Fig 1: Clinical validation of the IPS. (A) H&E staining results of IPS-identified high- and low-risk ccRCC samples. Arrows indicate some macrophage examples. (B) IHC results of the top four most hazardous IGPs. All scale bars are 50 µm. (C) IGP expression and IPS score of each patient with ccRCC. (D) Quantification of IHC results of the top four most hazardous IGPs. ccRCC, clear cell renal cell carcinoma; IPS, inflammatory prognostic signature. CCL7, C-C motif chemokine ligand 7; LTB4R2, leukotriene B4 receptor 2. **P<0.01 and ***P<0.001.
Fig 2: Stress and chemokine response in ALS degenerating motor neurons(A–E) RT-qPCR was used to examine motor neuron gene expression after Rpl22HA IP from 1-, 3-, and 5-month-old RiboG93A mice relative to RiboWT littermate controls. Neither ChAT (A) nor the ?MN marker Esrrg (C) was altered at any time point; however, a decrease in NeuN was observed at the 5-month time point (B) and an increase in Atf3 (D) and Fgf21 (E) were observed at both 3 and 5 months of age in RiboG93A motor neurons. Asterisks above the different RiboALS time points in A-E indicate statistical significance as compared to the average of RiboWT littermate controls from all time points.(F) Increased Fgf21 was confirmed by FISH. Fgf21 transcript (blue) colocalized with Chat+ (red), degenerating motor neurons.(G) Fgf21 transcript levels were scored as negative (Fgf21-), low (Fgf21lo), or high (Fgf21hi) and the percentage of Chat+ motor neurons was quantified in RiboWT (each time point, n = 3), RiboALS mice (1-, 4-mo, n = 3; 3-mo, n = 2; 5-mo, n = 4).(H) Ccl7 and Ccl12 expression were similarly examined by FISH. At 4- and 5-month time points, rare Chat+ (red) motor neurons in RiboG93A mice exhibited Ccl7 (green) expression. Ccl12 (blue) was generally increased, however expression was not observed in motor neurons.(I) The percentage of Ccl7 positive neurons were scored as in (G) Scale bar represents 50 µm.Error bars represent the mean ± s.e. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.
Fig 3: Chemokines are upregulated in injured motor neurons(A and B) INJ and UNJ lumbar hemispheres were isolated from RiboHO;ChATCre mice D1-D50 after nerve crush or 21 days after nerve transection (D21-t). After Rpl22HA IP, RT-qPCR was used to examine Atf3 (A) and Gap43 (B) gene expression in both the IP and the input relative to the UNJ control. For simplicity, asterisks indicate statistical significance as compared to the D50 when reinnervation is largely complete.(C) Quantification of the percentage of Gap43+ motor neurons that co-expressed Ccl4, Ccl2, or Ccl7 at D3 and D7 after injury based on the data in D and E (n = 5).(D) Fluorescent in situ hybridization (FISH) was performed on WT lumbar spinal cord sections at D3 (n = 3) and D7 (n = 2) after sciatic nerve crush. Ccl7 (red) or Ccl12 (blue) were not expressed in the UNJ contralateral spinal cord, were upregulated by injury at both D3 and D7 time points, and only Ccl7 was observed in Gap43+ (green) injured motor neurons.(E) Similar analysis as in D demonstrated that both Ccl2 (blue) and Ccl4 (red) were observed in D3 and D7 injured Gap43+ (green) motor neurons. Scale bar represents 50 µm.Error bars represent the mean ± s.e. ****p = 0.0001, **p = 0.01.
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