group). Neu neutrophil, Macro macrophage, DC dendritic cell. d IL-6 and IL-10 depletion decreased PD-L2 expression of lung neutrophils in 4TO7-Lin28B mice. The 4TO7-Lin28B or the control mice were intratracheally delivered with anti-IL-6 and anti-IL-10 neutralizing Abs or normal IgG as control once every 3 days starting 2 weeks after tumor inoculation. 1 week later, the lung neutrophils were evaluated for PD-L2 expression ( n = 6 mice in each group). e Neutrophils were stimulated with the indicated cytokines, and the mRNA expression of PD-L1 and PD-L2 were determined. GAPDH served as an internal control ( n = 3 mice in each group). f , g PD-L2 blocking attenuated N2 neutrophil-induced T cell proliferation and activation inhibition ( n = 6 culturing experiments). h Naïve CD4 + T cells were incubated with the neutrophil supernatants in the presence of indicated antibodies or cytokines. The expression of IFN-γ was analyzed by intracellular staining ( n = 6 culturing experiments). The lung cells (neutrophils, macrophages, and DCs) were isolated from the pre-metastatic lung of tumor-bearing mice 3 weeks after tumor inoculation. Data were presented as the mean ± SEM, and p values were calculated using a two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Expressing, Blocking Assay, Activation Assay, Inhibition, Incubation, Staining, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a Let-7s and miR-125s family members were specially reduced in the circulation exosomes in mice bearing 4TO7-Lin28B tumor ( n = 3 mice in each group). b The experiment was the same as in panel a , except that let-7s and miR-125s family members were measured in the serum exosomes of PyMT mice (11-week old) ( n = 3 mice in each group). c Let-7a inhibited increased production of IL-6 in neutrophils mediated by Lin28B-exosome. Naïve mice were i.v injected with Control-exosome or Lin28B-exosome in the presence of the indicated agomir. IL-6 expression was determined in lung neutrophils by FACS ( n = 6 mice in each group). d The experiment was the same as for panel c , except that the IL-10 was detected in macrophages ( n = 6 mice in each group). e Let-7a inhibited neutrophil N2 conversion induced by Lin28B-exosomes. The exosome treatment was the same as for panel c. GAPDH served as an internal control ( n = 6 mice in each group). f Let-7a rescued T cell inactivation induced by Lin28B-exosome ( n = 6 mice in each group). g Let-7a restored Lin28B-exosome-mediated inhibitory Th1 differentiation of CD4 + T cells ( n = 6 mice in each group). h Let-7a attenuated Lin28B-exosome-mediated lung metastasis ( n = 6 mice in each group). i ALDH + and ALDH − subsets of 4TO7-Lin28B cells isolated from primary tumors were analyzed for exosomal miRNA expression. U6 served as an internal control ( n = 3 mice in each group). j The ALDH + and ALDH − subset exosomes were collected as for panel i , and the exosome education and the I.V. injection of tumor cells were the same as in Fig. . Shown are images and BLI quantification of lung metastasis of 4TO7-Control cells ( n = 6 mice in each group). Data were presented as the mean ± SEM, and p values were calculated using a two-tailed Student’s t -test. For panels ( h ) and ( j ), boxes represent data within the 25th to 75th percentiles. Whiskers depict the range of all data points. Horizontal lines within boxes represent mean values. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Injection, Expressing, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression

doi: 10.1038/s41467-022-28438-x

Figure Lengend Snippet: a Exosome protein concentrations purified from sera of healthy donors and primary breast cancer patients ( n = 30 patients). b Let-7a, let-7d, and let-7g were quantified by qRT-PCR in exosomes prepared as in panel a . MiR-16 served as an internal control ( n = 30 patients). c Mir-15a and U6 expression in exosomes prepared as in panel a . MiR-16 served as an internal control ( n = 30 patients). d Pearson correlation between the relative Lin28B mRNA level of primary tumors and the relative let-7a amount of serum exosomes in the same breast cancer patient ( n = 91 patients). e Relative expression of let-7a in serum exosomes in different breast cancer subtypes ( n = 91 patients). f Pearson correlation between the relative let-7a level of primary tumors and the relative let-7a amount in serum exosomes in the same breast cancer patient ( n = 91 patients). g Representative images of IHC staining of Lin28B along with in situ hybridization of let-7a on TMA Cohort II (140 cases). Bars: 500 µm (upper panels) and 50 µm (lower panels). h Correlation between the expression of Lin28B and let-7a as in panel g was assessed via linear regression ( n = 140 patients). i Overall survival probabilities were calculated using the Kaplan–Meier method and analyzed using a log-rank test in a retrospective cohort of patients (139 cases) with breast cancer (Cohort II) with low ( n = 102 patients) or high ( n = 37 patients) expression of let-7a. j Overall survival probabilities were calculated using the Kaplan–Meier method and analyzed using a log-rank test in a cohort of GDC TCGA breast cancer patients (1073 cases), by let-7a expression. k Metastasis-free survival probabilities were calculated by let-7a expression using the Kaplan–Meier method and analyzed using a log-rank test in a clinical cohort of 204 breast cancer patients from GEO dataset GSE12276 with let-7a expression. In a – c and e , data were presented as the mean ± SEM, and p values were calculated using a two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, ns no significance. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry of the TMA chips was performed with a primary antibody against Lin28B (Cat #LS-B3424-200; LifeSpan BioSciences, Inc.).

Techniques: Purification, Quantitative RT-PCR, Expressing, Immunohistochemistry, In Situ Hybridization, Two Tailed Test

Journal: Pharmaceutical Biology

Article Title: Peptides from the croceine croaker ( Larimichthys crocea ) swim bladder attenuate busulfan-induced oligoasthenospermia in mice

doi: 10.1080/13880209.2022.2034895

Figure Lengend Snippet: The effects of SBP on the expression of genes involved in germ cell development. (A) Relative Lin28b mRNA level. (B) Relative Igf2bp1 mRNA level. (C) The protein level of Lin28b and grayscale analysis. (D) The protein level of Igf2bp1 and grayscale analysis. * p < 0.05, ** p < 0.01 vs. Control group; # p < 0.05, ## p < 0.01 vs. Busulfan group. Data are expressed as mean ± SDE, with n = 3 for each group.

Article Snippet: Primary antibodies against Lin28b (24017-1-AP) and Igf2bp1 (22803-1-AP) were obtained from Proteintech (Rosemont, IL).

Techniques: Expressing, Control