Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

doi: 10.1080/02656736.2022.2029585

Figure Lengend Snippet: Figure 4. The validation experiment of hub genes. (A) The differences in gene expression of 10 hub genes were validated with RT-qPCR. (B) Western blot analysis of FOS, CREB5, MAPK8 and NFKB1 protein level. (C) The efficiency of siRNA to knockdown the expression of FOS and CREB5. (D) Cell proliferation in the si-FOS and si-CREB5 group was faster compared with that in the control group, using the CCK-8 assay. (E). Half-inhibition rate of adriamycin in MCF-7/ADR cells treated with si-FOS and si-CREB5. (F) Different expression of FOS and CERB5 between invasive breast cancer and normal breast tissue by TCGA database. Error bars represented the mean ± SD of at least three independent experiments, p < .05, p < .01, p < .001.

Article Snippet: The membrane was then probed with primary antibodies at 4 C overnight and with species-specific secondary antibodies at room temperature for 1 h. The primary antibodies, including FOS antibody (1:1000, Proteintech, 66590-1-Ig), NF-jB antibody (1:1000, Proteintech, 14220-1-AP), MAPK8 antibody (1:1000, Proteintech, 66210-1-Ig), CREB5 antibody (1:1000, Proteintech, 14196-1-AP), GAPDH antibody (1:5000, Proteintech, 60004-1-Ig), CD9 antibody (1:1000, Abcam, ab223052), CD63 antibody (1:1000, Abcam, ab68418), TSG101 Figure 1.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Control, CCK-8 Assay, Inhibition

Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: Hyperthermia promotes exosome secretion by regulating Rab7b while increasing drug sensitivity in adriamycin-resistant breast cancer.

doi: 10.1080/02656736.2022.2029585

Figure Lengend Snippet: Figure 5. Hyperthermia promotes the secretion of exosome in MCF-7/ADR cells. (A) The expression level of FOS and CREB5 in MCF-7/ADR exosomes after hyper- thermia. (B) Half-inhibition rate of adriamycin in MCF-7/ADR cells incubated with the exosomes which produced by MCF-7/ADR cells after hyperthermia. (C) The expression level of FOS and CREB5 in MCF-7/ADR after incubated with hyperthermia ADR/exo. (D) Particles concentration of exosomes derived from breast cancer cells analyzed by NTA. (E) Total protein amounts of exosomes derived from breast cancer cells analyzed by bicinchoninic acid (BCA) protein kit. (F) Transmission electron micrographs (TEM) of cells before or 6 h after hyperthermia. Intense extracellular vesicle shedding occurred 6 h after hyperthermia. Scale bar ¼1 lm. (G) Confocal microscope analysis of exosome uptake by MCF-7/ADR cells with or without hyperthermia treatment. Scale bar ¼20 lm. Error bars represent the mean ± SD of at least three independent experiments, p < .05, p < .01, p < .001.

Article Snippet: The membrane was then probed with primary antibodies at 4 C overnight and with species-specific secondary antibodies at room temperature for 1 h. The primary antibodies, including FOS antibody (1:1000, Proteintech, 66590-1-Ig), NF-jB antibody (1:1000, Proteintech, 14220-1-AP), MAPK8 antibody (1:1000, Proteintech, 66210-1-Ig), CREB5 antibody (1:1000, Proteintech, 14196-1-AP), GAPDH antibody (1:5000, Proteintech, 60004-1-Ig), CD9 antibody (1:1000, Abcam, ab223052), CD63 antibody (1:1000, Abcam, ab68418), TSG101 Figure 1.

Techniques: Expressing, Inhibition, Incubation, Produced, Concentration Assay, Derivative Assay, Transmission Assay, Microscopy

Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 3. vlPAG activity is required for systemic morphine antinociception and drives spinal NA–dependent antinociception. (A) Bilateral vlPAG AAV-DIO-hM4Di- mCherry injections in Vglut2-cre mice with representative image of viral expression. Scale bar, 500 μm. (B) Left: Hot plate withdrawal latencies of vlPAGVglut2-cre::hM4Di mice administered CNO (3 mg/kg i.p.) versus saline without (light green bars) and with morphine (5 mg/kg s.c.) (dark green bars) (two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 13 mice; saline versus CNO effect, P < 0.0001, F1,12 = 56.93). Right: Withdrawal latencies of non–virus-injected controls (two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 9 mice; saline versus CNO effect, P = 0.32, F1,8 = 1.143). (C) Bilateral vlPAG AAV-DIO-hM3Dq- mCherry injections in Vglut2-cre mice, combinations of systemic CNO with intrathecal antagonists, and representative image of viral expression. Scale bar, 500 μm. (D) Left: Withdrawal latencies after systemic saline or CNO and intrathecal saline, phentolamine (5 μg), or naltrexone (5 μg) (Friedman test with Dunn’s multiple comparisons test, n = 12 subjects, P < 0.0001, Friedman statistic = 26.40). Right: Von Frey mechanical thresholds (Friedman test with Dunn’s multiple comparisons test, n = 12 subjects, P = 0.0001, Friedman statistic = 21.00). (E) Representative images of c-Fos immunohistochemistry in TH-positive LC neurons of vlPAGVglut2-cre::hM3Dq mice after systemic injection of saline (top) or CNO (bottom). Scale bars, 150 μm. Right: % of TH-positive LC neurons that colocalize with green c-Fos signal (five to eight images analyzed per mouse; n = 6 saline, n = 5 CNO, two-sided Mann-Whitney test). Data in each graph reported as means ± SEM.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Activity Assay, Expressing, Saline, Virus, Injection, Immunohistochemistry, MANN-WHITNEY

Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 4. Anatomical characterization of inputs to the LC from the vlPAG. (A) vlPAG injection of AAV-DIO-tdTom in Vglut2- or Vgat-cre mice. (B) Vglut2-cre injection site. (C) vlPAGVglut2-cre terminals (magenta) and TH (green) in the peri-LC. Bar, Barrington’s nucleus. (D) Vgat-cre injection site. (E) vlPAGVgat-cre terminals. Scale bars, 300 μm [(B) to (E)]. (F) Quantification of magenta and green pixel intensity in the peri-LC of vlPAGVglut2-cre::tdTom mice normalized by z score (n = 6 LC slices from three mice). (G) Same as (F) for vlPAGVgat-cre::tdTom. (H) Left: Injections of AAVretro-cre in the LC and AAV-DIO-mCherry in the vlPAG of wild-type mice. Middle: Image of mCherry+ vlPAG neurons. Right: Resulting terminals in the LC and RVM. Scale bars, 500 μm. (I) Same as (H) for vlPAG→RVM neurons. Scale bars, 500 μm. (J) Left: Orthogonal recombinase strategy to label vlPAG neurons that project to the RVM and LC. Middle: Representative image of mCherry (magenta) and YFP (green). Arrows indicate double-labeled (white) neu- rons. Scale bar, 300 μm. Right: Quantification of mCherry and YFP in the vlPAG.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Injection, Labeling

Journal: Science advances

Article Title: Inputs to the locus coeruleus from the periaqueductal gray and rostroventral medulla shape opioid-mediated descending pain modulation.

doi: 10.1126/sciadv.adj9581

Figure Lengend Snippet: Fig. 8. Pathway-specific modulation of vlPAG and RVM terminals in the LC modulates nociceptive behavior. (A) Left: Bilateral cannula placement over the LC in uninjected control mice. Right: Hot plate withdrawal latencies of control mice microinfused in the LC with saline (150 nl) versus CNO (3 μM, 150 nl) without (light gray) and with morphine (5 mg/kg s.c.) (dark gray; two-way repeated-measures ANOVA with Tukey’s multiple comparisons test; n = 8 mice; saline versus CNO effect, P = 0.4178, F1,7 = 0.7412). (B) Left: Bilateral viral injection of AAV-DIO-hM4Di-mCherry in the vlPAG of Vglut2-cre mice with bilateral cannula placement over the LC. Right: Hot plate withdrawal latencies after microinfusion with saline versus CNO without (light green) and with morphine (5 mg/kg s.c.) (dark green; two-way repeated-measures ANOVA with Sidak’s multiple comparisons test; n = 12 mice; saline versus CNO effect, P = 0.0022, F1,11 = 15.72). (C) Left: Bilateral viral injection of AAV-DIO-hM4Di-mCherry in the RVM of Vgat-cre mice with bilateral cannula placement over the LC. Right: Hot plate withdrawal latencies after microinfusion of saline versus CNO (blue bars, n = 12 mice), microinfusion of CNO with intrathecal injections of saline versus phentolamine (5 μg, light blue bars, n = 9 mice), and microinfusion of saline versus CNO with morphine (5 mg/kg s.c.) (dark blue, n = 12 mice; mixed-effects analysis with matching across row and Tukey’s multiple comparisons test, P < 0.0001, F2.560,25.09 = 27.36). (D) Circuit diagram of DPMS inputs to the LC and their opioid sensitivity. Data in each graph reported as means ± SEM.

Article Snippet: The following were made and titered in- house using Addgene plasmids: AAVDJ- hsyn- DIO- mCherry (Addgene 50459, titer 3.5 × 1012), AAVDJ- ef1a- fDIO- EYFP (Addgene 55641, titer 2 × 1011), AAVDJ- Ef1a- mCherry- IRES- cre (Addgene 55632, titer 4.3 × 1012), and AAVDJ- ef1a- DIO- ChR2(H134R)mCherry (Addgene 20297, titer 2.55 × 1013).

Techniques: Control, Saline, Injection