Journal: Nature Communications

Article Title: Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

doi: 10.1038/s41467-024-47601-0

Figure Lengend Snippet: a Seminiferous tubule sections in WT testis (8 weeks old) were immunostained as indicated. pL preleptotene, L Leptotene, Z Zygotene, ePa early Pachytene, P Pachytene, M I Metaphase I, int Interkinesis, rS round Spermatid, eS elongated Spermatid. Boundaries of the seminiferous tubules are indicated by white dashed lines. * indicates a non-specific cross-reactivity of the gunia pig anti-SYCP3 antibody to sperm tail. HSF5 was immunostained by HSF5-C antibody. The same immunostaining pattern of HSF5 was confirmed by other HSF5-N1 and HSF5-N2 antibodies, as shown in Fig. . Roman numbers indicate the seminiferous tubule stages. Biologically independent mice ( n = 3) were examined in three separate experiments. Scale bar: 25 μm. b The schematic of the Hsf5-3xFLAG-HA knockin allele. c Testis extracts from Hsf5-3xFLAG-HA knockin and negative control WT mouse testis (5 weeks old) were immunoblotted as indicated. Red arrow indicates HSF5-3xFLAG-HA protein derived from the knockin allele. Blue arrow indicates HSF5 protein derived from WT allele. d Seminiferous tubule sections in Hsf5-3xFLAG-HA knockin ( n = 1) and negative control WT mouse testis ( n = 1) at 5 weeks old were immunostained as indicated. Scale bar: 25 μm. e Seminiferous tubule sections in WT testis (P16) were immunostained as indicated. Arrow and arrowhead indicate HSF5-positive/H1t-positive and HSF5-negative/H1t-positive pachytene spermatocytes, respectively. Scale bar: 25 μm. A single experiment was performed. f The schematic of expression of HSF5 (green), H1t (blue), HSF1(red) , in the stages of the seminiferous tubules.

Article Snippet: For counting seminiferous tubules, immunostaining images were captured with BIOREVO BZ-X710 (KEYENCE), and processed with BZ-H3A program.

Techniques: Immunostaining, Knock-In, Negative Control, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

doi: 10.1038/s41467-024-47601-0

Figure Lengend Snippet: a The targeted Hsf5 allele with deletion of Exon1-6 is shown. 5’- and 3’- homology sequences in the ssODN are shown in green and red, respectively. Arrowheads: synthetic gRNAs. Arrows: PCR primers for genotyping. b Immunoblot analysis of testis extracts prepared from mice with the indicated genotypes (P17). The arrow indicates a band of HSF5. * indicates nonspecific bands. Two technically independent experiments from two pairs of WT and Hsf5 KO siblings were repeated and showed similar results. c Seminiferous tubule sections (8 weeks old) were stained for SYCP3, HSF5, and DAPI. pL preleptotene, Pa pachytene spermatocyte, rS round spermatid, eS elongated spermatid. Boundaries of the seminiferous tubules are indicated by white dashed lines. Roman numbers indicate the seminiferous tubule stages. Biologically independent mice ( n = 3) for each genotype were examined. Scale bars: 25 μm. d Testes from WT, Hsf5 +/- and Hsf5 KO (upper left: 4 weeks old, upper right: 8 weeks old left). Testis/body-weight ratio (mg/g) of WT, Hsf5 +/-, and Hsf5 KO mice (lower left: 4 weeks old, lower right: 8 weeks old) are shown below (Mean with SD). n: the number of animals examined. Statistical significance is shown by p value (Two-tailed t-test). Scale bar: 5 mm. e Hematoxylin and eosin staining of the sections from WT, Hsf5 +/- and Hsf5 KO testes (upper: 4 weeks old, lower: 8 weeks old). Biologically independent mice ( n = 3) for each genotype were examined. Scale bar: 100 μm. f Hematoxylin and eosin staining of the sections from WT, Hsf5 +/- and Hsf5 KO epididymis (8 weeks old). Biologically independent mice ( n = 3) for each genotype were examined. Scale bar: 100 μm. g Seminiferous tubule sections (8 weeks old) were stained for PNA lectin and DAPI. Scale bar: 25 μm. A single experiment was performed. h Number of pups born by mating Hsf5 + /- and Hsf5 KO males with WT females to examine fertility. Hsf5 +/- males and Hsf5 KO males were initially mated with WT females (all 4 weeks old at the start point of mating). This cage was observed for 24 weeks from the start of mating.

Article Snippet: For counting seminiferous tubules, immunostaining images were captured with BIOREVO BZ-X710 (KEYENCE), and processed with BZ-H3A program.

Techniques: Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions

doi: 10.1038/s41467-024-47601-0

Figure Lengend Snippet: a Seminiferous tubule sections (4 weeks) were stained as indicated. Scale bar: 25 μm. Yellow arrowhead indicates H1t-positive spermatocyte with abnormal SYCP3 staining. pL: preleptotene, Pa: pachytene, rS: round spermatid. Shown on the right is the quantification of the seminiferous tubules that have H1t + /SYCP3+ cells per the seminiferous tubules that have SYCP3+ spermatocyte cells in Hsf5 +/- and Hsf5 KO testes (Mean with SD). n: the number of animals examined. Statistical significance is shown (p = 0.0051, unpaired two-tailed t-test). b – f Chromosome spreads of Hsf5 +/- and Hsf5 KO spermatocytes (P21) were immunostained as indicated. b Lep: leptotene, Zyg: zygotene, Pac: pachytene, Dip: diplotene. Pac* indicates pachytene spermatocyte with high level of γH2AX signals remained on autosomes. Scale bar: 10 μm. Shown on the right is quantification of stages per total SYCP3+ spermatocytes. n: the number of cells examined. c Hsf5 +/- ( n = 60) Hsf5 KO ( n = 55). Scale bar: 10 μm. Enlarged images of the XY body are shown on the bottom. Scale bar: 5μm. d ~ 17.8% of Hsf5 KO pachytene spermatocytes ( n = 62) exhibited BRCA1 along autosomes with γH2AX signals, whereas none of Hsf5 +/- pachytene spermatocytes ( n = 51) did except for XY chromosome. Scale bar: 10 μm. e The number of DMC1 foci is shown in the scatter plot with median (right). Statistical significance is shown ( p < 0.0001, two-sided Mann-Whitney U-test). Lep leptotene, Zyg Zygotene, Pac Pachytene. Scale bar: 10 μm. f The number of MLH1 foci is shown in the scatter plot with median (right). Statistical significance is shown (Mann-Whitney U-test). n: number of spermatocytes examined. Statistical significance is shown ( p < 0.0001, two-sided Mann–Whitney U-test). Scale bar: 5 μm. g Seminiferous tubule sections (4 weeks) were subjected to TUNEL assay. Whole testis sections (left, Scale bar: 500 μm) and closeup view of seminiferous tubule sections (middle, Scale bar: 25 μm) are shown. Shown on the right is the quantification of the seminiferous tubules that have TUNEL+ cells per total tubules in Hsf5 +/- ( n = 3) and Hsf5 KO ( n = 3) testes (Mean with SD). Statistical significance is shown by p -value ( p = 0.0244, unpaired two-tailed t-test).

Article Snippet: For counting seminiferous tubules, immunostaining images were captured with BIOREVO BZ-X710 (KEYENCE), and processed with BZ-H3A program.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, TUNEL Assay

Journal: PLoS Biology

Article Title: Negative Regulation of Active Zone Assembly by a Newly Identified SR Protein Kinase

doi: 10.1371/journal.pbio.1000193

Figure Lengend Snippet: (A) The srpk79D gene region is shown, including the srpk79D atc transposon insertion site and deleted regions in mutants used for genetic analyses (black bars). Gene loci for srpk79D and the adjacent Csp gene are shown in blue. (B–J) Immunofluorescence images of larval nerves demonstrating large Brp accumulations in srpk79D loss-of-function mutants. Each image shows a section of a single larval nerve, photographed at the same relative position approximately 100 µm from where the nerve exits the CNS. Each nerve contains approximately 85 total axons, including approximately 35 motor axons. Images are shown at two exposures. Images in (B, E, and H) and (C, F, and I) were taken at an exposure in which small, infrequent anti-Brp puncta could be resolved in wild-type nerves. This resulted in overexposure of the Brp puncta in srpk79D mutant nerves. Images (D, G, and J) are identical to (B, E, and H, and C, F, and I) except taken at a lower exposure such that no puncta are found in wild type, and the puncta intensities are not saturated in the srpk79D mutant. (K) Total Brp fluorescence integrated over the nerve area is dramatically increased with srpk79D disruption, whereas loss of Csp does not increase nerve Brp levels. Each bar graph represents data collected from a total of 30 nerves from 12 different larvae. (L) Cumulative frequency plots of individual Brp punctum fluorescence intensities are shifted toward larger values with srpk79D loss of function (gray and blue lines representing srpk79D atc and srpk79D atc /Df , respectively, are shifted to the far right whereas other genotypes are clustered to the left). Each curve represents data collected from a total of 30 nerves from 12 different larvae. Sample size for wild type, srpk79D atc /+, srpk79D atc , srpk79D atc /Df , and srpk79D atc /Csp X1 = 479, 1,715, 3,387, 3,718, and 2,714, respectively. Significance is indicated according to the following: * = p <0.05, ** = p <0.01, *** = p <0.001, and ns = not significant; Student t -test. Scale bar indicates 10 µm. Error bars indicate ±SEM. au = arbitrary units; Brp = anti-Bruchpilot; HRP = anti-horseradish peroxidase.

Article Snippet: The intensity of anti-BRP immunostaining was quantified as follows: Each series of 0.5-µm optical nerve sections was deconvolved (nearest-neighbors; Intelligent Imaging Innovations).

Techniques: Immunofluorescence, Mutagenesis, Fluorescence

Journal: PLoS Biology

Article Title: Negative Regulation of Active Zone Assembly by a Newly Identified SR Protein Kinase

doi: 10.1371/journal.pbio.1000193

Figure Lengend Snippet: (A–D) Immunofluorescence images of wild-type and srpk79D atc mutant NMJ reveal a synaptic Brp deficit in srpk79D atc mutants. NMJs are stained with anti-HRP (red) to label the presynaptic membrane and anti-Brp (green). Larvae in wild type and srpk79D atc were stained in the same reaction tube and imaged identically. (E) The total synaptic Brp fluorescence is decreased in srpk79D atc mutants. Each bar graph represents data collected from a total of 30 synapses from nine different larvae. (F) Cumulative frequency plots of synaptic Brp puncta fluorescence intensities are shifted toward smaller values with srpk79D loss of function. Each curve represents data collected from 30 synapses from nine different larvae. n for wild type, srpk79D atc , and srpk79D atc /Df = 6,554, 4,713, and 5,952, respectively. au = arbitrary units; HRP = anti-horseradish peroxidase. (G and H) Disruption of srpk79D affects neither synaptic Brp puncta number (G) nor synaptic bouton number (H). Each bar graph in (G) and (H) represents data collected from a total of 38 synapses taken from 15 different larvae. Scale bar indicates 10 µm. Significance is indicated according to the following: *** = p <0.001 and ns = not significant; Student t -test. Error bars indicate ±SEM.

Article Snippet: The intensity of anti-BRP immunostaining was quantified as follows: Each series of 0.5-µm optical nerve sections was deconvolved (nearest-neighbors; Intelligent Imaging Innovations).

Techniques: Immunofluorescence, Mutagenesis, Staining, Fluorescence

Journal: PLoS Biology

Article Title: Negative Regulation of Active Zone Assembly by a Newly Identified SR Protein Kinase

doi: 10.1371/journal.pbio.1000193

Figure Lengend Snippet: (A and B) Whole-mount in situ hybridizations demonstrate that srpk79D is widely expressed but is enriched in the CNS. Anterior is to the left. (C and D) Immunofluorescence images of a control nerve (C) and a nerve expressing srpk79D -specific double-stranded RNA (dsRNA) ( UAS - srpk79D RNAi ) in neurons (D). Expression of dsRNA causes accumulation of Brp puncta. (E and F) Total Brp fluorescence is increased and cumulative frequency plots are shifted toward larger values when srpk79D RNAi is expressed in neurons, but not when it is expressed in glia using the glia-specific Repo-GAL4 driver. Each bar graph and curve in (E) and (F) represents data collected from a total of 30 nerves from nine different larvae. In (F), n for C155/+ , C155/+;;UAS-srpk79DRNAi/+ , Repo/+ , and Repo / UAS - srpk79D RNAi = 1,034, 2,451, 893, and 862, respectively. (G–J) Expression of a Venus-tagged srpk79D transgene ( UAS - v-srpk79D-rd *) rescues Brp accumulations in srpk79D mutant nerves. (G and H) Nerves are stained with anti-Brp and imaged identically. Brp accumulations are present in the srpk79D mutant (G), and these accumulations are less intense following rescue of the srpk79D mutant with the UAS - srpk79D transgene (H). (I and J) Quantification of Brp fluorescence intensity (I) and puncta intensities (J), comparing control ( C155/+ ), srpk79D mutant ( C155/+;srpk79D atc ), and rescue animals ( C155/+ ; UAS-v-srpk79D-rd*(28)/+;srpk79D atc ). Each bar graph and curve in (I) and (J) represent data collected from a total of 36 nerves from nine different larvae. In (J), n for C155/+ , C155/+;;srpk79D atc , and C155/+;UAS - v-srpk79D-rd*(28)/+ ; srpk79D atc = 2,452, 2,696, and 3,645, respectively. Scale bar = 10 µm. Significance is indicated according to the following: *** = p <0.001 and ns = not significant; Student t -test. Error bars indicate ±SEM. au = arbitrary units.

Article Snippet: The intensity of anti-BRP immunostaining was quantified as follows: Each series of 0.5-µm optical nerve sections was deconvolved (nearest-neighbors; Intelligent Imaging Innovations).

Techniques: In Situ, Immunofluorescence, Expressing, Fluorescence, Mutagenesis, Staining

Journal: PLoS Biology

Article Title: Negative Regulation of Active Zone Assembly by a Newly Identified SR Protein Kinase

doi: 10.1371/journal.pbio.1000193

Figure Lengend Snippet: (A and B) Immunofluorescence images of wild-/type and srpk79D mutant nerves. (C) Similar Brp accumulations appear when GFP-Brp is overexpressed in motor neurons using the motoneuron-specific GAL4 driver OK371-GAL4 . (D) Brp accumulations persist in srpk79D mutants when one copy of brp is deleted by placing a heterozygous brp 69 /+ mutation in the homozygous srpk79D mutant background. (E and F) Immunofluorescence images of wild-type and srpk79D mutant muscle 4 NMJ stained with anti-Brp. (G) Synaptic Brp is increased following GFP-Brp overexpression. (H) Removal of one copy of brp ( brp 69 /+) in a homozygous srpk79D mutant background causes a further decrease in synaptic Brp levels compared to srpk79D mutants alone, but does not cause altered distribution of Brp immunoreactivity. (I) Quantifications of total Brp fluorescence for each indicated genotype normalized to wild type (wt). Each bar graph represents data collected from a total of 29 synapses from 14 different animals. (J) Axonal Brp fluorescence is increased in srpk79D atc /+ heterozygotes ( OK371 /+; srpk79D atc /+) and when Brp is overexpressed in motor neurons ( OK371 /+; UAS-g-brp ). An additive effect is seen when these two perturbations are combined ( OK371 /+; UAS-brp/srpk79D atc ). In all cases, total synaptic anti-Brp fluorescence is significantly less than that seen in srpk79D mutants ( srpk79D atc ). Each bar graph represents data collected from a total of 32 synapses from eight different larvae. Scale bars indicate 10 µm. Significance is indicated according to the following: *** = p <0.001 and ns = not significant; Student t -test. Error bars indicate ±SEM.

Article Snippet: The intensity of anti-BRP immunostaining was quantified as follows: Each series of 0.5-µm optical nerve sections was deconvolved (nearest-neighbors; Intelligent Imaging Innovations).

Techniques: Immunofluorescence, Mutagenesis, Staining, Over Expression, Fluorescence

Journal: PLoS Biology

Article Title: Negative Regulation of Active Zone Assembly by a Newly Identified SR Protein Kinase

doi: 10.1371/journal.pbio.1000193

Figure Lengend Snippet: (A–D) Representative muscle 4 NMJ and individual bouton from control ( C155 /+) and SRPK79D-RD*-overexpressing larvae demonstrating diffuse Brp staining and reduced total Brp fluorescence. Image offset and gain in (A) and (B) are identical. (C) Example of type-1b boutons from control animals demonstrating typical punctate anti-Brp staining (green) and anti-HRP staining (red) to elucidate the nerve terminal membrane. (D) Examples of diffuse Brp staining observed at type-1b boutons within the NMJ of an SRPK79D overexpressing animal. Image offset and gain in (C) and (D) are identical. (E) SRPK79D-RD* overexpression causes a decrease in total synaptic Brp fluorescence. (F) Overexpression of SRPK79D-RD* causes a highly significant decrease in EPSP amplitude. There is a trend toward an increase in the average amplitudes of spontaneous miniature events comparing SRPK79D-RD*-overexpressing animals to wild type ( p = 0.06); representative mEPSPs are shown. (G) Quantification of average EPSP amplitude and quantal content (QC) in SRPK79D-RD*-overexpressing larvae show a greater than 50% decreases in both measures relative to control. C155 /+ and C155 /+; UAS-v-srpk79D-rd*(F) /+ bar graphs represent data collected from a total of 15 synapses from six different larvae and 12 synapses from five different larvae, respectively. Scale bar in (A) indicates 10 µm, and in (C) indicates 5 µm. Significance is indicated according to the following: *** = p <0.001; Student t -test. Error bars indicate ±SEM. HR = anti-horseradish peroxidase.

Article Snippet: The intensity of anti-BRP immunostaining was quantified as follows: Each series of 0.5-µm optical nerve sections was deconvolved (nearest-neighbors; Intelligent Imaging Innovations).

Techniques: Staining, Fluorescence, Over Expression

Journal: iScience

Article Title: ExIR enables prioritizing driver and biomarker genes from omics data in a reference free manner

doi: 10.1016/j.isci.2026.116303

Figure Lengend Snippet: Differential immunoreactivity of known and novel ExIR-predicted LUAD biomarkers between normal and cancer samples (A–E) Immunohistochemical (IHC) data from the Human Protein Atlas database in LUAD and normal lung tissue for top five known LUAD biomarkers. (A) SFTPC–ExIR rank #1, LUAD (negative intensity; patient ID: 1847) and normal pneumocytes (quantity: 75%–25%; strong intensity; patient ID: 2268). (B) SPP1—ExIR rank #117, LUAD (quantity: >75%; moderate intensity; patient ID: 537) and normal pneumocytes (not detected; patient ID: 2268). (C) CBLC—ExIR rank #140, LUAD (quantity: >75%; moderate intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2417). (D) MDK—ExIR rank #247, LUAD (quantity: 75%–25%; strong intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2222). (E) MRC1 – ExIR rank #471, LUAD (undetected; patient ID: 1932) and normal macrophages (quantity: 75%–25%; strong intensity; patient ID: 2208). (F–I) IHC data of top five ExIR-predicted LUAD biomarkers (excluding SFTPC rank #1 already in a). (F) AGER – ExIR rank #2, LUAD (not detected; patient ID: 3144) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 4840). (G) EMP2 – ExIR rank #3, LUAD (not detected; patient ID: 1847) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 2101). (H) CAV1 – ExIR rank #4, LUAD (not detected; patient ID: 1249) and normal pneumocytes (quantity: >75%; strong intensity; patient ID: 2208). i, RTKN2 – ExIR rank 5, LUAD (not detected; patient ID: 3003) and normal pneumocytes (quantity: <25%; moderate intensity; patient ID: 2268).(J–N) IHC data of top six ExIR-predicted LUAD up-regulated biomarkers (excluding FAM83A rank #2, for which no IHC data was available). (J) PYCR1 – ExIR rank #1, LUAD (quantity: >75%; moderate intensity; patient ID: 2777) and normal pneumocytes (not detected; patient ID: 2208). (K) TOP2A – ExIR rank #3, LUAD (quantity: 75%–25%; strong intensity; patient ID: 3003) and normal pneumocytes (quantity: 75%–25%; weak intensity; patient ID: 2101). (L) MMP11 – ExIR rank #4, LUAD (quantity: >75%; weak intensity; patient ID: 1847) and normal pneumocytes (not detected; patient ID: 2438). (M) TEDC2– ExIR rank 5, LUAD (quantity: 75%–25%; moderate intensity; patient ID: 4208) and normal pneumocytes (not detected; patient ID: 1470). (N) IQGAP3– ExIR rank 6, LUAD (quantity: >75%; strong intensity; patient ID: 3048) and normal pneumocytes (not detected; patient ID: 1470). Per the Human Protein Atlas database usage guidelines, the link to the immunostaining images of all of the selected proteins in normal pneumocytes and LUAD samples are included as hyperlinks within the figure legend. Ab: antibody; LUAD: lung adenocarcinoma.

Article Snippet: Per the Human Protein Atlas database usage guidelines, the link to the immunostaining images of all of the selected proteins in normal pneumocytes and LUAD samples are included as hyperlinks within the figure legend.

Techniques: Immunohistochemical staining, Immunostaining